interactions and subcellular distribution of human sun2

Aus dem Zentrum für Biochemie (Medizinische Fakultät) der Universität zu Köln

Institut für Biochemie I

Geschäftsführende Direktorin: Frau Universitätsprofessor Dr. rer. nat. A. A. Noegel

Interactions and subcellular distribution

of human SUN2

Inaugural-Dissertation zur Erlangung der Doktorwürde eines

Doctor rerum medicinalium

der Hohen Medizinischen Fakultät

der Universität zu Köln

vorgelegt von

Eva Mawina Vaylann

Promoviert am: 05.10.2011

Dekanin/Dekan:

Universitätsprofessor Dr. med.Th. Krieg

1. Berichterstatterin: Frau Universitätsprofessor Dr. rer. nat. A. A. Noegel

2. Berichterstatterin: Frau Universitätsprofessor Dr. rer. nat. B. Wirth

Erklärung

Ich erkläre hiermit, dass ich die vorliegende Dissertationsschrift ohne unzulässige Hilfe

Dritter und ohne Benutzung anderer als der angegebenen Hilfsmittel angefertigt habe;

die aus fremden Quellen direkt oder indirekt übernommenen Gedanken sind als solche

kenntlich gemacht. Bei der Auswahl und Auswertung des Materials sowie bei der

Herstellung des Manuskriptes habe ich keine Unterstützungsleistungen bzw.

Unterstützungsleistungen von folgenden Personen erhalten:

Frau Universitätsprofessor Dr. rer. nat. A. A. Noegel.

Weitere Personen waren an der geistigen Herstellung der vorliegenden Arbeit nicht

beteiligt. Insbesondere habe ich nicht die Hilfe einer Promotionsberaterin/eines

Promotionsberaters in Anspruch genommen. Dritte haben von mir weder unmittelbar

noch mittelbar geldwerte Leistungen für Arbeiten erhalten, die im Zusammenhang mit

dem Inhalt der vorgelegten Dissertationsschrift stehen. Die Dissertationsschrift wurde

von mir bisher weder im Inland noch im Ausland in gleicher oder ähnlicher Form einer

anderen Prüfungsbehörde vorgelegt, und ist abgesehen von den angegebenen

Teilpublikationen noch nicht veröffentlicht worden.

Cologne/Köln: 24.02.2011 Signature/Unterschrift: Eva Vaylann

Dedicated to Munhu Mutema

Nyarara uzive ndini Mwari! Rwiyo 46:10

(Shona)

Danksagung

Ich möchte mich vor allem bei Frau Prof. Dr. A. A. Noegel für die Gelegenheit, an ihrem

renommierten Institut meine Dissertation anfertigen zu dürfen sowie für ihre Bereitschaft

meine Arbeit zu korrigieren, bedanken.

Größter Dank gilt meinen lieben Laborkollegen Tanja, Rashmi, Vivek, Karthic S., Xin

Napoleon und Ilknur, die dazu beigetragen haben, dass ich immer gerne an die Zeit im

Labor und die gemeinsam gemeisterten Höhen und Tiefen zurückdenken werde.

Insbesondere danke ich Sonja, Rosi, Berthold, Maria, Rolf und Martina für ihre

Unterstützung im Laboralltag. Ein herzlicher Dank geht auch an Budi und Gudrun für die

stetige und freundliche Hilfe bei organisatorischen und EDV-Angelegenheiten.

Ganz herzlich möchte ich mich auch bei allen meinen lieben aktuellen und ehemaligen

Kollegen bedanken: Kalle, Margit, Raphael, Sascha, Anja, Claudia, Sajid, Bhagyashri,

Karthic T., Liu, Sandra, Juliane, Christoph, Jan, Sze Man, Lin, Khalid, Mary, Georgia,

Verena und Surayya.

Allen anderen Mitgliedern des Instituts möchte ich für ihre Kollegialität und die wirklich

tolle Atmosphäre danken.

Ein besonderer Dank geht an meinen Mann Jens und an meine Freunde und Familie.

Table of contents

Abbreviations

1 Introduction ................................................................................................................. 1

1.1 The LINC complexes ....................................................................................... 1

1.1.1 Nesprins - ONM components of LINC complexes ...................................... 2

1.1.2 SUN proteins, emerin and lamins- INM components of LINC complexes ... 3

1.2 Cellular functions of LINC complexes ........................................................... 5

1.3 LINC complexes and human diseases .......................................................... 6

1.4 Aim of this study ........................................................................................... 10

2 Materials and methods ............................................................................................. 11

2.1 Materials ........................................................................................................ 11

2.2 Methods ......................................................................................................... 14

2.2.1 Molecular biological methods ................................................................... 14

2.2.1.1 Cloning strategies .................................................................................. 14

2.3 Protein chemical and immunological methods .......................................... 19

2.3.1 Protein extraction from E.coli and human cells ......................................... 19

2.3.2 Gel electrophoresis and immunoblotting .................................................. 20

2.3.4 Western blot stripping ............................................................................... 21

2.3.5 Cell fractionation ....................................................................................... 21

2.3.6 Preparation of GST fusion proteins .......................................................... 21

2.3.6 GST pull-down assay ............................................................................... 22

2.3.7 In vitro binding assay ................................................................................ 22

2.3.8 Immunofluorescence microscopy ............................................................. 22

2.4 Cell culture .................................................................................................... 24

2.4.1 Human cell lines and media ..................................................................... 24

2.4.2 Cultivation of mammalian cell lines ........................................................... 24

2.4.3 Freezing and thawing of mammalian cells ................................................ 25

2.5 Cell biological assays ................................................................................... 25

2.5. 1 Transient transfection by electroporation of mammalian cells ................. 25

2.5.2 Senescence-associated β-galactosidase assays ..................................... 26

2.5.3 Focal adhesion assay ............................................................................... 26

2.5.4 Cell synchronization ................................................................................. 27

2.6 Generation of a monoclonal antibody ......................................................... 27

2.6.1 Immunization of Balb/c mice ..................................................................... 27

2.6.2 Generation of hybridoma cells .................................................................. 27

2.6.3 Selection of monoclonal antibodies .......................................................... 28

2.6.4 Purification of IgG from hybridoma supernatant ....................................... 29

3 Results....................................................................................................................... 30

3.1 Human SUN2 protein .................................................................................... 30

3.2 Generation of a mouse monoclonal antibody against the N- terminal

region of human SUN2 ....................................................................................... 31

3.2.1 Determination of an epitope in the N-terminal sequence of human SUN2

suitable for antibody production ......................................................................... 31

3.2.2 Expression and detection of the SUN2NT protein .................................... 32

3.2.3 Identification of positive hybridoma clone K80-207-11 by immunoblot and

immunofluorescence analysis ........................................................................... 33

3.2.4 Transfection of POP10 cells with pJG129SUN2 full length, tagged with

V5*6xHis ............................................................................................................ 35

3.3 Distribution of endogenous SUN2 protein during the cell cycle .............. 38

3.4 Putative interaction partners of SUN2Nt ..................................................... 40

3.5 Direct interaction of SUN2Nt protein with LMNC polypeptides ................ 47

3.6 Characterization of fibroblasts from Duchenne muscular dystrophy

(DMD), Emery-Dreifuss muscular dystrophy/ Charcot-MarieTooth syndrome

(EDMD/CMT) and Stiff skin syndrome (SSS) patients ...................................... 50

3.6.1 Case report of Duchenne muscular dystrophy (DMD), and Emery-Dreifuss

muscular dystrophy/Charcot-Marie-Tooth syndrome (EDMD/CMT) and Stiff skin

syndrome (SSS) patients ................................................................................... 50

3.6.2 Patient fibroblasts show nuclear defects .................................................. 52

3.6.3 Proliferative ability of patient fibroblasts is restricted ................................ 54

3.6.4 Increased senescence is induced in patient fibroblasts ............................ 55

3.6.5 SUN2 gene expression is down-regulated in senescent patient cells ...... 56

3.6.6 Cell adhesion is altered in patient fibroblasts ........................................... 58

3.6.7 Distribution of nuclear envelope proteins in control fibroblasts and patient

fibroblasts .......................................................................................................... 61

3.6.8 Nucleus-centrosome distance is increased in EDMD/CMT, DMD and Stiff

skin syndrome fibroblasts .................................................................................. 68

3.6.9 Precipitation profile in EDMD/CMT fibroblast cells differ from control

fibroblast cells .................................................................................................... 70

4 Discussion ................................................................................................................ 73

4.1 Generation of a monoclonal antibody ......................................................... 73

4.2 Subcellular localization of endogenous SUN2 during the cell cycle ........ 73

4.3 Protein networks formed by SUN2 .............................................................. 74

4.4 Direct interactions of LMNA/C with the N-terminus of SUN2 in vitro ....... 78

4.5 Characterization of fibroblast from Stiff skin syndrome (SSS), Duchenne

muscular dystrophy (DMD) and Emery-Dreifuss muscular dystrophy /

Charcot-Marie-Tooth syndrome (EDMD/CMT) patients ................................... 80

4.5.1 Stiff skin syndrome (SSS) patients ........................................................... 80

4.5.2 Duchenne muscular dystrophy (DMD) and Emery-Dreifuss muscular

dystrophy / Charcot-Marie-Tooth syndrome (EDMD/CMT) ................................ 82

Summary ...................................................................................................................... 92

Zusammenfassung (deutsch) ..................................................................................... 88

References ................................................................................................................... 90

Preliminary Puplications ........................................................................................... 103

Curriculum vitae/Lebenslauf .................................................................................... 104

Abbreviations % aa Amp

APS

Aqua dest. ATP

bp

BSA

C

ca. CIP

Cm CoREST DMEM

DMSO

DNA DTT E. coli EDTA

ER

EtBr EtOH FCS

g

g

GAPDH GbM GST hr HEPES INM

KASH kb

kDa

λ M

mAb

MDa min

µg

ml µm

µl mM

mRNA NCoR

NE

ng

Percent Amino acid(s) Ampicilline Ammonium persulfate Aqua destillata, destilled water Adenosine triphosphat Base pair(s) Bovine serum albumine Celsius or nucleotide Cytosine Circa, approximately Calf intestine alcaline phosphatase Centimetre Corepressor for RE1 silencing transcription factor Dulbecco´s Modified Eagle Medium Dimethylsulfoxide Deoxyribonucleic acid Desoxyribonucleotidetriphosphat Dithiothreitol Escherichia coli Ethylen-Diamine-Tetra-acetate endoplasmatic reticulum Ethidiumbromide Ethanol Fetal calf serum Gramm Relative centrifugation force Glycerinaldehydephosphate dehydrogenase glioblastoma multiforme glutathione S-transferase Hour(s) N-(2-Hydroxyethyl)piperazin-N´-2-ethansulfonsäure Inner nuclear membrane Klarsicht/Anc-1/Syne homology kilo base kilo Dalton Wave length Molar Monoclonal antibody Mega Dalton Minute Microgramm Milliliter Micrometer Microliter Millimolar Messenger Ribonucleic acid nuclear receptor co-repressor Nuclear envelope Nanogramm

nm NURD ONM

ORF

pAb

PAGE

PBS

PCR

PNS

RNA

rpm

RT

SDS

sec SMRT

SUN

TAE

TE

Taq TEMED

Tris

U

UV

V

v/v w/v X-Gal

Nanometer Nucleosome remodeling and histone deactylation Outer nuclear membrane Open reading frame Polyclonal antibody Polyacrylamide gel electrophoresis Phosphat buffered saline Polymerase chain reaction Perinuclear space Ribonucleic acid Rounds per minute Room temperature Sodium dodecyl sulfate Second Silencing Mediator of Retinoid acid and Thyroid hormone receptor Sad1/UNC-84 homology Tris-Acetate-EDTA Tris-EDTA Thermus aquaticus N,N,N´,N´-Tetramethyl-ethylendiamin Trishydroxyaminomethan Unit Ultraviolet light Volt Volume per volume Weight per volume 5-Bromo-4-chlor-3-indolyl-β-D-galactopyranoside

Introduction

1

1 Introduction

1.1 The LINC complexes

The nucleus is separated from the cytoplasm by a double membrane, the outer

nuclear membrane (ONM) and the inner nuclear membrane (INM). The lumen

between both membranes is the perinuclear space (PNS). Linker of the

nucleoskeleton and cytoskeleton (LINC) complexes physically connect the nuclear

interior with the cytoskeleton. They consist of an INM transmembrane protein and an

ONM transmembrane protein which physically interact with each other in the PNS.

The INM LINC component interacts on the nucleoplasmic side with either the lamina,

a meshwork of intermediate filaments, or with an INM-associated protein. The ONM

LINC component on the other hand contacts on the cytoplasmatic side components

of the cytoskeleton. In mammals, the LINC complexes include nesprins NESPRIN1/2

(Nuclear envelope Spectrin repeat), SUN (Sad1p, UNC-84) domain proteins, emerin,

F-actin, microtubules, intermediate filaments, plectin, laminA/C (LMNA/C) and

chromatin (Fig. 1), (Crisp et al., 2006; Tzur and Gruenbaum, 2006).

Fig. 1: The LINC complex facilitates the coupling of the nuclear lamina to cytoplasmic cytoskeletal

systems comprised of SUN proteins in the INM that binds to the nuclear lamina via interactions with

laminA/C and potentially smaller nesprin isoforms and emerin. The KASH domain of the larger

isoforms of NESPRIN1/2 at the ONM associates with the SUN-domain of the SUN proteins within the

perinuclear space to tether the NE to either cytoplasmic actin, IFs or microtubule network and links the

MTOC. In the muscle sarcomere, nesprins are present in the sarcoplasmic reticulum, Z-line and A/I

junction and potentially link these structures and the actin cytoskeleton (Zhang et al, 2007). Question

marks indicate suggested but not proven interactions.

I-band

A-band

Z Z

emerin

LMNA/C

chromatin

actin- filaments/myosin

plasmamembrane

ONM

INM

NESPRIN 1/2

actin/myosin titin

SUN2

NESPRIN1α

α-actinin vinculin

talin

α/β intergrins

sarcoplasmatic reticulum

intermediate filaments

microtubules

MTOC

?

?

nucleus

Introduction

2

1.1.1 Nesprins - ONM components of LINC complexes

In mammalian cells, two giant (up to 1 MD) actin-binding proteins have been

identified (variously termed NUANCE, NESPRIN2 Giant [NESP2G], SYNE2,

NESPRIN1, ENAPTIN, SYNE1, and MYNE1) as integral proteins of the ONM. They

are encoded by the genes SYNE1 and SYNE2 and belong to type II membrane

proteins (Zhang et al., 2001; Mislow et al., 2002; Zhen et al., 2002; Padmakumar et

al., 2004). Both proteins are composed of an N-terminal alpha-actinin-like actin

binding domain, a long rod domain which harbors spectrin repeats and a highly

conserved C-terminal KASH (Klarsicht/ANC-1/Syne homologue) domain which is

sufficient for nuclear envelope targeting of these proteins. Via their N-terminal actin

binding domains NESPRIN1 and 2 bind to filamentous actin (F-actin) whereas the

KASH and transmembrane domains mediate their localization to the NE (Zhen et al.,

2002; Zhang et al., 2005). The KASH domains of NESPRIN1 and 2 directly bind to

SUN domain proteins at the INM stabilizing their interaction with the inner NE

(Padmakumar et al., 2005).

Mammalian NESPRIN1 and NESPRIN2 genes display enormous complexity,

generating a wide variety of transcripts that differ in length, domain composition,

expression pattern and probably in their functional properties (Zhang et al., 2002).

Multiple nesprin isoforms are produced by alternative splicing and transcription

initiation (Fig. 2). Moreover, the number of individual nesprin genes increased to four

with NESPRIN3 and 4 being significantly smaller proteins (Zhen et al., 2002;

Wilhelmsen et al., 2005; Warren et al., 2005; Roux et al., 2009).

Nesprins are also characterized by variable N-terminal motifs that enable interactions

with different components of the cytoskeleton. Giant isoforms of NESPRIN1 (~1

MDa) and NESPRIN2 (~800 kDa) have N-terminal calponin homology (CH) domains

that link to F-actin. NESPRIN3 (~110 kDa) contains a plectin-binding motif that

permits interactions with cytoplasmic intermediate filaments (IFs) while NESPRIN4

interacts indirectly with microtubules (Crisp et al, 2009). Via the KASH domain

nesprins localize to both the INM and ONM and are partitioned to these domains

depending on their size and binding partners. Thus, nesprins can form structural

connections on either face of the NE (Shanahan et al, 2010).

Introduction

3

Fig.2: Scheme of multiple nesprin isoforms of variable length and with different SR domains. Picture

was taken from Shanahan et al., 2010.

1.1.2 SUN proteins, emerin and lamins- INM components of LINC complexes

Mammalian SUN1 and SUN2 proteins were first identified by bioinformatic analysis

as homologues of C. elegans UNC84. Later, they have been confirmed in screens for

NE components (Dreger et al., 2001; Malone et al., 1999; Schirmer et al., 2003).

Based on shared homology between sad1 in Schizosaccharomyces pombe and

UNC84 in Caenorhabditis elegans the evolutionarily highly conserved sad1p-UNC84

SUN domain was identified suggesting that this family of proteins has crucial nuclear

and cellular roles.

Several SUN domain proteins have been identified in mammals, including SUN1,

SUN2, SUN3, SPAG4 and SPAG4L 46. SUN3 expression seems to be restricted to

testes and its localization is limited to the ER (Crisp et al., 2006). SPAG4 is only

Introduction

4

expressed in spermatids, pancreas and testes (Shao et al., 1999). SUN1 and 2 are

widely expressed. All SUN proteins are conserved type-II INM proteins and contain at

least one transmembrane domain and a C-terminal SUN domain localized inside the

lumen of the NE. KASH-domain proteins are recruited to the NE by binding to SUN

domain proteins SUN1 and SUN2 within the perinuclear space, forming the LINC

complex. Overexpression of dominant-negative KASH domain constructs and

knockdown of LINC components NESPRIN1/2, SUN1/2 or LMNA uncouples the INM

from the ONM, detaches the nucleus from the cytoskeleton and decreases

mechanical stiffness (Hodzic et al, 2009).

Emerin is together with LAP2 (lamina associated polypeptide) and MAN1 a LEM

(LAP2, Emerin, MAN1) domain-containing integral membrane protein of the nuclear

membrane in vertebrates. The LEM domain is composed of a motif of about 43

amino acids that is exposed to the nucleoplasm and interacts with BAF (barrier to

autointegration factor), an abundant chromatin-associated protein (Lin et al., 2000;

Laguri et al., 2001; Shumaker et al., 2001). Emerin is known to interact with nuclear

lamins and NESPRIN1α, and stabilizes and promotes the formation of a nuclear actin

cortical network. Emerin links centrosomes to the nuclear envelope via a microtubule

association. It is also reported to be involved in β-catenin inhibition by preventing its

accumulation in the nucleus (Holaska et al., 2004; Markiewicz et al., 2006;

Salpingidou et al., 2007).

Nuclear lamins are type V intermediate filament proteins containing a central α-

helical rod flanked by N- and C-terminal non-helical domains (Fig. 3). Most lamins,

except for LMNC, are farnesylated at their carboxy termini via a CaaX motif. LMNA

further contains a site for endoproteolytic cleavage that is recognised by ZMPSTE24-

FACE1 enzyme (P2) which cleaves the protein and removes the farnesylated

cysteine. LMNC, on the other hand does not undergo such post-translational

modifications (Hutchison et al., 2001).

Based on their primary sequences and biochemical features, lamins are subdivided

into A-type and B-type lamins (Gerace et al., 1978). Both major (A and C) and minor

(A∆10 and C2) A-type lamin species are encoded by a single developmentally

regulated gene (LMNA) and arise through alternative splicing (Fisher et al., 1986). By

contrast, the main B-type lamins (B1 and B2) are encoded by two separate genes

Introduction

5

LMNB1 and LMNB2 (Hoeger et al., 1990). A single minor B-type lamin (B3) is a

splice variant of LMNB2 (Furukawa and Hotta, 1993).

Fig.3: Basic structure of mammalian lamins; light blue: N-term. head domain (left); C. term. domain

(right), dark blue: coiled-coil domain; green: NLS (nuclear localisation signal); CaaX: posttranslational

modification motif

1.2 Cellular functions of LINC complexes

By linking the nuclear lamina with the cytoskeleton, LINC complexes play key roles in

many crucial cellular functions including cell proliferation, cytoskeleton organization

and organelle positioning. The conserved SUN domain proteins and the KASH

domain proteins of the nuclear envelope (NE) have been identified as molecular

linkers, which position the nucleus on actin filaments, intermediate filaments,

microtubules and the centrosome. Several studies revealed that SUN1 and SUN2

also form a physical interaction between the NE and the centrosome (Zhang et al.,

2009, Koizumi and Gleeson, 2009).

Proper nuclear positioning relative to the cell body is important for many cellular

processes during mammalian development. It has been shown that SUN-KASH

protein complexes function in synaptic and nonsynaptic nuclear anchorage.

Organization of synaptic and nonsynaptic nuclei and the localization of NESPRIN1 to

the NE of muscle cells are disrupted in Sun1/2 double-knockout mice. This indicates

prelamin A

mature LMNA

prelamin B

mature LMNB

prelamin C

mature LMNC

Introduction

6

critical functions for SUN1 and SUN2 in skeletal muscle cells for NESPRIN1

localization at the NE, which is essential for proper myonuclear positioning (Zhang et

al., 2008, Gundersen et al., 2011).

SUN-KASH protein complexes are also required for alignment of homologous

chromosomes, their pairing and recombination in meiosis. Mammalian SUN domain

proteins SUN1/2 and KASH-domain proteins NESPRIN1/2 are involved in nuclear-

centrosome coupling during cortical neuronal migration and interkinetic nuclear

migration during neurogenesis (Zhang et al., 2009). Beside this, certain roles in the

regulation of apoptosis and maturation and survival of the germline have been

proposed (Daboussi et al., 2005; Prasanth et al., 2004; Prasanth et al., 2002). SUN-

KASH-linkages contribute to the structural integrity of the NE in maintaining the

precise separation of the two membranes. Moreover, the SUN-KASH links provide

direct molecular connections between the actin cytoskeleton and the nuclear interior

due to the fact that giant nesprins are F-actin binding. This mechanical link not only

provides structural continuity within and between cells but it also allows for a direct

physical signaling pathway from the cell surface to the nucleus, potentially facilitating

rapid and regionalized gene regulation (Lifeng et al., 2007; Hassold et al., 2007;

Hassold and Hunt, 2001; Linge et al, 2001).

1.3 LINC complexes and human diseases

The centrosome-nucleus attachment is a prerequisite for faithful chromosome

segregation during mitosis, and centrosome abnormalities may therefore cause

chromosome missegregation promoting genome instability such as aneuploidy,

which are the hallmarks of all solid tumors. Recent studies reveal that centrosome

defects, including an excess number of centrioles, increased microtubule nucleation

capacity, and inappropriate phosphorylation of centrosomal proteins, are features of

malignant breast tumors and solid tumors in general. Presently two models for the

origin of centrosome defects in the development of cancer are being discussed. In

the first model, centrosome amplification arises through the failure of cytokinesis and

the consequent failure of equal partition of sister chromatids and spindle poles into

daughter cells. Therefore, a single 4N daughter cell inherits both spindle poles,

instead of just one, to yield two functional centrosomes. The two centrosomes double

again in the next cell cycle to yield four functional spindle poles and multipolar

Introduction

7

mitosis. In the second model, centrosome amplification arises through a deregulation

of the centriole duplication cycle leading to centrosomes with supernumerary

centrioles. Disruption of key cell and/or centrosome cycle regulators may play a

causative role. These models are not mutually exclusive and may operate

independently or sequentially in the development of cancer. Centrosome

amplification leads to an increased frequency of multipolar mitosis and consequent

chromosomal instability, and therefore, is one mechanism by which aneuploidy and

phenotypic variability arise in the development of cancer (Hassold et al., 2007; Lingle

et al., 2001; Salisbury et al., 2005; Fukasawa et al., 2005; Nigg et al, 2006).

Numerous mutations in the genes encoding the nuclear envelope proteins were

found to cause a wide range of human diseases, known collectively as nuclear

envelopathies or laminopathies. Examples are the autosomal-dominant form of

Emery-Dreifuss muscular dystrophy (AD-EDMD), dilated cardiomyopathy with

conduction system defects disease (DCMCD), Limb-girdle muscular dystrophy 1B

(LGMD1B), Dunnigan-type familial partial lipodystrophy (FPLD), atypical Werner

syndrome, Charcot-Marie-Tooth syndrome 2B (CMT2B) and Hutchinson-Gilford

progeria syndrome (HGPS). Muscle defects are common amongst the laminopathies

despite the large spectrum of affected tissues and disease phenotypes and more

than 80% of LMNA mutations lead to cardiac and/or skeletal muscle pathologies

(Muchir et al, 2000; Vytopil et al 2002; Verstraeten et al., 2007; Rankin et al 2008).

Laminopathies affecting specifically either striated muscles, the peripheral nerves, or

the adipose tissues are classified as “tissue-specific” laminopathies. In contrast, if

several tissues are affected concomitantly like in premature ageing syndromes, they

are tentatively classified as “systemic laminopathies”. Additionally to these two main

categories, a still expanding class of laminopathies corresponds to clinical

heterogeneous situations which are characterized by the coexistence of two or more

tissue involvements. These “overlapping laminopathies” suggest the existence of a

real continuum within all the different types of laminopathies (The UMD-LMNA

database: http://www.umd.be/LMNA/W_LMNA/).

For many of these disorders including EDMD, there is variable penetrance and

phenotypic heterogeneity which suggest that mutations in other, presently unknown

Introduction

8

modifier genes and their products, may contribute to the variable phenotypic

expression of the diseases (Politano et al, 2003). Also, approximately 60% of

patients with EDMD or EDMD-like phenotypes do not have mutations in either EMD

encoding for emerin, or LMNA suggesting the involvement of other genes and/or

gene products which are likely binding partners of emerin and LMNA/C at the INM,

particularly those highly expressed in muscle tissue (Zhang et al, 2007; Puckelwartz

et al., 2009; 2010).

EDMD is typically characterized by the clinical triad of 1) early contractures of the

achilles tendons, elbows and postcervical muscles (with subsequent limitation of

neck flexion, but later forward flexion of the entire spine becomes limited); 2)

progressive skeletal muscle weakness and wasting with a humero-peroneal

predominance at the onset of the disease (i.e. proximal in the upper limbs and distal

in the lower limbs) and 3) a life threatening cardiac disease where conduction defects

coexist with ventricular and supraventricular arrhythmias, chamber dilation and heart

failure.

Charcot-Marie-Tooth disease (CMT) constitutes a clinically and genetically

heterogeneous group of hereditary motor and sensory neuropathies. On the basis of

electrophysiological criteria, CMT are divided into two major types: type 1, the

demyelinating forms, characterized by a motor median nerve conduction velocity less

than 38 m/s; and type 2, the axonal form, with a normal or slightly reduced nerve

conduction velocity (The UMD-LMNA database).

Similar to mutations affecting the LINC complex proteins, disruption of signal

transmission pathways that occur upstream of the LINC complex in cytoskeletal

proteins can contribute to the wide spectrum of laminopathies. These pathways

reach from the extracellular matrix to the nuclear envelope. At the plasma membrane

cell-cell and cell-extracellular matrix interactions are mediated by integrin receptors

and dystroglycan which transduce signals coming from the matrix and link the

extracellular matrix to the actin cytoskeleton (Moore et al, 2010). The cytoskeleton is

connected to the internal nuclear envelope through the LINC complex which in turn is

connected with chromatin binding nuclear lamins (Fig. 1). Thus, mechanical forces

Introduction

9

can be transmitted directly from the extracellular matrix to the nuclear interior (Mejat

and Misteli, 2010).

Responsible for the connection of the cytoskeleton of each muscle fiber to the

dytsroglycan complex and the extracellular matrix is dystrophin. Mutations of the

dystrophin gene at locus Xp21 causes Duchenne muscular dystrophy, the most

common form of muscular dystrophies, which is an X-linked disorder characterized

by progressive wasting of skeletal muscles. First, limb-girdle muscles show

weakness by the age of 3 to 5 years, followed by an inability to walk by the age of 8

to 12 years. Other findings include elevated creatine kinase levels,

pseudohypertrophic calf muscles, and cognitive impairment in some patients.

Weakness of respiratory muscles leads to restrictive lung disease and eventual

respiratory failure in severe cases. Histopathological findings include absence of

dystrophin from the membrane of muscle fibers, increased adipose and connective

tissue between muscle fibers, increased variability in muscle fiber size, infiltration of

inflammatory cells, and centrally located nuclei, which are indicative of degenerating

and regenerating muscle fibers (Ehmsen et al., 2002; Lovering et al., 2004; Porter et

al., 2005).

Stiff Skin syndrome is characterized by an early onset of stony-hard skin, with

associated contracture like joint restriction, hypertrichosis, and postural and thoracic

wall abnormalities. Occasional findings include focal lipodystrophy and muscle

weakness. Histopathologic findings consist of either fascial sclerosis or increased

fibroblast cellularity with sclerotic collagen bundles in the deep reticular dermis and/or

subcutaneous septa (Liu et al., 2008). It is suggested that the defect is a heritable

disorder of the autosomal dominant type. The possibility of an autosomal recessive

pattern of inheritance can not be excluded since many of the reported cases are

progeny of consanguineous marriage (Esterly et al., 1971, Jablonska et al., 2000).

Cells from a patient suffering from the syndrome have been included in this study

based on findings that nuclear envelope proteins are involved in skin disease (Youn

et al., 2010).

Introduction

10

1.4 Aim of this study

To address the function and subcellular distribution of SUN2 and its possible role in

muscle dystrophies in different patient cell lines, a newly generated monoclonal

antibody against the N-terminal region was used. Its use in immunoblot and

immunofluorescence applications and the identification of interaction partners of

SUN2 should deliver novel insights into the function and subcellular distribution of

SUN2 during the cell cycle.

Furthermore, a characterization of three different primary cell lines from laminopathy

patients affected by either Stiff Skin syndrome (SSS), Duchenne muscular dystrophy

(DMD) which originally had been described as Emery-Dreifuss muscular dystrophy

(EDMD), and Emery-Dreifuss muscular dystrophy/Charcot-Marie-Tooth syndrome

(EDMD/CMT) was carried out.

In case of the DMD and EDMD/CMT patient cell lines it was initially reported that

they carry mutations in the LINC complex component NESPRIN1 which were then

claimed to be responsible for the disease (Zhang et al., 2007). Based on these

description components of the LINC complex and characteristics of the cells that are

associated with the complex were analysed. After completion of the project further

mutations were discovered and were taken into account in the discussion.

Materials and methods

11

2 Materials and methods

2.1 Materials

Standard laboratory reagents and materials were obtained from local suppliers, fine

chemicals from Sigma if not otherwise indicated and instruments were supplied by the

departmental facility.

Kits

M-MLV reverse transcriptase RNase H Minus-kit Promega

NucleoSpin Extraction Kit Macherey Nagel

pGEM-T easy Cloning Kit Promega

Pure YieldTM Plasmid System Promega

Qiagen RNeasy Mini Kit Qiagen

Enzymes

Calf intestinal alkaline phosphatase (CIAP) Boehringer

Lysozyme Sigma

Restriction endonucleases Life technologies, NEB

RNAse Boehringer

T4 DNA ligase Boehringer

Taq polymerase Boehringer

Thrombin Amersham

Trypsin Invitrogen

Inhibitors

Complete mini protease inhibitor cocktail Sigma

nocodazole Sigma

Antibiotics

Ampicillin Sigma

Kanamycin Sigma

Penicillin/Streptomycin Biochrom


12

Primary antibodies

Rabbit-anti-GST pAb Institute of Biochemistry

Goat-anti-emerin pAb Santa Cruz

Mouse-anti-SUN2Nt mAb (K80-207-11) this study, E. Vaylann

Mouse-anti-LAP2 mAb BD Biosciences

Mouse-anti-tubulin WA3 mAb U. Euteneuer (thesis Y.

Lücke)

Mouse-anti-V5 mAb Invitrogen

Mouse-anti-vinculin mAb Sigma

Rabbit-anti-His mAb Invitrogen

Rabbit-anti-LMNB1 pAb Abcam

Rabbit-anti- LMNA/C pAb Santa Cruz

Rabbit-anti-NESPRIN1SpecII pAb S. Abraham, Thesis,

2004

Rabbit-anti-NESPRIN2abd pAb Libotte et al., 2005

Rabbit-anti-pericentrin pAb Abcam

Secondary antibodies

Anti-mouse IgG, peroxidase-coupled Sigma

Anti-rabbit IgG, peroxidase-coupled Sigma

Anti-goat IgG, peroxidase-coupled Sigma

Anti-mouse IgG, Alexa488-conjugated Sigma

Anti-mouse IgG, Alexa568-conjugated Sigma

Anti-goat IgG, Alexa568-conjugated Sigma

Bacterial host strains

E. coli XL1 Blue Bullock et al., 1987

E. coli BL21 (Hanahan, 1983)

Vectors and plasmids

pJG129SUN2 human FL Dr. J. Gotzmann

(Biocenter, Vienna).

pGEMTeasy Promega

pGEX-4T1 GE Healthcare


13

pGST-LMNC-N-term/laminA (aa 1-127) Libotte et al., 2005

pGST-LMNC-coil1B-∆ (aa 128-218) Dreuillet et al., 2002

pGST-LMNC-coil2 (aa 243- 387) Dreuillet et al., 2002

pGST-LMNC-tail (aa 384-566) Dreuillet et al., 2002

pGST-∆LMNC (aa 128-572) Dreuillet et al., 2002

Oligonucleotides

Oligonucleotides for PCR were purchased from Roth GmbH (Karlsruhe), Germany

SUN2Nt 1-139.3´ CGCGAATTCATGTCCCGAAGAAGCCAGCGC-3´

SUN2Nt 1-139.5´ CGCCTCGAGGTCGTCCTCAGAGGAGTAGCC-5´

GAPDH3’ GCCGTCTAGAAAAACCTGCCAAATATGATG-3’

GAPDH5’ GTGAGGGTCTCTCTCTTCCTCTTGTGCTCT-5’

Anesthetics

Isoflurane (2-chloro-2-(difluoromethoxy)-1, 1, 1-trifluoro-ethane)


14

2.2 Methods

2.2.1 Molecular biological methods

Standard molecular biology techniques were performed as described in "Laboratory

Manual", Cold Spring Harbor Laboratory Press, NY, Vol. 1-3 (Sambrook et al., 1989).

All media, solutions and reagents that have been used are given in the corresponding

sections. Media and buffers were prepared using deionized water, filtered through an

ion-exchange unit (Membrane Pure). All media and buffers were sterilized by

autoclaving at 120 °C; the antibiotics were added to the media after cooling to approx.

50 °C. Agar plates were prepared using a semi-automatic plate-pouring machine

(Technomat).

2.2.1.1 Cloning strategies

Plasmid pJG129 containing V5·6xHis-tagged full-length human SUN2 was kindly

provided by Dr. Josef Gotzmann (Biocenter, Vienna). It was used for SUN2Nt

amplification by SUN2Nt 1-139.3´ and SUN2Nt 1-139.5´ primers designed from

published human SUN2 DNA sequences (accession No. AY682988) purified by gel-

extraction and cloned into pGEMTeasy vector. After verification by sequencing SUN2Nt

was cloned into EcoRI and XhoI cut pGEX4T-1 vector.

2.2.1.2 Polymerase chain reaction (PCR)

Reaction mixture: PCR buffer:

× µl Template DNA (10 ng cDNA or plasmid DNA) 100 mM Tris/HCl; pH 8.3

1 µl Oligonucleotides (primer) A (10 pmol/ml) 500 mM KCl

1 µl Oligonucleotides (primer) B (10 pmol/ml) 20 mM MgCl2

1 µl dNTP mix (10mM)

5 µl 10 × PCR buffer

1 µl Taq polymerase (3-4U)

Add aqua dest. to 50 µl


15

2.2.1.3 Ligation of vector and DNA fragments

Ligation buffer Ligation reaction

150 mM Tris/HCl; pH 7,8 Linearized vector (200-400 ng)

50 mM MgCl2 DNA-fragment (1-2-µg)

50 mM DTT 4 µl 5x Ligation buffer

5 mM ATP 1µl T4-ligase (1U/µl)

25% PEG-6000 Add H2O to 10 µl

Ligation reaction of vector/SUN2Nt with a ratio of 5:1 was catalyzed by T4-DNA-ligase

incubated overnight 12-16 hours at 8 °C.

2.2.1.4 Dephosphorylation of DNA fragments

10x CIAP-Puffer (pH 9.0)

5 M Tris/HCl (pH 9.0)

10 mM MgCl2

10 mM MgCl2

10 mM spermidin

To prevent religation, 5´-ends of the linearized plasmid were dephosphorylated by calf

intestinal alkaline phosphatase (CIAP). Therefore, 1-5 µg of the vector were incubated

with 1 U of CIAP in a 50 µl reaction volume (37 °C, 10 min). The enzyme was removed

by extraction with phenol/chloroform and precipitated with 1/10 volume sodium acetate,

pH 5.2 and 2.5 volume of 96% ethanol.

2.2.1.5 Restriction digestion of DNA

Digestion of DNA with restriction endonucleases was performed in buffer systems

provided by the manufacturers at the recommended temperatures.

2.2.1.6 Plasmid DNA preparation from E. coli

2.2.1.6.1 DNA-Mini-preparation (Birnboim et al., 1979)

With this DNA isolation method plasmid DNA from small amounts of bacterial cultures

was prepared.

An overnight E. coli culture was centrifuged for 2 minutes (5000 g). The pellet was

suspend in 300 µl B1, 300 µl B2 was added, mixed, incubated (5 min, RT), 300 µl B3


16

was added, mixed again and centrifuged for 20 minutes (14.000g, 4 °C). The

supernatant was precipitated with 0.8 ml isopropanol. Then, the DNA was pelleted by

centrifugation (14.000 g, 4 °C, 20 min). Finally, the precipitate was washed with 200 µl

of 70% ethanol. After a further centrifugation step the ethanol was discarded and the

plasmid DNA was dried on air and suspend in 10-20 µl Tris/HCl; pH 8.0-8.5.

B1 B2 B3

50 mM Tris/HCl; pH 8,0 0,2 N NaOH 3 M KAc; pH 5,5

10 mM EDTA 1% SDS

100 µg/ml RNAse

2.2.1.6.1 DNA-Midi/Maxi preparation – Pure YieldTM Plasmid System

Buffer and Solutions:

Cell Suspension Solution Cell Lysis Solution

50 mM Tris/HCl; pH 7.5 10 mM EDTA; 8.0 0.2 M NaOH 1% SDS

5 10 mM EDTA; 8.0

100 µg/ml RNase A

neutralization solution column wash

4.09 M Guanidinium hydrochloride 60% ethanol

759 mM Potassium acetate 60 mM Potassium acetate

2.12 M Glacial acetic acid 8.3 mM Tris/HCl; pH 7.5

0.04 mM EDTA

endotoxin removal wash

nuclease free water

Instruments/Materials:

clearing column, binding column, vacuum station

High amounts of plasmid DNA were needed (10-15 µg plasmid DNA/transfection) for the

transfection of eukaryotic cells by electroporation. Therefore, 50-100 ml midi/250 ml

maxi, of an overnight E. coli culture were centrifuged (5000 g, 5 min) and subsequently


17

the pellet was suspend in 3 ml/6ml cell suspension solution. After the addition of 3 ml/6

ml cell lysis solution the mixture was carefully mixed followed by an incubation step at

room temperature for 2 minutes. The reaction was then stopped by adding 5 ml/10 ml

neutralization solution. Furthermore, the mixture was incubated at room temperature for

3 minutes, and subsequently centrifuged (15.000g, 10 min). DNA was bound to the resin

of a binding-column, washed with 5 ml endotoxin removal wash followed by a washing

step with 20 ml column wash solution and eluted with 600 µl nuclease free water.

2.2.1.7 Phenol/chloroform extraction and ethanol precipitation of DNA

In order to separate DNA from proteins in a DNA-containing solution phenol-chloroform

extraction was used. To the aqueous solution one volume phenol was added, shaken

and centrifuged at 14.000 g for 5 min. One volume of chloroform was added to the DNA

containing aqueous phase, mixed and centrifuged at 14.000 × g for 5 minutes. DNA

precipitation was performed by adding 1/10 volume of sodium acetate, subsequent short

shaking, and further addition of 2.5 volume of 96% ethanol. The solution was mixed,

incubated (−70 °C, 15 min) and centrifuged (16.000 g, 4 °C, 15 min). The pellet was

washed with 50-100 µl 70% ethanol and after renewed pelleting at 16.000 g, 4 °C for 5

min the DNA was suspended in 20-30 µl de-ionized and sterile H2O.

2.2.1.8 Isolation of DNA fragments from agarose gels

Bands containing DNA fragments of interest were excised from the agarose gel and

subsequently purified using a gel elution kit (NucleoSpin Exctraction kit); the DNA was

bound to a silica matrix, washed several times and eluted with a low salt solution.

2.2.1.9 Measurement of DNA and RNA concentrations

Concentrations of DNA and RNA were estimated by determining the absorbance at a

wavelength of 260 nm. A ratio of OD260/OD280 >2 higher than 2.2 indicates that a

contamination with phenol could have happened whereas a ratio under 1.8 suggests a

contamination with proteins.

2.2.1.10 Total RNA isolation and cDNA generation for RT-PCR analysis

Total RNA was extracted from cells grown in a monolayer in cell culture dishes with a kit

following the instructions. The concentration of the RNA was measured with a UV

spectrophotometer. First-strand cDNA synthesis was performed using M-MLV reverse


18

transcriptase RNase H Minus-kit from Promega. Briefly, total RNA was reverse

transcribed using oligo (dT) primers and reverse transcriptase (Promega) according to

the manufacture’s instructions. The cDNA (2 µg) was amplified with sets of specific

primers for human SUN2 for 36 cycles using the following conditions: 94°C for 30

seconds, 71, 5°C for 45 seconds and 72°C for 45 seconds, resulting in a 417-bp cDNA

coding for human SUN2. GAPDH was amplified as control using GAPDH specific

primers. Different concentrations of Annexin DNA were used as internal calibration

alignment.

2.2.1.11 Transformation of plasmids in E. coli

SOC-medium

2% Bacto-Trypton

0.5% Yeast extract

10 mM NaCl

2.5 mM KCl

10 mM MgCl2

10 mM MgSO4

20 mM Glucose

5 ng of plasmid DNA were mixed with 200 µl competent E. coli cells and incubated on

ice for 5 minutes. The cells were then heat-shocked (42 °C, 60 sec) and immediately

transferred to ice for 5 minutes. 1 ml SOC-medium was added and the cells were

shaken (600 rpm, 37 °C, 1 h). Finally, 50-150 µl of the transformation mix was plated

onto selection plates and the transformants were grown overnight (37 °C).

2.2.1.12 Blue-white selection

If plasmid vectors with a multiple cloning site located in the ß-galactosidase ORF were

used such as pGEMTeasy, a blue-white selection could be performed. For this selection

plates were coated with 30 µl of the substrate X-Gal and 10 µl of the lac-operon inductor

IPTG (isopropyl-thiogalactosid) in addition to the according antibiotics. In cells carrying

the original vector the enzyme ß-galactosidase was expressed and could subsequently

convert the substrate X-Gal (Br-Cl-Indoxyl-ß-D-Galactoside) into Br-Cl-Indoxyl that

shows a blue colouring. In contrast, colonies carrying plasmids with insert appeared

white.


19

2.2.1.13 Stocks of E. coli cultures

Glycerol stocks of all bacterial strains/transformants were prepared for long-term

storage. The colony of interest was grown in LB-medium containing the selective

antibiotic. 850 µl of an overnight culture was added to 150 µl of sterile glycerol and

stored at −80 °C.

2.3 Protein chemical and immunological methods

2.3.1 Protein extraction from E.coli and human cells

E.coli cells were lysed with prokaryotic lysis buffer (PLB). Human cells were rinsed with

icecold PBS and lysed either in hypotonic lysis buffer (HLB) or RIPA buffer. To obtain

cell lysates for subsequent incubation with GST-Sepharose 4B, 1% Triton X-100 was

added to the respective lysis buffer. After sonification the soluble fraction of the lysate

was obtained by centrifugation (13000 g, 4 °C, 20 min).

RIPA buffer HLB PLB

1M Tris/HCl; pH7.5 1 M HEPES, pH7.9 50 mM Tris-HCl, pH 7.5

5 M NaCl 1 M MgCl2 150 mM NaCl

10% NP 40 2.5 M KCl lysozyme

10% deoxycholate 1 M DTT 1% Sarcosyl

Protease inhibitor cocktail

PIC (Sigma)


PIC (Sigma)


PIC (Sigma)

PBS (pH 7.2)

10 mM KCl

10 mM NaCl

16 mM Na2HPO4

32 mM KH2PO4


20

2.3.2 Gel electrophoresis and immunoblotting

SDS-loading buffer was added to protein samples and the proteins separated on 10%

polyacrylamide gels (SDS-PAGE), then either stained with Coomassie Brillant Blue

R250 and subsequent destained using Destain solution, or transferred onto

nitrocellulose membranes (Schleicher and Schuell) for semi-dry or wet blotting transfer,.

After transfer, the membranes were blocked with 5% (w/v) milk powder in 1x NCP prior

to the appropriate antibody detections. The primary antibodies were detected using the

according peroxidase-conjugated secondary antibodies and visualized by enhanced

chemiluminescence (ECL) reactions. ECL reactions on the nitrocellulose membranes

were documented on X-ray films.

5 × SDS loading buffer 10 × SDS-PAGE running buffer

2.5 ml 1M Tris/HCl; pH 6.5 0.25 M Tris

4.0 ml 10% SDS 1.9 M Glycin

2.0 ml Glycerol 1% SDS

1.0 ml 14.3 M ß-Mercaptoethanol

200 µl 10% Bromophenol blue

Coomassie Blue R 250 Destain solution

0,1% Coomassie brilliant blue R 250 10% Ethanol

50% Ethanol 7% Acetic acid

10% Acetic acid

Transfer buffer

39 mM Glycine

48 mM Tris/HCl; pH 8.0

0.0375% SDS

20% Ethanol

Ponceau staining solution NCP buffer (pH 8.0)

2 g Ponceau S 100 mM Tris/HCl

100 ml 3% Trichloroacetic acid 1.5 M NaCl


21

0.04% Tween 20 5 ml Tween 20

2.0 g sodium azide

ECL solution:

2 ml 1 M Tris/HCl (pH 8.5)

200 µl Luminol (0.25 M in DMSO) 3-aminonaphthylhydrazide

89 µl (0.1 M in DMSO) p-coumaric acid

18 ml dH2O

6.1 µl 30% H2O2

2.3.4 Western blot stripping

Western blot stripping allows an already immunolabelled nitrocellulose membrane to be

repeatedly treated with antibodies. Primary and secondary antibodies were removed by

shaking the membrane in 1% SDS for 1 hour. Prior to new immunolabelling, the

membrane was washed in NCP for 10 minutes and unspecific binding sites were

blocked with 4% milk powder in NCP for 1 hour.

2.3.5 Cell fractionation

For nuclei preparation, cells were lysed in HLB and nuclei were sedimented (1,000 g,

4°C, 15 min.). For further fractionation the supernatant was centrifuged at (100,000 g,

30 min. 4°C). Both fractions were resuspended in HLB and analyzed on immunoblots

with the according antibodies.

2.3.6 Preparation of GST fusion proteins

Recombinant GST-SUN2Nt and GST-LMNC polypeptides expression was induced in E.

coli strain BL21 (0.5 mM IPTG, 4h, 37°C). Cells were lysed as described and the

proteins were isolated from the supernatant by incubating with glutathione agarose

beads (4 h, 4°C). Glutathione agarose beads coupled with GST-SUN2Nt was washed

five times with PBS (500 g, 4°C, 10 min) before performing thrombin cleavage (4°C, 24-

48 h) or incubating with the according human total cell lysate.


22

LB-medium

10 g Bacto-Trypton

5 g Yeast extract

5 g NaCl

Add H2O to 1l

Thrombin cleavage buffer

20 mM Tris/HCl; pH 7.4

10 mM CaCl2

2.3.6 GST pull-down assay

Total cell lysate of human cells was prepared as described above. Glutathione agarose

beads coupled with GST-SUN2Nt (see above) were incubated with total cell lysate

(100000 g supernatant) at 4°C for 6 h. Beads were washed three times with PBS (500 g,

4°C, 1 min) and boiled in SDS sample buffer (95°C, 5 min). Samples were analyzed

using 12% SDS polyacrylamide gels and stained with Coomassie Brilliant Blue. Protein

bands of interest were excised from the gels and subjected to LCMS analysis.

2.3.7 In vitro binding assay

Total cell lysate of cultured human cells was prepared as described above. Glutathione

agarose beads coupled with GST-LMNC polypeptides (see above) were incubated with

total cell lysate (100000 g supernatant, 4°C, 6 h). Beads were washed three times with

PBS (500 g, 4°C, 1min) and then analyzed by SDS-PAGE using 15% SDS

polyacrylamide gels and concomitant western blot analysis.

2.3.8 Immunofluorescence microscopy

If not mentioned otherwise, standard immunofluorescence stainings were carried out

using 3% paraformaldehyde (PFA) as fixative (5 min, RT) prior to lysis using 1% Triton-

X100 in PBS (5 min, RT) and subsequent incubation with blocking solution (30 min, RT).

The appropriate antibodies were diluted in the blocking solution to the working

concentration and applied (12 h, 4°C for mAb K80-207-11; RT for remaining antibodies).

The excess of antibodies was removed by washing with PBS prior to the incubation with


23

the according secondary antibodies (1 h, RT). Nuclear DNA was stained with 4’-6-

diamidino-2-phenylindole (DAPI). Coverslips were embedded in Gelvatol and left at

room temperature for polymerization overnight. Images of immunolabelled cells were

acquired by a confocal laser scanning microscope TCS-SP (Leica) equipped with

TCSNT software. A 488-nm argon-ion laser for excitation of GFP and Alexa 488

fluorescence and a 568-nm krypton-ion laser for excitation of TRITC, Cy3 and Alexa 568

fluorescence were simultaneously used. For the proper acquisition of both signals the

emission signals for green and red fluorophores were separated by using appropriate

wavelength settings for each photo multipler. Image processing was done with Leica

LAS AF Lite software and Microsoft office picture manager, respectively.

Gelvatol PBG (pH 7.4):

4.8 g Polyvinyl alcohol (87%-89%, Sigma P 8136) 0.5% BSA

12 g Glycerol 0.045% fish gelatine

Add 12 ml de-ionized water, stir (RT, 10h)

24 ml 0.2 M Tris/HCl; pH 8.5; stir (50 °C, 20-40 min)

cenrifugation (15 min, 5000 g)

2.5% Diazabicyclooktan (DABCO)

Aliquot-storage: – 20°C


24

2.4 Cell culture

2.4.1 Human cell lines and media

name species tissue

HeLa Homo sapiens Human cervical cancer

HaCaT Homo sapiens Human keratinocytes

Pop10 Homo sapiens Human hepatocellular carcinoma

U373 Homo sapiens Human glioblastoma-astrocytoma

SSS Homo sapiens Human primary fibroblasts

DMD Homo sapiens Human primary fibroblasts

EDMD/CMT Homo sapiens Human primary fibroblasts

control Homo sapiens Human primary fibroblasts

Media:

Dulbecco`s modified Eagle`s medium (DMEM): 4.5 g/l Glucose, 10% Fetale Bovine

Serum (FBS), 2 mM L-glutamine, 1 mM pyruvate, 100 U/ml Penicillin G, 100 µg/ml

Streptomycin, Non Essential Amino Acids (see table 2.2)

Composition of Non Essential Amino Acids (mg/l):

L-Alanine 890

L-Asparagin 1500

L-Asparatic acid 1330

L-Glutamic acide 1470

Glycien 750

L-Proline 750

L-Serine 1050

2.4.2 Cultivation of mammalian cell lines

Trypsin/EDTA PBS pH 7.4

0.05%/0.02% 137 mM NaCl

2.7 mM KCl

8.1 mM Na2HPO4

1.5 mM KH2PO4


25

Mammalian cells were cultivated in petri dishes kept in an incubator at 5% CO2 and

water-satured atmosphere at 37°C in the corresponding medium. To passage

subconfluent cell cultures, cells were incubated with 0.05% Trypsin/EDTA to detach

cells from the plates after rinsing with PBS. Trypsin reaction was stopped by adding

serum containing media, diluted in ratios of 1:2-1:10 according to their growth, and

plated onto new petri dishes.

2.4.3 Freezing and thawing of mammalian cells

Freezing medium

80% DMEM or RPMI-1640

10% FBS

10% DMSO

To store cells as DMSO stocks in liquid nitrogen, cells were pelleted at 250 g for 5

minutes and transferred to the corresponding freezing medium. The cells were quickly

aliquoted into cryotubes and placed into a styropor box at −80 °C. After 24 hours

cryotubes were transferred to liquid nitrogen. Cryopreserved cells were thawed rapidly

and plated at a relatively high density to optimize recovery.

2.5 Cell biological assays

2.5. 1 Transient transfection by electroporation of mammalian cells

Media: DMEM

HEPES (Biochrom, L1613): 1 M; pH 7.2

Hanks, 10 × (Biochrom, L2023) Electroporation medium

137 mM NaCl 1 ml Hepes

5 mM KCl 5 ml Hanks (10 × )

0.8 mM MgSO4 44 ml de-ionized water

0.33 mM Na2HPO4

0.44 mM KH2PO4

0.25 mM CaCl2


26

1 mM MgCl2

1 mM Sodium butyrate

0.15 mM Tris-HCl; pH7.4

Approximately 107 cells were pelleted at 1000 g at 4 °C, resuspended in 800 µl

electroporation medium and transferred to an electroporation cuvette containing 10-12

µg plasmid DNA. After incubated on ice for 10 minutes, cells were electroporated using

a Gene pulser (Bio-Rad) set at 975 µF and 200 V. Finally, cells were seeded on petri

dishes in fresh medium.

2.5.2 Senescence-associated β-galactosidase assays

Cells were seeded on cover slips; the next day cover slips were washed with PBS and

fixed with 3% PFA (5 min, RT). Cells were washed twice with PBS and incubated at

37°C with freshly prepared senescence-associated β-Gal (SA-β-Gal) staining solution.

Examination for staining was done after 4-8 hours under bright field microscopy at 40x

magnification.

SA-β-Gal staining solution

1 mg/ml 5-bromo-4-chloro-3-indolyl β-D-galactoside (X-Gal

40 mM citric acid/sodium phosphate pH 6.0

5 mM potassium ferrocyanide K4Fe(CN)6

5 mM potassium ferricyanide K3Fe(CN)6

150 mM NaCl

2 mM MgCl2).

2.5.3 Focal adhesion assay

Trypsinized cells were seeded on coverslips in culture dishes with an initial cell number

of 1x103 and subjected to immunofluorescence as described above. Analysis was

carried out with a confocal laser scanning microscope TCS-SP (Leica) equipped with

TCSNT software. All pictures were taken in the same z-plane so that the spreading of

focal adhesions on the surface of the coverslip is comparable. LAS-AF Lite Application

Suite software from Leica was used to quantify the spread area in µm2.


27

2.5.4 Cell synchronization

At 40% confluency HaCaT cells were incubated with 2 mM thymidine for 24 h. Cells

were released from the thymidine block by washing the culture plates with PBS and

adding fresh medium for 3 h. Then 100 ng/ml nocodazole was added to the media. After

the 12 h nocodazole block, cells were harvested and lysed as described above and the

supernatant was used for subsequent GST pull down assays.

2.6 Generation of a monoclonal antibody

2.6.1 Immunization of Balb/c mice

Immunization-solutions:

FC: Freund`s adjuvant complete (Sigma, F-5881)

FCI: Freund`s adjuvant incomplete (Sigma, F-5506)

For the generation of a monoclonal antibody four female Balb/c mice were immunized

with the according antigen. The antigen was suspended in PBS, so that a concentration

of 1 µg/µl was achieved. The antigen was injected 6 × in a time interval of 3 days. For

the immunization 50 µl of the antigen were added to 50 µl of FC-solution in the first

immunization, to 50 µl FCI-solution in the second immunization and to 50 µl PBS in the

last four immunizations. Isolated macrophages were fused with the myeloma cell lines

AG8 and PAI.

2.6.2 Generation of hybridoma cells

Cultivation solution Fusion solution; pH 7.4

RPMI 1640 (PAA) PEG-solution (SIGMA)

4 mM L-Glutamine

10% FBS

1 mM ß-mercaptoethanol

Bri: Hybridoma cloning medium (NICB)


28

Prior to the fusion, macrophages were extracted from 12 male Balb/c mice aged 10

weeks. Mice were anesthetized with Isoflurane (2-chloro-2-(difluoromethoxy)-1,1,1-

trifluoro-ethane) and killed by neck translocation, fixed and after removal of the

abdominal wall 10 ml of cold cultivation medium was injected into the peritoneum

without injuring the internal organs in order to solubilise the macrophages. Macrophage

containing medium was centrifuged twice (400 g, 4 °C, 5 min) and the pellet was

suspended in cultivation medium and transferred onto 40 × 24 well plates (500 µl/well)

and cultivated at 37 °C and 5% CO2 in the incubator.

The four female immunized mice were anesthetized and killed by neck translocation at

the day of fusion and lymph nodes localized at the back of the knee joint were removed

and transferred into cultivation medium. After removal of lymph tissue, single

lymphocytes were transferred into fresh medium, subdivided into four fractions and

pelleted (200 g, 5 min), added to Ag8 and Pai myeloma cells and centrifuged (200 g, 5

min). The resulting pellet was warmed to 37 °C and 1 ml PEG-solution (37 °C) was

added slowly within 60 seconds at room temperature. The mixture was again incubated

for 60 seconds at 37 °C and subsequently 20 ml cultivation medium (37 °C) were

applied within 2 minutes by light shaking; 1 ml within 30 seconds, further 3 ml within 30

seconds and the residual 16 ml within 60 seconds. After further incubation (37 °C, 5

min) the pellet was suspended in 125 ml cultivation medium and distributed onto 24 well

plates (500 µl/well). This procedure was done for all four fractions.

2.6.3 Selection of monoclonal antibodies

Cultivation medium Selection-medium HAT Selection-medium HT

RPIM-1640 medium Cultivation medium Cultivation medium

4 mM L-Glutamine 0.1 mM Sodium hypoxanthin 0.1 mM Sodium hypoxanthin

10% FBS 0.4 µM Aminopterin 16 µM Thymidine

1 mM ß-mercaptoethanol 16 µM Thymidine

Applying of the HAT selection medium after the fusion of lymphocytes and myeloma

cells led to the survival of only hybridoma cells. Hybridoma cells were cultivated in

selection medium HAT which was changed every third day for three times, at the fourth

time selection medium HT was used and afterwards only Bri-containing culturing


29

medium was used. Western blot strips of E.coli cell lysates containing GST-SUN2Nt

were incubated with collected hybridoma supernatants followed by horseradish-

peroxidase (POD) conjugated secondary antibody and chemiluminescence analysis. As

a negative control the strips were incubated with RPMI medium. Positive clones were

selected according to the detection of a signal at ~45 kDa and subcloned by dilutions of

the desired hybridoma clones. Using a thin capillary tube, single cells were distributed

onto 96 well plates and macrophages containing Bri medium was added. Supernatant of

positive subclones was collected according to the detection of GST-SUN2Nt in E.coli

lysates, recombinant SUN2Nt protein after thrombin cleavage and the protein in whole

HeLa cell lysates transferred onto a nitrocellulose membrane. Positive tested mother-

and subclones were stored as DMSO stocks in liquid nitrogen:

2.6.4 Purification of IgG from hybridoma supernatant

For the purification of the antibody 500 ml hybridoma supernatant was subjected to a

Protein-A-Sepharose-column (1 ml Protein-A-Sepharose) equilibrated with 50 ml PBS /

2 mM sodium azide and circulated for 24-36 hours at 4°C with a pumping system

(BioRad). After incubation the column was washed with 50 ml PBS / 2 mM sodium azide

until the eluate exhibited a constant OD280 < 0.01. The elution of the bound antibody

was performed by adding 10 × 1 ml 0, 2 M glycine (pH 2.7) and immediate neutralization

with NaHCO3. The OD280 of the collected eluate was checked against glycine-buffer.

Antibody containing fractions identified by SDS-PAGE were dialyzed against PBS.

Results

30

3 Results

3.1 Human SUN2 protein

According to the UniGene program, the SUN2 ( also termed KIAA0668) gene

(LocusLink 25777) is located on the human chromosome 22 (cytoband: 22q13.1),

chromosome location 39130730- 39190148 and could be found in the following cDNA

sources (tissue expression): adipose tissue, adrenal gland, ascites, bladder, blood,

bone, bone marrow, brain, cervix, connective tissue, embryonic tissue, eye, heart,

intestine, kidney, larynx, liver, lung, lymph node, mammary gland, mouth, muscle, nerve,

ovary, pancreas, parathyroid, pharynx, placenta, prostate, skin, spleen, stomach, testis,

thymus, thyroid, tonsil, trachea, uterus, vascular adrenal tumor, bladder, carcinoma

breast (mammary gland) tumor, cervical tumor, chondrosarcoma, colorectal tumor,

gastrointestinal tumor, germ cell tumor, glioma, head and neck tumor, kidney tumor,

leukemia, liver tumor, lung tumor, non-neoplasia, normal ovarian tumor, pancreatic

tumor, primitive neuroectodermal tumor of the CNS, prostate cancer, retinoblastoma

skin tumor, soft tissue/muscle tissue tumor, uterine tumor, embryoid body blastocyst,

whole embryo (The Human Protein Atlas). KIAA0668 is expressed in all tissues at a

moderately high level, and at a particularly high level in heart, brain, testis and ovary.

Homo sapiens SUN2 consists of 717 amino acids and contains according to SMART

one predicted transmembrane (TM) domain (213-233 aa), three potential coiled-coil

regions and one C-terminal SUN domain (601-717 aa) (Fig. 4). SUN2 also has one

serine-rich region, one poly-arginine region and two poly-glycine regions. The human

SUN2 shares 66% identity and 71% similarity with its mouse homolog (NP_919323).

The identity of the SUN domain between these two species of SUN2 protein is 95%.

Fig.4: structural domains of human SUN2 protein: TM domain (dark blue), C-terminal SUN domain

(yellow) serine-rich (turquoise), poly-arginine (orange), coiled-coil (light blue) and poly-glycine (green)

region. Antibody epitope is indicated by black bar.

coiled-coil

poly-gly

poly-arg

ser-rich

TM SUN

1 717

K80 207-11

Results

31

3.2 Generation of a mouse monoclonal antibody against the N-

terminal region of human SUN2

3.2.1 Determination of an epitope in the N-terminal sequence of human SUN2

suitable for antibody production

For the generation of a mouse monoclonal antibody directed against the N-terminus of

human SUN2 protein, a short sequence stretch specific for SUN2 protein was

determined. Alignment of the N-terminal region of human SUN2, murin Sun2, human

SUN1 and murin Sun1 by protein BLAST (Basic Local Alignment Search Tools)

revealed a unique sequence localized in the N-terminus of the SUN2 protein with a

homology of 76% between the human and the murin sequence. This specific region is

located in the nucleoplasm and consists of 414 base pairs or 138 amino acids (aa 1-

138), respectively (Fig 5). The corresponding peptide has a molecular mass of 15 kDa

and an isoelectric point of 9.4. In the following, this epitope region will be named as

SUN2Nt.

HsSUN2 --------------------------------------------------MSRRSQRLTR 10

MmSun2 --------------------------------------------------MSRRSQRLTR 10

HsSUN1 MDFSRLHMYSPPQCVPENTGYTYALSSSYSSDALDFETEHKLDPVFDSPRMSRRSLRLAT 60

MmSun1 MDFSRVHTYTPPQCVPENTGYTYALSSSYSSDALDFETEHKLEPVFDSPRMSRRSLRLVT 60

***** **.

HsSUN2 YSQGDDDG-SSSSGGSSVAGSQSTLFKDSPLRTLKRKSSNMKRLSPAPQLGPSSDAHTSY 69

MmSun2 YSQDDNDGGSSSSGASSVAGSQGTVFKDSPLRTLKRKSSNMKHLSPAPQLGPSSDSHTSY 70

HsSUN1 TA-CTLGD--GEAVGADSGTSSAVSLKNRAARTTKQRRSTNKSAFSINHVSRQVTSSGVS 117

MmSun1 TASYSSGD--SQAIDSHISTSRATPAKGRETRTVKQRRSASKPAFSINHLSGKGLSSSTS 118

: .. ..: : . * .. *. ** *:: * * . ::. . :

HsSUN2 YSESLVHESWFPP-------RSSL--EELHGDANWGEDLRVRRRRGTGGSESSRASGLVG 120

MmSun2 YSESVVRESYIGSPRAVSLARSALLDDHLHSEPYWSGDLRGRRRRGTGGSESSKANGLTA 130

HsSUN1 YGGTVSLQDAVTRRP--PVLDESWIREQTTVDHFWGLDDDGDLKGGNKAAIQGNGDVGVA 175

MmSun1 HDSSCSLRSATVLRH--PVLDESLIREQTKVDHFWGLDDDGDLKGGNKAATQGNGELAAE 176

:. : .. .: :. : *. * : *. .: ..... .

HsSUN2 R-KATEDFLGSSSGYSSE------------------------------------------ 137

MmSun2 ESKASEDFFGSSSGYSSE------------------------------------------ 148

HsSUN1 AATAHNGFSCSNCSMLSERKDVLTAHPAAPGPVSRVYSRDRNQK---------------- 219

MmSun1 VASS-NGYTCRDCRMLSARTDALTAHSAIHGTTSRVYSRDRTLKPPHLGHCGRMTAGELS 235

.: :.: .. *

HsSUN2 --------DDYVGYSDVDQQSS---------------------------------SSRLR 156

MmSun2 --------DDLAG----------------------------------------------- 153

HsSUN1 -------CDDCKGKRHLDAHPG----------RAGTLWHIWACAGYFLLQILRRIGAVGQ 262

MmSun1 RVDGESLCDDCKGKKHLEIHTATHSQLPQPHRVAGAMGRLCIYTGDLLVQALRRTRAAGW 295

** *

Results

32

HsSUN2 SAVSRAGSLLWMVATSPGRLFRLLYWWAGTTWYRLTTAASLLDVFVLTR--RFSS-LKTF 213

MmSun2 ------------------RLFGLLYWWIGTTWYRLTTAASLLDVFVLTRSRHFSLNLKSF 195

HsSUN1 AVSRTAWSALWLAVVAPGKAASGVFWWLGIGWYQFVTLISWLNVFLLTR------CLRNI 316

MmSun1 SVAEAVWSVLWLAVSAPGKAASGTFWWLGSGWYQFVTLISWLNVFLLTR------CLRNI 349

: :** * **::.* * *:**:*** *:.:

HsSUN2 LWFLLPLLLLTCLTYGAWYFYPYGLQTFHPALVSWWAAKDSRRPDEGWEARDSSPHFQAE 273

MmSun2 LWFLLLLLLLTGLTYGAWHFYPLGLQTLQPAVVSWWAAKESRKQPEVWESRDASQHFQAE 255

HsSUN1 CKFLVLLIPLFLLL-AGLSLRGQGNFFSFLPVLNWASMHRTQRVDDPQDVFKPTTSRLKQ 375

MmSun1 CKVFVLLLPLLLLLGAGVSLWGQGNLFSLLPVLNWTAMQPTQRVDDSKGMHRPGPLPPSP 409

.:: *: * * .. : * .::.* : : ::: : .

Fig.5: Sequence homology of SUN-domain proteins: HsSUN2: human SUN2 (AAT905001); MmSun2:

murin Sun2 (NP919323); HsSUN1: human SUN1 (NP079430); MmSun1: murin Sun1 NP077771. Grey

arrows mark the region used for antibody-generation. Colours are according to the physiochemical

characteristics of the amino acids: nonpolar, neutral (red); polar, neutral, noncharged (green); polar, acid,

charged (blue); polar, basic, charged (pink). Identical residues are shown with an ‘*’, conserved with ‘:’

and semi-conserved with a ‘.’ .

3.2.2 Expression and detection of the SUN2NT protein

The DNA sequence encoding the N-terminal domain (SUN2Nt) was cloned into the

bacterial expression vector pGEX-4T1. The 45 kDa fusion protein GST-SUN2Nt (Fig.6A)

confirmed by western blot using GST specific antibodies (Fig.6B) was purified from the

Escherichia coli strain XL1-Blue and the GST tag was subsequently removed by

thrombin cleavage and a concentration of 1 µg/µl was achieved (Fig.6C). To confirm the

peptide identity prior to immunization, the 15 kDa gel band and the smaller products

suspected to be degradation products of the SUN2Nt peptide were subjected to PMF

(Peptide Mass Fingerprinting) analysis and the identity of SUN2Nt protein and its

degradation products were confirmed.

Results

33

Fig. 6: A: Coomassie Blue stained polyacrylamide (PAA) gels (12% acrylamide) of uninduced (T0) and

induced (T1) E.coli lysates expressing GST-SUN2Nt fusion protein; supernatant (SN) and pellet (P)

fraction of E.coli lysates. B: immunoblot using GST-specific antibodies to detect the GST-SUN2Nt fusion

protein. C: Coomassie Blue stained PAA gel (12%) of decreasing BSA concentrations (1µg; 0 8µg; 0, 6

µg; 0,4 µg; 0,2 µg/µl ) for calibration; 1µl input of recombinant SUN2Nt protein after thrombin cleavage.

Black arrow: main SUN2Nt protein product; grey arrows: degradations products of SUN2Nt.

3.2.3 Identification of positive hybridoma clone K80-207-11 by immunoblot and

immunofluorescence analysis

Antibody producing hybridoma clones were identified by westernblot analysis.

Supernatant of more than 800 growing mother clones were tested for the successful

production of monoclonal antibody against human SUN2Nt. Subclones K80-207-4, K80-

207-11, K80- 739-3, K80-650-3 and K80-845-4 were able to recognize the recombinant

SUN2Nt peptide and the GST-SUN2Nt protein expressed in E.coli (Fig. 7A) as well as

the endogenous SUN2 in HeLa cell lysates. Subclone K80-207-11 was chosen due to

specific staining observed in immunofluorescence experiments as described in the

A B T0 T1 SN P

97

66

45

30

14

66

45

30

anti- GST

C

kDa

kDa

1,0 0,8 0,6 0,4 0,2

BSA µg/µl

SUN2Nt degradation products

SUN2Nt:

1µl

Results

34

following. Subclones K80-207-4, K80-207-11, K80-739-3, K80-650-3 and K80-845-4

recognized the 15 kDa recombinant SUN2Nt peptide and its degradation products (Fig.

7A). Subclones K80-207-4 and K80-207-11 additionally detected endogenous SUN2 in

HeLa cell lysates (Fig. 7B). Staining of two bands of approximately 80 kDa (Fig. 7B,

upper band) and 70 kDa (Fig. 7B, lower band) occurred of which the smaller one might

be a degradation product or a splice variant. Subclone K80-207-11 was chosen for

further studies as it recognized the protein also in immunofluorescence analysis as

described in the following. The antibodies were purified from the hybridoma supernatant

by affinity chromatography using Protein A Sepharose beads and by subsequent salt

removal through dialysis against PBS. A dilution range test of K80-207-11 indicated that

a dilution of 1:50 for western blot analysis is applicable.

Fig.7: Detection of recombinant SUN2Nt protein and its degradation products (grey arrows) blotted on

nitrocellulose membrane by hybridoma supernatant of different mother clones and their subclones. A: E.

coli lysate expressing recombinant SUN2Nt. B: HeLa lysate expressing endogenous SUN2. Detection:

hybridoma supernatant as primary antibody of different mother- and subclones; secondary antibody:

peroxidase (POD), detection was done by enhanced chemiluminescence (ECL). Negative control: RPMI

medium.

Since endogenous SUN2 is reported as inner nuclear envelope component (Hodzic et

al., 2004; Turgay et al., 2010), cellular components of HeLa cell lysates were isolated by

fractionation. Samples were mixed with sample buffer and heated for five minutes at 37,

42, 56, 72, 82 and 98°C and analyzed on western blots. Using various heating

temperatures to achieve different denaturing levels of the protein components, the ability

K80-207- 11 4

ctrl 739- 3

207- 4 11

650- 3

15

11

kDa

845- 4

A B

72

kDa

Results

35

of K80-207-11 to recognize mild to fully denatured endogenous SUN2 was tested. To

verify the accuracy of the fractionation procedure, samples from the cytoplasmic fraction

and the nuclear fraction were incubated with antibodies against emerin and α- tubulin

(Fig.8).

K80-207-11 detected the endogenous SUN2 protein in fractionated cell lysates heated

at 37, 42, 56, 72, 82 and 98°C at the same intensity levels. Again, two bands differing in

approximately 5 kDa were detected by mAb K80-207-11.

As a marker for the inner nuclear envelope, the majority of endogenous emerin was

predominantly localized to the nuclear fractions. Comparable to emerin endogenous

SUN2 was recovered by K80-207-11 in the nuclear fractions and was not detected in

the cytoplasmic fraction. α-tubulin was predominantly seen in the cytoplasmic fraction

but also to some extend in the nuclear fraction.

Fig.8: Western blot analysis of the nuclear and cytoplasmic fraction of HeLa cell lysates using K80-207-

11 antibody and WA3 (anti−tubulin) and anti-emerin as control for the separation of the cellular fractions.

3.2.4 Transfection of POP10 cells with pJG129SUN2 full length, tagged with

V5*6xHis

To further verify the selected antibody produced by the subclone K80-207-11, Pop10

cells, which are derived from hepatocellular carcinoma, were transfected with pJG129

encoding V5*6xHis tagged SUN2 full length and subjected to immunoblotting and

α-tubulin

nuclear fraction

cytoplasmic fraction

37°C 42°C 56°C 72°C 82°C 98°C

72

37°C 42°C 56°C 72°C 82°C 98°C

55

36

emerin

K80-207-11

Results

36

immunofluorescence. Primary antibodies anti-His, anti-V5 and K80-207-11 hybridoma

supernatant detected the V5*6xHis- fusion protein SUN2 in cell lysates from transfected

cells, mAb K80-207-11 detected two bands which corresponds to endogenous and

tagged SUN2 which should be ~2kDa larger than the endogenouse protein. α-Tubulin

was used as loading control. In confocal immunofluorescence analysis K80-207-11

staining colocalized with anti-His-tag antibody staining in transfected cells (Fig. 9B,

white arrows). Because of the low SUN2 expression in hepatocytes (annotated by The

Human Protein Atlas), non transfected cells showed a week nuclear envelope staining.

B

Fig.9: A: Immunoblotting of Pop10-cells transfected with pJG129SUN2 full length V5*6xHis; anti- His,

anti-V5 and K80-207-11 hybridoma supernatant as primary antibody, POD as secondary antibody,

detection by ECL. B: Confocal immunofluorescence of Pop10-cells transfected with pJG129SUN2 full

length V5*6xHis; K80-207-11, anti-His–tag as primary antibody, Alexa Flour 568, 488 as second antibody,

DAPI for nuclear staining; scale bar 10µm. White arrows indicate successfully transfected cells.

merge

DAPI

K80-207-11

anti- His-tag

V5*6xHis ctrl

95

kDa

A

V5*6xHis ctrl ctrl V5*6xHis

anti-tubulin

anti- His anti-V5 K80-207-11

55

Results

37

In immunofluorescence analysis the subclone K80-207-11 localized the endogenous

SUN2 to the nuclear envelope and to some extent also in the cytoplasm in Pop10 (Fig.

9) and HeLa cells (Fig. 10). To further confirm the nuclear envelope localization, HeLa

cells were stained with DAPI to visualize the DNA, with anti-emerin as inner nuclear

envelope marker and with K80-207-11. Anti-emerin localized emerin exclusively to the

nuclear envelope. In a merged image, SUN2 can be observed surrounding the nucleus

in a rim-like pattern, clearly colocalizing with emerin, demonstrating that K80-207-11

recognizes specifically endogenous SUN2. The strongly stained spots inside the

nucleus might represent nucleoplasmic reticulum (Malhas et al., 2011) (Fig. 10).

Subsequently, mAb K80-207-11 was used for all cell biological analyses.

Fig.10: Confocal immunofluorescence of HeLa cells, anti-emerin and K80-207-11 as primary antibody,

Alexa Flour 568, 488 as conjugated secondary antibodies, DAPI for nuclear staining; scale bar 5µm.

merge DAPI K80-207-11 emerin

Results

38

3.3 Distribution of endogenous SUN2 protein during the cell cycle

During interphase endogenous SUN2 protein was associated with the nuclear envelope

and colocalized with lamins. By contrast, in mitotic cells no colocalisation with LMNA/C

was observed (Fig. 11A).

At the onset of mitosis, the nuclear envelope becomes disrupted by spindle

microtubules during mid-late prophase and intranuclear contents are released. The

lamina depolymerizes and the nuclear membranes disperse into the endoplasmic

reticulum network during prometaphase. Nuclear rim staining by K80-207-11 persisted

during prophase. At metaphase, when the nuclear envelope is completely

disassembled, SUN2 associated with the condensed chromosomes. SUN2 staining

could also be detected in two dot-like structures presumably the centrosomes. In

Schizosaccharomyces pombe endogenous sad1 (homolog of mammalian SUN2)

localizes to the spindle pole body (SPB) (Hagan et al., 1995). Costaining of HeLa cells

with pericentrin (an integral component of the pericentriolar material) revealed

colocalisation with SUN2 and confirmed its centrosomal localization (Fig. 11 C).

In anaphase the K80-207-11 antibody localized the SUN2 protein still associated with

the condensed chromosomes but in a more distributed and in a vesicular manner (Fig.

11A; B, panel four). The SUN2 protein appeared to accumulate at distinct chromosome

regions of the condensed chromatids which might be telomeres as described in meiotic

cells for sad1 in S. pombe (Hagan et al., 1995; Alsheimer et al., 2006).

The envelope reassembles onto chromosomes during late anaphase and telophase and

K80- 207-11 detected the SUN2 protein reconstituted in a rim like pattern.

A

sun2

merge

DAPI K80-207-11 LMNA/C merge

merge

non-mitotic

mitotic

Results

39

B

DAPI K80-207-11 merge

interphase

prophase

metaphase

telophase

DAPI K80-207-11

anaphase

metaphase

Results

40

C

Fig.11: Distribution of SUN2 during mitosis: A: Immunofluorescence of non- mitotic (upper panel) and

mitotic (bottom panel) HeLa cells stained with mAb K80-207-11 and anti-LMNA/C as primary antibodies,

Alexa Flour 568, 488 as conjugated secondary antibodies, DAPI for nuclear staining; scale bar 2 µm. B:

Immunofluorescence analysis of HeLa cells during mitosis (interphase-telophase), mAb K80-207-11 as

primary antibody, Alexa Flour 568 as conjugated secondary antibodies, DAPI for nuclear staining; scale

bar 5 µm. C: localization of SUN2 and pericentrin in telophase: Immunofluorescence analysis of HeLa

cells stained with mAb K80-207-11 and anti-pericentrin as primary antibody, Alexa Flour 568, 488 as

conjugated secondary antibodies, DAPI for nuclear staining; scale bar 5 µm.

3.4 Putative interaction partners of SUN2Nt

To identify protein interaction partners of SUN2Nt, pull down experiments with GST-

SUN2Nt and HaCaT cells lysates and lysates from HaCaT cells arrested in

prometaphase (mHaCaT) were performed. To analyze possibly changing interaction

partners of SUN2 during nuclear envelope breakdown, pulldown experiments were

performed using GST-SUN2Nt as bait and incubated with either normal grown,

untreated HaCaT total cell lysates or mitotically arrested HaCaT total cell lysates

(mHaCaT).

For the mitotic arrest cells were treated with nocodazole and arrested with a G2- or M-

phase DNA content. Microscopic analysis of nocodazole-treated cells showed that they

do enter mitosis but can not form metaphase spindles because microtubules cannot

polymerize. The absence of microtubule attachment to kinetochores activates the

spindle assembly checkpoint, causing the cells to arrest in prometaphase. Therefore,

lysates of HaCaT cells arrested in prometaphase were used to perform a pulldown

experiment. DAPI staining and microscopic analysis confirmed the prometaphase arrest

of more than 90% of the cells (Fig. 12).

pericentrin DAPI merge K80-207-11 merge

Results

41

Using recombinant GST-SUN2Nt immobilized on Sepharose beads several proteins

have been isolated from HaCaT total cell lysates, that bind either directly or indirectly to

SUN2Nt. In control reactions, HaCaT cell lysates were incubated with GST immobilized

on GST-Sepharose beads. GST-SUN2Nt immobilized on Sepharose beads was loaded

for comparison. The cell lysates were obtained from asynchronously growing (HaCaT)

and mitotically arrested HaCaT cells (mHaCaT). Analysis was done by separating the

proteins by SDS-PAGE (12% acrylamide) (Fig. 13), cutting out protein bands and

characterization by LC-MS (Liquid chromatography-mass spectrometry). Protein

separation was performed by nano-liquid chromatography and the proteins were

introduced into a mass spectrometer via an ionization interface. Identification of the

proteins by mass spectrometry after reduction and alkylation of Cys residues by

proteolysis with trypsin, V8, or other endopeptidase, is coupled online (HCT ESI-ion

trap) or offline (UltrafleXtreme MALDI-TOF-TOF) with MS1 detection (molecular mass)

and MS2 fragmentation. Combined information from molecular mass and the

corresponding fragment spectra is used for Mascot searches in virtual digests and MS

fragmentation libraries.

Fig.12: HaCaT cell arrested in

prometaphase; DNA visualized with DAPI

Results

42

Fig. 13: A: Coomassie Blue stained PAA-gel (12%) loaded with GST-SUN2Nt Sepharose beads

incubated with PBS as first control (first lane); GST-SUN2Nt Sepharose beads incubated with HaCaT cell

lysates (second lane); GST-Sepharose beads incubated with HaCaT cell lysates as second control (third

lane). B: Coomassie Blue stained PAA-gel (12%) probed with GST-SUN2Nt Sepharose beads incubated

with PBS as first control (first lane); GST-Sepharose beads incubated with mHaCaT cell lysates as

second control (second lane); GST-SUN2Nt Sepharose beads incubated with mHaCaT cell lysates (third

lane). Grey arrows and brackets indicate areas of interest for protein analysis.

The mass spectrometry results were further analysed according to the following criteria

(Table 1 A-D): The size of the detected protein matched the size of the band in the

Coomassie stained SDS-gel; the protein has a similar cellular localization like SUN2; the

Probability Based MOWSE (MOlecular Weight SEarch) Score are >32 indicating identity

or extensive homology (significant threshold p<0.05). MOWSE is a method for

identification of proteins from the molecular weight of peptides created by proteolytic

digestion and measured with mass spectrometry. Mascot is a software search engine

that uses mass spectrometry data to identify proteins from primary sequence databases.

Normally, the protein length is taken into account. In Mascot, a protein can be correctly

scored even though it is substantially shorter than the database entry because in any

database entry which exceeds the full mass of the protein, the code searches for the

A B GST+ mHaCaT -lysate

GST SUN2Nt +mHaCaT - lysate

GST-SUN2Nt +PBS

95

130

72

250

55

kDa

97

66

30

GST- SUN2Nt +PBS

GST-SUN2Nt + HaCaT -lysate

GST + HaCaT -lysate

45

kDa

Results

43

highest scoring set of matches which occur within the full mass window of the protein.

Therefore, also proteins have been added to the list which had sequence coverage less

than 10%.

Tab.1: Proteins detected in total HaCaT or mHaCaT cell lysates precipitated by GST-SUN2Nt categorized

in gene regulatory (A), RNA processing (B), architectural complex (C) and signaling (D) proteins. Identifier

indicates the SwissProt accession number if not declared otherwise.

Identifier Name Category HaCaT mHaCaT

Q16576.1 RBBP7 A + +

Q9Y265.1 RuvBL1 A + +

Q9Y230.3 RuvBL 2 A + -

Q9NVE4 (UniProtKB) CCDC87 A + +

P12956.2 XRCC A + -

P62805.2 H4 A + -

Q09028.3 RBBP4 A + -

9606 (NCBI) H3-like A + -

P19338.3 nucleolin A + +

Q13547.1 HDAC1 A - +

O94776.1 MTA2 A - +

YLBM1_HUMAN YLB-motif containing protein1 A - +

P56545 CTB2 A - +

Q8N7H5.2 PAF1 A, B - +

Q14498.2 RNA-binding protein 39 A, B - +

O15371.1 EIF3D B + -

NP_991247.1 HNRNPH1 B + -

O00571.3 RNA helicase 3X B + -

O00148.2 RNA helicase 39 B + -

P17844.1 RNA helicase 5 B + -

P52272 HNRNPM B + -

O00148.2 HNRNPK B + +

P52597 HNRNPF B - +

Q8N9N8 EIF1G B - +

P60842 (UniProtKB) EIF4A1 B - +

P60228.1 EIF3E B - +

NP_060145.2 protocadherin C + -

O43707.2 α-actinin1 C - +

Results

44

P12814.2 α-actinin4 C - +

P41351 tubulin a C + +

P05219 tubulin ß C + +

P46940 Ras-GTPase activating like protein C - +

Q9ULV4.1 coronin1C C - +

P02549 spectrin α C, D - +

P11277 spectrin β C, D - +

P68032 actin C, D - +

P35579 myosin9 C, D - +

P35221.1 β-catenin C, D - +

P16144 integrin β 4 C, D - +

P02545.1 laminA/C C, D + +

P67775.1 ser/thr protein phosphatase 2A C, D + +

P35222.1 α-catenin C, D + +

CAI12454.1 coatomer subunit α D + -

P53618.3 coatomer subunit β D + +

AAH20498.2 coatomer subunit γ D + +

NP_001646.2 coatomer subunit δ D + +

Q2NKX8.1 ERCCL6 D + -

Q9NZM1 myoferlin D - +

Q05655 PRKCD D - +

B5BU72 PICALM D - +

Table 1 presents a list of proteins which are categorized in proteins taking part in gene

regulatory processes e.g. DNA-transcription and chromatin remodeling (A) and in

proteins contained in RNA processing complexes (B). Furthermore they can be

classified in proteins of architectural complexes (C) and proteins participating in

signaling (D). Figure 14 summarizes in a diagram the distribution of proteins in either

total HaCaT or mHaCaT cell lysates precipitated by GST-SUN2Nt as bait. Proteins

found in both lysates are illustrated in the intersection.

Proteins contributing to gene regulation e.g. DNA replication and chromatin remodeling

can be found in almost equal amounts in HaCaT and mHaCaT cell lysates.

Approximately half of these proteins appeared in HaCaT and in mHaCaT cell lysates as

Results

45

well. Both lysates contained predominantly histone modification and transcription

repressing proteins such as RBBP7, RuvBL1 and nucleolin.

In the protein pool participating in RNA processing, an equal number of proteins such as

several translation factors and RNA-Pol II associated factor1, could be detected. Only

HNRNPK was detected in both cell lysates. Lysates of mHaCaT cells contained three

translation factors absent from the HaCaT cell lysates. Translation initiation factors were

listed in this category as they appear to have diverse roles i.e. in RNA biogenesis

(Alexandrov et al., 2011).

In mHaCaT cell lysates more than twice the number of proteins contributing to the cell

architecture could be detected which were absent from the HaCaT cell lysates. Proteins

found only in the mHaCaT cell lysates participate mainly in the actin cytoskeleton e.g.

actin, myosin and intergrinβ4, but also take part in signaling events. Accordingly, the

number of proteins involved in signaling events is increased in mHaCaT cell lysates

compared to lysates from HaCaT cells.

Fig. 14: Distribution of proteins in either total HaCaT or mHaCaT cell lysates precipitated by GST-SUN2Nt

as bait. Proteins found in both lysates are illustrated in the intersection.

protocadherin RuvBL2, XRCC,

H4, RBBP4, H3-like, EIF3D, HNRNPH1, RNA helicase 3X, RNA helicase 39,

HNRNPK, RNA- helicase 5,

HNRNPM, ERCCL6

HDAC1, MTA2, YLB-motif containing protein1, CTB2,

PAF1, RNA-binding protein 39, HNRNPK,

HNRNPF, EIF1G, EIF4A1,

EIF3E, α-actinin1,

α-actinin4, spectrinα,

spectrinβ, Ras-GTPase activating like protein,

integrinβ4, β- catenin, coronin1C, ser/thr protein

kinaseβ, myoferlin, actin, myosin9, PRKCD,

PICALM, β-catenin

RuvBL1, nucleolin, RBBP7,

CCDC87, ser/thr protein

phosphatase 2A,

α-catenin,

coatomerβ,

coatomerδ,

coatomerγ,

laminA/C,

tubulinα,

tubulinß

HaCaT

mHaCaT

Results

46

The F-actin binding protein coronin2A has been identified as a component of the co-

repressor complex NcoR and recent studies revealed that it is required for recruiting

further factors to the co-repressor complex such as the transrepressor LXRα/β. For this,

its actin binding activity is required (Huang et al., 2011). In previous studies coronin1C,

also termed coronin3 or CRN2, was detected in the nucleus (Spoerl et al., 2002). In the

present study, coronin1C was detected in pull down assays of mHaCaT cell lysates

using SUN2Nt-GST. Since coronin1C expression is altered in diffuse glioblastomas

(Roadcap et al., 2008; Xavier et al., 2009), the human glioblastoma-astrocytoma cell line

U373 was stained with K80-207-11. SUN2 staining was significantly increased in U373

cells compared to the moderate staining in HeLa cells and the typical rim staining of

SUN2 was expanded and significantly broadened, in some cells appearing in lobulations

of the NE reaching into the cytoplasm (Fig. 15, third picture from the left). These findings

are consistent with strong SUN2 staining of malignant glioma tissue annotated by The

Human Protein Atlas.

Fig. 15: Immunofluorescence analysis of HeLa and U373 cells stained with mAb K80-207-11 as primary

antibody, Alexa Flour 568 as conjugated secondary antibodies, DAPI for nuclear staining, scale bar 5 µm.

U373

K80-207-11

HeLa U373 U373

Results

47

3.5 Direct interaction of SUN2Nt protein with LMNC polypeptides

LMNA/C was present in the pulldowns from asynchronously growing and mitotically

arrested cells. A direct interaction with the N-termini of SUN1 and SUN2 has been

reported recently (Haque et al., 2010).The LMNA gene codes for four proteins: LMNA,

LMNC, LMNA_10, testis-specific LMNC2, which are generated through alternative

splicing. Lamins A and C differ in that LMNA possesses additional 90 amino acids at its

C-terminus. To map the respective LMNA/C interacting domains, five GST-LMNC

fragments have been tested for interaction with the N-terminus of recombinant SUN2 by

GST pulldown assays: GST-LMNC N-term/LMNA (aa 1-127), GST-LMNC coil1B-∆ (aa

128-218), GST-LMNC coil2 (aa 243- 387), GST-LMNC tail (aa 384-566), GST-∆LMNC

(aa 128-572). Fig. 16 B shows a schematic overview of the GST-LMNC constructs used

for the in vitro pull down assay.

Fig. 16 A: Overview of the domain architecture of the LMNA/C protein. B: Schematic overview of LMNA/C

protein structure and the GST-lamin fusion proteins used for the in vitro GST-pull down assays: GST-

LMNC N-term/LMNA (aa 1-127), GST-LMNC coil1B-∆ (aa 128-218), GST-LMNC coil2 (aa 243- 387),

GST-LMNC tail (aa 384-566), GST-∆LMNC (aa 128-572).

N-term. globular domain central rod domain (dimerization)

C-term. globular domain

N-term/LMNA (1-127)

coil1B∆ (128−218)

coil2 (243-387)

tail (384-566)

∆LMNC (128-572)

LMNC 1

34 70 80 218 242 383 574

LMNA 1

34 70 80 218 242 383 574 664

Results

48

Equal amounts of uninduced (T0) and induced (T1) E. coli cell lysates expressing each

one of the five different GST-LMNC fusion proteins were subjected to SDS-PAGE. The

expression of the correct fusion proteins and their degradation products was verified by

western blot analysis using anti-GST antibodies (Fig.17 A and B). For all proteins there

was some expression already in uninduced cells. The amount of the proteins increased

after IPTG induction. The antibody detected also degradation products.

Approximately equal amounts of five different GST-LMNC fusion proteins were

immobilized on sepharose beads and incubated with equal amounts of recombinant

human SUN2 protein after thrombin cleavage. The samples were centrifuged and the

supernatant and the pellet fractions were subjected to SDS-PAGE followed by

Coomassie blue staining to detect the GST-LMNC fusion proteins (Fig. 17 C, top panel)

and western blot analysis to detect the recombinant SUN2 protein (Fig. 17 C, bottom

panel).

The N-terminus of SUN2 was coprecipitated by all five GST-LMNC fusion proteins with

variable binding affinities. The GST-fusion constructs LMNNt and coil1B∆ which are

contained within ∆LMNC could precipitate high amounts of SUN2Nt whereas in the

supernatant of ∆LMNC decreased amounts of SUN2Nt could be detected. GST alone

as control was not able to precipitate SUN2Nt. These observed differences in the

binding affinity for ∆LMNC compared with LMNNt might be due to folding and

dimerization processes of overexpressed recombinant LMNC polypeptides in E. coli.

kDa

36

55

72

kDa

LMNNt coil1B∆ coil2 ∆LMNC tail

anti-GST

T0 T1 T0 T1 T0 T1 T0 T1 T0 T1

28

T0 T1 GST

anti-GST

A B

Results

49

Fig. 17: A: Western blot analysis of uninduced (T0) and induced (T1) E. coli XL2 blue cell lysates

expressing the five different GST-LMNC fusion proteins GST-LMNNt, GST- coil1B∆, GST- coil2, GST- tail,

GST-∆LMNC using anti- GST antibody. B: Western blot of uninduced (T0) and induced (T1) E. coli cell

lysates expressing GST alone as control. Anti-GST as primary antibody, POD as secondary antibody,

detection by ECL. C: Coomassie blue stained PAA gel (15%) shows the GST-LMNC fusion proteins (top

panel); western blot using mAb K80-207-11 detecting the recombinant SUN2Nt protein (bottom panel),

POD as secondary antibody, detection by ECL. D: recombinant SUN2Nt protein as control.

17 207-11

SN P SN P SN P SN P SN P

LMNNt coil1B∆ coil2 ∆LMNC tail

SN P

GST

72

55

28

36

kDa

SUN2Nt

207-11

17

kDa

C

D

Results

50

3.6 Characterization of fibroblasts from Duchenne muscular

dystrophy (DMD), Emery-Dreifuss muscular dystrophy/ Charcot-

MarieTooth syndrome (EDMD/CMT) and Stiff skin syndrome (SSS)

patients

In this study, control fibroblasts from a healthy donor individual and three patients

suffering from three different phenotypes related to laminopathies were subjected to a

basic characterization. At the start of this study the patients have been diagnosed by

their symptoms as Emery-Dreifuss muscular dystrophy (EDMD), Emery-Dreifuss

muscular dystrophy/ Charcot Marie-Tooth syndrome (EDMD/CMT) or Stiff skin

syndrome (SSS). In the EDMD and EDMD/CMT patients mutations in the NESPRIN1

gene SYNE1 had been identified and were thought to cause the disease (Zhang et al.,

2007). After the completion of this study, new genetic information resulted in a

redefinition of previous Emery-Dreifuss muscular dystrophy for one patient to Duchenne

muscular dystrophy (DMD) as a mutation in the dystrophin gene was found. Further, for

the EDMD/CMT case the mutation in NESPRIN1 also may not be responsible for the

disease.

3.6.1 Case report of Duchenne muscular dystrophy (DMD), and Emery-Dreifuss

muscular dystrophy/Charcot-Marie-Tooth syndrome (EDMD/CMT) and Stiff skin

syndrome (SSS) patients

Following information (current stage) concerning the patients was obtained from Prof.

Dr. M. Wehnert, Institute for Human Molecular Genetics, Greifswald; Germany.

Patient 1: This patient harbors a pathogenic nonsense E1137X mutation in the DMD

gene coding for dystrophin (c.3409C>T, p. E1137X). Based on this data, the phenotype

of this patient is now classified as Duchenne muscular dystrophy. Prior to genetic

background information the phenotype of this patient was recognized as Emery-Dreifuss

muscular dystrophy.

Notably, this patient harbors a 29A>G mutation in the 5’UTR of the NESPRIN1α2

isoform. As 4% of an unaffected asian reference population reveal these 29A>G

mutation in the 5’UTR of the NESPRIN1α2 isoform, these described nesprin mutation is

probably not responsible for the clinical phenotype but might contribute to the clinical

outcome.

Results

51

Patient 2: The second patient is suffering from EDMD and Charchot Marie Tooth

syndrome (CMT) and has a N323H mutation in a spectrin repeat of NESPRIN1α1

isoform inherited from a clinical healthy asian mother. Similar to the first described

patient, this nesprin mutation seems not to be responsible for the clinical phenotype but

might contribute to another, presently unknown mutation in these patient cells.

Patient 3: This patient is suffering from a phenotype recognized as Stiff skin syndrome

(SSS) (Jablonska et al., 1984; 2000).

Recently it was shown that a mutation in the fibrillin gene FBN1 causes SSS (Loeys et

al., 2010). Fibrillin is a glycoprotein, which is essential for the formation of elastic fibers

found in connective tissue (Kielty et al., 2002). Based on this Dr. Robinson, Charitè,

Berlin, sequenced all exons of the FBN1 gene of the patient with the exception of the

last exon. As no pathogenic mutations were detected an analysis of the coding exons

and intron-exon boundaries of the ZMPSTE24 (FACE1) and the LMNA gene were done.

None of these genes carried disease associated mutations either. ZMPSTE24 (FACE1)

encodes a zinc metalloproteinase involved in the two step post-translational proteolytic

cleavage of carboxy terminal residues of farnesylated prelamin A to form mature LMNA.

Both genes are frequently affected in restrictive dermopathy (RD), a condition which

overlaps with SSS (Youn et al., 2010).

Therefore, underlying mutations in this patient are presently unknown, yet the patient’s

phenotype is categorized as SSS. In addition to a recent publication where a disease in

dogs resembling SSS was due to a mutation in the metalloprotease ADAMTSL2 (Bader

et al., 2010), mutations in FBN1 were also not detected in human SSS patients

(personal communication Dr. P. Robinson, Charité, Berlin) indicating that this condition

may be caused by mutations in different genes as well.

Since laminopathies exhibit variable penetrance and phenotypic heterogeneity and the

fact that about 60% of patients suffering from EDMD or EDMD-like phenotypes do not

have mutations in either emerin or lamin, the involvement of mutations in other,

presently unknown genes and their products is suggested (Politano et al, 2003; Zhang

et al, 2007). It is conceivable that the here discussed NESPRIN1 mutations may

contribute to the disease state in the patients although individuals carrying the

NESPRIN1 mutations are phenotypically normal. Therefore, the patient’s clinical

Results

52

phenotype might diverge from the one expected for the corresponding genotype,

explaining the difficulty to clearly classify the patient’s phenotype.

3.6.2 Patient fibroblasts show nuclear defects

Several abnormalities affecting nuclear shape and distribution of nuclear envelope

proteins could be observed in nuclei from patient cells. To quantify these observations

100 nuclei of control and patient fibroblasts with the EDMD/CMT and DMD phenotype

and the Stiff skin syndrome cells were evaluated at passage 8, 12 and 16. Each

experiment was carried out twice.

Nuclei of control cell generally had a round or ovoid shape. In patient fibroblasts the

number of cells with nuclear defects was increased compared to control cells and

micronuclei and a variety of nuclear shape defects including folds, lobulations and blebs

were observed (Fig. 18). Remarkably, the DMD patient cells exhibited the most

significant nuclear shape alterations. At an average, 30% of DMD cells showed

misshapen nuclei whit a slight increase from passage 8 to passage 16, while in Stiff skin

syndrome cells ~20% of the nuclei were misshapen and no significant increase of

altered nuclei shape was observed in higher passages. In contrast, EDMD/CMT

fibroblasts exhibited 15 to 19% misshapen cells in passage 8 and passage 12,

respectively, and at passage 16 increase to 32 % of altered shaped cells were

observed.

The relation between micronuclei and misshapen cells correlated and remained almost

constant.

Results

53

D

Fig.18: A-C: 100 nuclei per control and patient cells were evaluated at passage 8, 12 and 16. Each

experiment was carried out twice. D: Immunofluorescence of DMD and EDMD/CMT patient fibroblasts.

Nuclei stained with DAPI. Arrows indicate the observed defects.

EDMD/CMT EDMD/CMT DMD

A

C

B

DMD

Results

54

3.6.3 Proliferative ability of patient fibroblasts is restricted

To evaluate the effect of the nuclear envelope defects observed in the EDMD/CMT,

DMD and stiff skin syndrome patient fibroblast on cellular functions the proliferative

ability was analyzed. To determine cell growth rates 1x105 cells of each patient cell line

and the control cells were seeded and counted every 48 hours for a period of 6 days

(Fig. 19). For the DMD patient cells the cell number in the beginning was 1x104 due to

very slow growth rate and therefore the limited availability of these cells. The experiment

was carried out twice.

Patient cell lines exhibited decreased proliferation when compared with control cells.

The growth curve of the patient cell line with the EDMD/CMT shows a remarkably

reduced growth compared to the control whereas the stiff skin cells revealed only a

slightly reduced growth. In contrast, fibroblasts from the patient suffering from DMD

showed significantly reduced growth compared to control cells and to the other two

patient cells.

-1

0

1

2

3

4

5

6

7

8

1 2 3 4 5 6

cell

num

ber

x 1

05

time (d)

ctrlEDMD/CMTstiff skinDMD

Fig. 19: Growth curves of control and patient fibroblasts.

Results

55

3.6.4 Increased senescence is induced in patient fibroblasts

A decline in the growth rate is often associated with cell senescence. To examine

whether cellular senescence was induced in patient fibroblasts the expression of a

specific isoenzyme of β galactosidase, which is referred to as senescence-associated β-

galactosidase (SA-β-gal) was analysed. Cells fixed in 4% PFMA were incubated with

freshly prepared SA-β-Gal staining solution at 37°C for 12 hours and subsequently the

percentage of stained cells under bright field microscopy determined. The experiment

was carried out twice with 200 cells each. In control fibroblasts less than 1% of the cells

were positive for β-galactosidase. In contrast, 8% fibroblasts from the patient with the

Stiff skin syndrome expressed β-galactosidase and a blue staining was observed. Cells

from EDMD/CMT patient revealed a positive staining in 11% of the cells whereas in

DMD patient fibroblasts 14% of the cells were stained (Fig. 20).

0

2

4

6

8

10

12

14

16

ctrl DMD EDMD/CMT stiff skin

% o

f se

nescen

t cells

Fig. 20: Percentage of senescent cells in control and patient cells. The experiment was done in duplicate, 200 cells were counted each).

Results

56

3.6.5 SUN2 gene expression is down-regulated in senescent patient cells

To evaluate the gene expression level of the SUN2 protein in the patient fibroblast in

comparison to the control fibroblasts, total RNA was isolated (control fibroblasts at

passage 8 and 22, EDMD/CMT fibroblasts at passage 4 and 16, DMD fibroblasts at

passage 6 and 8, and Stiff skin syndrome fibroblasts at passage 5 and 17). The cDNA

was used for quantitative Real Time PCR (qRT-PCR) experiments. To normalize the

expression of the targeted gene the stably expressed housekeeping gene GAPDH was

chosen to correct possible differences in RNA quantity or quality across experimental

samples. Each experiment was performed in triplicate and repeated 3 times.

At lower passages transcript level of SUN2 is not up- or down- regulated in a statistically

significant way in the patient cells with the DMD and EDMD/CMT phenotype. Patient

fibroblasts with the Stiff skin syndrome show a regulation factor of 1.1 and therefore an

increased gene expression of SUN2 (Fig. 21). In the senescence stadium of the cells

the transcript level of SUN2 is remarkably decreased in the DMD and EDMD/CMT

patient cells. Compared to the control cells the transcript level of SUN2 is not

significantly decreased in the stiff skin fibroblasts at passage 17 but in comparison with

the expression levels measured in lower passages the gene expression of SUN2 is

remarkably decreased.

Fig. 21: SUN2 transcript levels in control and patient fibroblasts as determined by qRT-PCR. A: The

SUN2 mRNA level in control fibroblasts at passage 8 were taken for reference (1), EDMD/CMT fibroblasts

at passage 4, DMD fibroblasts at passage 6, Stiff skin syndrome fibroblasts at passage 5. B: control

fibroblasts at passage 22, EDMD/CMT fibroblasts at passage 16, DMD fibroblasts at passage 8, Stiff skin

syndrome fibroblasts at passage 17.

0,6

0,68

0,76

0,84

0,92

1

1,08

EMD/CMT p16 DMD p8 stiff skin p17

0,94

0,96

0,98

1

1,02

1,04

1,06

1,08

1,1

1,12

EMD/CMT p4 DMD p6 stiff skin p5

A B

Results

57

Immunoblot analysis of control and patient fibroblast using mAb K80-207-11 confirmed

the results obtained by quantitative PCR. Tubulin was used as loading control. Patient

fibroblast with the Stiff skin syndrome showed a higher amount of SUN2 protein at lower

passages compared to the control cells, and a decreased expression level in higher

passages. The tubulin-loading control was slightly decreased in DMD and EDMD/CMT

cell lysates; nevertheless, a decrease in the protein levels of SUN2 in the DMD and

EDMD/CMT patient cells at higher passages was observed.

Fig.22: Immunoblot analysis of lysates from control and patient fibroblasts at different passages: A:

control fibroblasts at passage 8, EDMD/CMT fibroblasts at passage 4, DMD fibroblasts at passage 6, Stiff

skin syndrome fibroblasts at passage 5. B: control fibroblasts at passage 22, EDMD/CMT fibroblasts at

passage 16, DMD fibroblasts at passage 8, Stiff skin syndrome fibroblasts at passage 17. mAb K80-207-

11 and WA3 (anti-tubulin) as loading control were used as primary antibodies, POD as secondary

antibody, detection was done by ECL.

anti-tubulin

SUN2

ctrl p22

SSSp17

EDMD/ CMTp16

ctrlp8

SSSp6

EDMD/ CMTp6

DMDp6

DMD p8

A B

Results

58

3.6.6 Cell adhesion is altered in patient fibroblasts

Cell adhesion makes an important contribution to the maintenance of tissue structure,

the promotion of cell migration, and the transduction of information about the cell

microenvironment across the plasma membrane. To evaluate the adhesion ability of the

patient cells to a substratum compared to the control fibroblasts, trypsinized cells were

seeded on coverslips in culture dishes with an initial cell number of 1x103. Every 15

minutes three coverslips of each cell line were rinsed with PBS and cells remaining

attached on the coverslips were subjected to immunofluorescence analysis with vinculin

specific antibodies (Fig. 23 A). Vinculin is a membrane-associated cytoskeletal protein in

focal adhesion plaques that is involved in the linkage of integrin adhesion molecules to

the actin cytoskeleton. The individual immunofluorescences shown in Fig. 23 A have the

same magnification (scale bar, 76.4 µm) and were taken in the same z-plane so that the

spreading of focal adhesions on the surface of the coverslip is comparable. To quantify

the observed differences in spreading, the area measured in µm2 was evaluated using

LAS-AF Lite Application Suite software from Leica (Fig.23B (b)).

As shown in the table in Fig. 23 B (a), all cell lines attached and the adhesions

increased progressively. Control cells had at every measured time point the largest area

of spreading on the substratum. Fibroblasts from the Stiff skin syndrome affected patient

exhibited the lowest spreading at all analysed time points. Notably, when settling of the

cells was completed after 75 min, spreading on the substratum of patient cells was

approximately two fold lower than the spreading observed for the control cells.

A statistic evaluation of the adhesion ability is shown in Fig. 23 C. After 15 minutes less

than 20% cells off the EDMD/CMT, DMD and Stiff skin syndrome patient cell lines were

attached to the surface of the coverslips. In comparison, 30% of the control cells were

attached to the coverslips after 15 minutes. Attachment to the substratum was

significantly increased in EDMD/CMT and DMD patient cells in comparison to the

control cells after 30 and 45 minutes. After 45 minutes the number of attached

EDMD/CMT and DMD patient cells was close to the number of attached control cells.

After 45 minutes the number of attached stiff skin cells was two fold lower than the

number of attached control cells. Attachment to the surface of Stiff skin syndrome

patient cells have been remarkably decreased at all evaluated time points. 90 minutes

after initial seeding the cells of all cell lines have been completely attached to the

surface of the coverslips.

Results

59

A

B (a)

15 min 30 min 45 min 60 min 75 min

ctrl 1277.5 2385.51 2389.04 3882.86 7165.07

SSS 722.37 865.16 834.83 1002.25 2865.36

EDMD/CMT 978.02 936.09 1453.69 3058.94 3193.63

DMD 989.18 1126.05 1800.52 2691.37 4012.26

B (b)

ctrl

SSS

EDMD/CMT

DMD

15‘ 30‘ 45‘ 60‘ 75‘

Results

60

C

0

20

40

60

80

100

120

15 30 45 60 75 90

min

% o

f a

tta

ch

ed

ce

lls

ctrl

stiff skin

EDMD/CMT

DMD

Fig. 23: A: Immunofluorescence analysis of control and patient fibroblasts 15, 30, 45, 60 and 75 min after

seedin stained for vinculin. Alexa Flour 488 conjugated secondary antibody was used, DAPI for nuclear

staining; scale bar 76.4µm. B (a): vinculin stained area on the surface of coverslips of control and patient

fibroblasts 15, 30, 45, 60 and 75 min after seeding, measured in µm2. (b): polygon drawn with LAS-AF

Lite Application Suite software from Leica indicates the encircled area in µm2. C: Adhesion ability of

control and patient fibroblasts 15, 30, 45, 60 and 75 min after seeding. After 90 min seeding attachment

was completed.

Results

61

3.6.7 Distribution of nuclear envelope proteins in control fibroblasts and patient

fibroblasts

The distribution of several nuclear envelope proteins was assessed by

immunofluorescence analysis. Control fibroblasts show a continuous nuclear rim

staining for SUN2 with mAb K80-207-11. EDMD/CMT, DMD and Stiff skin syndrome

fibroblasts staining for SUN2 with mAb K80-207-11 exhibit a similar staining pattern.

SUN2 was localized to the nuclear envelope but was also present in micro nuclei as well

as in blebs and foldings (Fig. 24-28, second column, arrowhead). Additionally, in

EDMD/CMT and Stiff skin syndrome cells the SUN2 staining appeared discontinuously

distributed and the rim staining was weaker than in the control cells. Especially for stiff

skin patient fibroblasts a broadened rim staining for SUN2 was observed. The same

observations could be made for emerin which was localized at the nuclear envelope but

also in blebs and folds and enriched in micronuclei in EDMD/CMT patient cells.

Especially EDMD/CMT and stiff skin patient fibroblasts exhibit more of an overall

nuclear emerin staining and a weaker rim staining compared to the control cells (Fig.

24).

Results

62

Fig. 24: Distribution of nuclear envelope proteins in control and patient fibroblasts. Control, EDMD/CMT,

DMD and Stiff skin syndrome fibroblasts were stained for SUN2 and emerin using mAb K80-207-11 and

anti-emerin as first antibodies; Alexa Flour 568 and 488 as conjugated secondary antibodies; DAPI for

nuclear staining. Arrowheads indicate SUN2 staining in nuclear shape defects; arrows indicate the

staining in nuclear shape defects described in the text. Scale bar 5 µm.

LMNA/C staining was localized to the nuclear envelope and to some extend distributed

within the nuclei in control cells and in the EDMD/CMT patient cells. SUN2 staining was

slightly diminished in these cells compared to the control cells.

Severely misshapen nuclei of DMD and stiff skin patient cells exhibited localization of

LMNA/C into foldings of the nuclear envelope (arrows). The strong cytoplasmic staining

in the bottom row is most probably unspecific staining due to cell fragments from dead

cells that have not been completely removed during the immunofluorescence

preparation (Fig. 25).

FR

Wt

emerin

-11

merge emerin DAPI 207-11

SSS

FR

Wt

emerin

-11

merge emerin DAPI

ctrl

DMD

207-11

EDMD/CMT

Results

63


DMD and Stiff skin syndrome fibroblasts were stained for SUN2 and LMNA/C using mAb K809-207-11

and anti-LMNA/C as first antibodies; Alexa Flour 568 and 488 as conjugated secondary antibodies; DAPI

for nuclear staining. Arrowheads indicate SUN2 staining in nuclear shape defects; arrows indicate the


LMNB1 was localized to the nuclear envelope in control cells and in all patient fibroblast.

Like SUN2, emerin and LMNA/C, LMNB1 is located in all regions of the nuclear surface,

extending into clefts and protuberances of the nuclear envelope in EDMD/CMT and

DMD patient cells (arrows). Compared to control cells SUN2 staining was slightly

weaker in EDMD/CMT cells.

LMNB1 was found in stiff skin patient cells colocalizing with SUN2. Note the protrusion

stained by LMNB1 observed in the lower area of the stiff skin fibroblasts (dotted arrow)

(Fig. 26).

WT

merge LMNA/C

DMD

ctrl.

SSS

K80-207-11 DAPI

EDMD/CMT

Results

64


DMD and Stiff skin syndrome fibroblasts were stained for SUN2 and LMNB1 using mAb K809-207-11 and

anti-LMNB1 as first antibodies; Alexa Flour 568 and 488 as conjugated secondary antibodies; DAPI for



To detect NESPRIN1 polyclonal antibodies directed against C-terminal spectrin repeats

of the protein (NESPRIN1SpecII) were used. In all cells, the NESPRIN1 staining was

week. In control cells NESPRIN1 was localized to the nuclear envelope. In EDMD/CMT

cells diminished staining for NESPRIN1 was detected in comparison to the control cells.

In DMD fibroblasts NESPRIN1 was localized to the nuclear envelope but also distributed

in the nuclei and found in the cytoplasm to some extend. In stiff skin patient fibroblasts

the arrow indicates an accumulation of NESPRIN1 staining (Fig. 27).

laminB w

20

-1

LMNB1

DMD

ctrl

l

SSS

K80-207-11 DAPI merge

EDMD/CMT

Results

65


DMD and Stiff skin syndrome fibroblasts were stained for SUN2 and NESPRIN1 using mAb K809-207-11

and anti-NES1SpecII as first antibodies; Alexa Flour 568 and 488 as conjugated secondary antibodies;

DAPI for nuclear staining. Arrowheads indicate SUN2 staining in nuclear shape defects; arrows indicate

the staining defects described in the text. Scale bar 5 µm.

To investigate the localization pattern of NESPRIN2, polyclonal antibodies directed

against the last two C-terminal spectrin repeats of human NESPRIN2 were used. The

patchy and distributed staining of NESPRIN2 in the nucleus that can be observed for

control cells could also be seen in all patient cell lines. Additionally, NESPRIN2

appeared to be enriched and evenly distributed in micro nuclei found in DMD patient

fibroblasts. Week cytoplasmic staining of NESPRIN2 was seen in control cells as well as

in all patient cells. Extended locations of NESPRIN2 into the cytoplasm were seen in

wt

merge

EDMD/CMT

DMD

ctrl.

NES1SpecII

SSS

DAPI K80-207-11

Results

66

Stiff skin syndrome cells. The strong cytoplasmic staining in the second row is

presumably an artefact (Fig. 28).


DMD and Stiff skin syndrome fibroblasts were stained for SUN2 and NESPRIN2 using mAb K809-207-11

and anti-NES2 as first antibodies; Alexa Flour 568 and 488 as conjugated secondary antibodies; DAPI for



The lamina-associated polypeptide-2 (LAP2) is also an inner nuclear membrane protein,

which has been shown to bind to A-type lamins and BAF-DNA complexes (Dechat et al.,

2000; Shumaker et al., 2001). The distribution of LAP2 was assessed using LAP2

antibodies. LAP2 distribution was seen at the nuclear envelope and to some extent to

the nucleoplasm in control cells as well as in all patient cells. In dysmorphic patient cell

merge NES2

DMD

EDMD/CMT

ctrl

SSS

DAPI K80-207-11

Results

67

nuclei LAP2 was present on the nuclear surface and in blebs and increased staining can

be seen in micro nuclei of DMD patient fibroblasts (arrow) (Fig. 29). Rim staining of

LAP2 was reduced in EDMD/CMT cells relative to control cells. Since LAP2 and K80-

207-11 are both mouse monoclonal antibodies, costaining for SUN2 was not possible.


DMD and Stiff skin syndrome fibroblasts were stained for SUN2 and LAP2 using mAb K809-207-11 and

anti-LAP2 as first antibodies; Alexa Flour 568 and 488 as conjugated secondary antibodies; DAPI for

nuclear staining. Arrow indicates the staining in nuclear shape defects described in the text. Scale bar 5

µm.

merge

ctrl

DAPI LAP2

SSS

DMD

EDMD/CMT

Results

68

3.6.8 Nucleus-centrosome distance is increased in EDMD/CMT, DMD and Stiff skin

syndrome fibroblasts

The centrosome plays a key role in cellular architecture by determining the position of

several associated organelles, including the nucleus. Previous published data indicate

that nuclear envelope proteins like LINC- proteins and emerin are participating in

centrosome-nucleus juxtaposition and mediate shuttling of nuclear and centrosomal

proteins between these organelles (Hutchison et al., 2007, Xiong et al., 2008).

Therefore, the localization of the centrosome relative to the nucleus was investigated

using antibodies against pericentrin (Fig. 30 A, B). Nucleus-centrosome distance was

measured for 50 cells for each patient cell line using Leica LAS AF Lite software. The

experiment was carried out once (Fig. 30 C).

In control cells each nucleus was located in close proximity of one centrosome during

the interphase. In control cells the centrosome was positioned adjacent to the

membrane or within 1.30 µm of the nuclear envelope. Misshapen nuclei from patient

cells as well as normal shaped nuclei exhibit a slightly increased distance of the

centrosome from the nuclei. The mean distance of the nucleus to the centrosome that

was measured for DMD cells was 3.25 µm, the centrosome was positioned between 0

µm and 8.67 µm and this cells showed therefore an increased nucleus-centrosome

distance in comparison to the control cells. In EDMD/CMT fibroblasts also an increased

distance of 4.01 µm was exhibited. The maximum distance that was measured was 9.05

µm. In stiff skin patient cells the mean centrosome-nucleus distance was 2.37 µm, with

a maximum distance of 8.42 µm (Fig. 30 C).

In regular-shaped control cells, the nucleus number correlated with that of the

centrosome. These observations were also true for regular-shaped and misshapen

nuclei from patient cells. For EDMD/CMT cells one cell with two centrosomes was seen.

The presence of micronuclei had no influence on the nucleus-centrosome distance (Fig.

30 B).

Results

69

C

0

0,5

1

1,5

2

2,5

3

3,5

4

4,5

ctrl 0-1.30 EDMD/CMT 0-9.05 DMD 0-8.67 stiff skin 0-8.42

mean d

ista

nce (

µm

)

Fig.30 A, B: Immunofluorescence of control, EDMD/CMT, DMD and stiff skin syndrome fibroblasts, anti-

pericentrin as primary antibody, Alexa Flour 488 as conjugated secondary antibodies, DAPI for nuclear

staining. Scale bar: 5µm. C: Nucleus–centrosome mean distances measured in µm with Leica LAS AF

Lite software, 50 cells for control, EDMD/CMT, DMD and Stiff skin syndrome fibroblasts were examined.

Minimal- and maximal values measured for each patient cell line are listed in the x-axis.

ctrl DMD SSS

A

2,62 µm

5,43 µm

8,67 µm

8,30 µm

2,32 µm

EDMD/CMT

616 nm

2.09µm

EDMD/CMT DMD

B

Results

70

3.6.9 Precipitation profile in EDMD/CMT fibroblast cells differ from control

fibroblast cells

As an initial screening assay for identifying possible differences in protein interaction

partners of SUN2Nt in control cells compared to patient cells, pull down experiments

with total control fibroblast cells lysates and total lysates from EDMD/CMT cells and

GST-SUN2Nt as bait were performed. Analysis was done by pre-separating the protein

complex by SDS-PAGE and followed by LC-MS (Liquid chromatography-mass

spectrometry) as described in 3.4.

Fig.31: Coomassie Blue stained PAA gel (12% acrylamide) showing proteins from pull down assays using

total control cell lysate and total EDMD/CMT cell lysate and GST-SUN2Nt as bait. Controls: beads

incubated with the indicated cell lysate. Arrows and grey brackets indicate areas of interest for protein

analysis. Black arrows: present in both samples; grey arrows: diminished in control cell/patient cell lysate;

red arrows: only found in control cell/patient cell lysate.

Table 2 represents a list of proteins which are categorized in proteins taking part in gene

regulatory events (A), in proteins present in RNA-processing complexes (B), in proteins

of architectural complexes (C) and proteins participating in signaling (D). The diagram in

figure 32 summarizes the proteins of each category and illustrates their distribution in

either total control cell lysates or EDMD/CMT cell lysates precipitated by GST-SUN2Nt

as bait. Proteins found in both lysates are illustrated in the intersection.

250 130

95

GST- SUN2Nt +EDMD/CMT- lysate

beads +EDMD/ CMT- lysate

beads +ctrl -lysate

GST- SUN2Nt +ctrl -lysate

72

55

Results

71

Tab. 2: Proteins detected in total control and patient fibroblast lysates precipitated by GST-SUN2Nt

categorized in gene regulatory (A), RNA processing (B), architectural complex (C) and signaling (D)

proteins. Identifier indicates the SwissProt accession number if not stated otherwise.

Identifier Name Category ctrl EDMD

/ CMT

Q9NVE4 (UniProtKB) CCDC87 A + +

P68104 (UniProtKB) EEF1A1 B - +

NP_991247.1 HNRNPH1 B + +

O00571 mRNA helicase 3X B + -

EEF2_HUMAN EEF2 B + -

P60842 (UniProtKB) EIF4A1 B + +

Q8N9N8 EEF1G B - +

Q9UHB6 LIMA1 C + -

Q8WZ42 titin C - +

O43707.2 α-actinin1 C + -

P12814.2 α-actinin4 C + -

P41351 tubulin α C + +

P05219 tubulin β C + +

Q9ULV4.1 coronin1C C + -

P02545.1 laminA/C C, D + +

P68032 actin C, D + -

P35579 myosin9 C, D + -

O00159 myosin1c C, D + -

Q8WXH0 nesprin2 C, D + -

P53618.3 coatomer β D + -

AAH20498.2 coatomer γ D + -

AAH20498.2 coatomer γ D - +

P22314 ubiquitin-like modifier-activating enzyme D - +

Only one protein taking part in DNA-processing events, the coiled coil domain

containing protein 87 (CCDC87), could be detected in lysates from control cells and in

lysates from EDMD/CMT affected patient cells. In both lysates equal amounts of

proteins participating in RNA-processing (B), like translation factors and HNRNPH1,

could be detected. In control cell lysates significantly more proteins contributing to the

cell architecture (C) and signaling (D) events could be detected which were absent from

the EDMD/CMT cell lysate. Missing proteins are mainly actin related proteins such as

Results

72

actin, myosin1C and myosin9. Notably, NESPRIN2 was only found in control cell

lysates. LMNA/C was identified in both lysates.

Fig. 32: Distribution of proteins in either total control fibroblasts or EDMD/CMT fibroblasts lysates

precipitated by GST-SUN2Nt as bait. Proteins found in both lysates are illustrated in the intersection.

tubulinα

tubulinβ LMNA/C

HNRNPH1 EIF4A1

coatomerγ

α-actinin1

α-actinin4 coronin1C

LIMA1 actin

myosin9 myosin1c

NESPRIN2

coatomerβ mRNA helicase 3X

EEF2 CCDC87

titin ubiquitin- like modifier-

activating enzyme EEF1A1 EEF1G

control

EDMD/CMT

Discussion

73

4 Discussion

4.1 Generation of a monoclonal antibody

In the present study mouse monoclonal antibodies directed against an N-terminal

peptide comprising amino acids 1-138 of human SUN2 protein were generated. Several

hybridoma clones showed a similar recognition pattern in western blot analysis.

Hybridoma subclone K80-207-11 detected a recombinant bacterially expressed protein,

the mammalian V5*6xHis-tagged fusion, and endogenous SUN2 protein in

immunoblotting and immunostaining and thus was selected for subsequent applications.

HeLa cells stained with emerin specific antibodies and K80-207-11 showed an

overlapping localization of the proteins at the nuclear envelope. In cell fractionation

studies, K80-207-11 recognized SUN2 exclusively in the nuclear fraction. These data

indicate that SUN2 is primarily located at the nuclear envelope. The cytoplasmic staining

that is observed in the immunofluorescence experiments may be derived from non-

specific interactions of mAb K80-207-11.

Westernblot analysis revealed the presence of 2 bands at ~75 and ~70 kDa in HeLa and

Pop10 cells. This could be due to differential splicing, although experimental evidence is

lacking so far (Hodzic et al, 2004). Alternatively, the faster migrating protein could be a

degradation product, or it might be a cross reactive protein.

4.2 Subcellular localization of endogenous SUN2 protein during the cell cycle

Immunostaining of HeLa cells revealed that SUN2 is largely segregated within the plane

of the NE in non mitotic cells. The nuclear rim staining by K80-207-11 persisted during

prophase. When the nuclear envelope is completely disassembled in metaphase, SUN2

associates with the condensed chromosomes. In anaphase SUN2 appears to

accumulate at distinct chromosome regions of the condensed chromatids which might

be telomere regions as described in meiotic cells for sad1 in S. pombe (Hagan et al.,

1995; Alsheimer et al., 2006).

In metaphase, SUN2 can be detected in two dot-like structures which colocalize with

pericentrin and therefore indicate a centrosomal localization of SUN2. Similar findings

have been reported by Wang et al. (2006). In C. elegans the KASH protein zyg1 and

sun1 are essential for tethering the centrosome to the NE and therefore for pronuclear

migration (Gönczy et al., 2004; Malone et al., 2003). In Dictyostelium, Sun1 is required

for centrosome attachment (Xiong et al. 2008), and mouse Nesprin1/2 double knockout

cells have a centrosome detachment phenotype (Zhang et al. 2009). The role of KASH-

Discussion

74

SUN bridges in centrosome attachment to the nucleus is less clear in other tissues and

systems. Since SUN2 colocalizes with pericentrin in mitotic HeLa cells, there is

evidence that SUN2 proteins associate with the centrosomes and provide a molecular

linkage between the NE and the centrosome. If this linkage is maintained via nesprins

and the microtubule cytoskeleton or if novel interaction partners are involved has to be

further elucidated.

Upon nuclear envelope reassembly during late anaphase and telophase, K80-207-11

detects the SUN2 protein in a rim like pattern. SUN2 is enriched at a distinct core region

of each newly separated chromatin mass. At late anaphase to early telophase,

reforming nuclei exhibit a distinct distribution of nuclear pore complexes (NPC) and NE

components and the core region is typically deficient in NPC reformation while NPC

assembly is initiated on the lateral margins of the chromatid masses (Liu et al., 2001).

Pore-free islands are directly connected with gene silent heterochromatin regions

beneath them (Casolari et al., 2004). Pore-rich and pore-poor subdomains on the

nuclear envelope might reflect gene activation and silencing states of the corresponding

nuclear surface of certain chromosome territories. So far LMNA/C has been shown to

play an essential structural and regulatory role in the formation of pore-free islands

(Maeshima et al., 2006). Higher proliferation ability and aggressiveness in human

malignancies including leukemias and lymphomas, small-cell lung carcinoma and skin

carcinomas is correlated with the absence or downregulation of LMNA/C (Stadelmann et

al., 1990; Broers and Ramaekers, 1993; Venables et al., 2001). These reports support

the hypothesis that LMNA/C has a negative influence on cell proliferation and that the

suppression of LMNA/C might contribute to tumorigenesis through increasing pore

density. SUN2 as a direct LMNA/C interactor might therefore be involved in the

regulation of cell cycle depending dynamics and nuclear envelope subdomain

organization by controlling the reorganization of inner nuclear structures.

4.3 Protein networks formed by SUN2

Since distribution and localization of SUN2 changes during the cell cycle, a change in

interaction partners during nuclear envelope breakdown is conceivable. In an initial

approach, proteins which interacted with the N-terminus of SUN2 from total cell lysates

of normally grown HaCaT cells (HaCaT cell lysate) and HaCaT cells arrested in

prometaphase (mHaCaT cell lysate) were analyzed by LC-MS and compared to each

other. The proteins identified in these experiments can be categorized in proteins

Discussion

75

participating in DNA-replication and chromatin remodeling, components of RNA

processing complexes, proteins of the architectural complex and proteins participating in

signaling.

Proteins contributing to DNA replication and chromatin remodeling are found in almost

equal amounts in the SUN2 precipitates from HaCaT and mHaCaT cell lysates. This

complex includes components of the nucleosome like H4 and H3-like and proteins that

mediate gene silencing via histone modifications like RNA-Pol II associated factor 1

(PAF1), Histone deacetylase (HDAC) and RUVB-like1/2 which are components of the

histone acetyltransferase (HAT)-complex. Also involved in histone modification and

transcription repression are C-terminus-binding protein 1 (CTB1), YLB-motif containing

protein 1 (YLB1) and metastasis associated protein 2 (MTA2). Both lysates contained

these proteins that predominantly participate in gene repression events in approximately

same amounts (Protein information obtained from www.ncbi.nlm.nih.gov).

Gene activation and repression are mostly regulated through DNA methylation,

chromatin remodeling and histone modifications. A variety of proteins are involved in

chromatin regulation including DNA methyltransferases, chromatin-remodeling

complexes, DNA transcription factors and chromatin-modifying complexes. Chromatin-

remodeling complexes modify nucleosome structure and modulate the accessibility of

DNA for transcription factors. Human Sin3-HDAC complex includes HDAC1, HDAC2

and the histone-binding proteins RbAp46 and RbAp48 (Perissi et al., 2010). RbAp48 is

also termed RBBP4 and was found in pull down experiments using the N-terminus of

SUN2 in both HaCaT cell lysates together with HDAC1. HDAC1 is also involved in the

CoREST and NURD complexes which are important transcription silencing machines

(Huang et al., 2011). CTB, a part of the CoREST complex, was also found in the lysates

while MTA2, a component of the NURD complex, was only detected in mHaCaT lysates.

A recent study reported the nuclear actin binding protein coronin2A as part of the NCoR

(nuclear receptor co-repressor) and SMRT (Silencing Mediator of Retinoid acid and

Thyroid hormone receptor) complex (Perissi et al., 2010). Here, a related coronin,

coronin1C, was found in pulldown experiments.

Links between NCoR and SMRT to several types of leukaemias including acute

promyelocytic leukaemia, acute myeloid leukaemia and paediatric b-cell acute

leukaemia have been reported (Karagianni and Wong, 2007). A correlation between

NCoR expression and the most common and aggressive type of primary brain tumour,

astrocyte-derived cancer glioblastoma multiforme (GbM), has been observed. In severe

Discussion

76

grades of astrocytomas NCoR is dramatically increased and correlates with progress

from WHO (World Health Organization) grade II to grade IV glioma (Lubensky et al.,

2006; Park et al., 2007). Likewise, coronin1C expression is significantly altered in

gliomas. In highgrade anaplastic astrocytomas, anaplastic oligodendrogliomas,

anaplastic oligoastrocytomas and glioblastomas high numbers of coronin1C-positive

tumor cells have been found, which is suggestive for a contribution of the protein in the

malignant progression of diffuse gliomas (Roadcap et al., 2008; Xavier et al., 2009). In

addition, SUN2 staining was significantly increased in the human glioblastoma-

astrocytoma cell line U373. Whether the observed interaction is direct or indirect is not

yet known. However a participation of the LINC complex protein SUN2 in the

development of tumorigenesis is intriguing. It is conceivable that increased SUN2

localization at the NE recruits increasing levels of coronin1C contributing to tumor

malignancy.

Altered mRNA synthesis and processing has been reported to be involved in a broad

spectrum of human diseases, including cancer, spinal muscular atrophy and

Hutchinson-Gilford Progeria syndrome (Morares, 2009). In the present study, putative

interactions with proteins of the RNA processing complex suggest a novel role for SUN2

in RNA processing and splicing. In the proteins categorized as RNA processing

components an equal number of proteins could be detected in both cell lysates. While

mainly heterogeneous nuclear ribonucleoproteins (hnRNPs) and splicing factors were

detected in lysates of asynchronously growing HaCaT cells, lysates of mHaCaT cells

contained three translation factors absent from the HaCaT cell lysates. Eukaryotic inition

factor 4A is a nuclear protein that specifically functions during nonsense-mediated

decay (NMD), a RNA surveillance mechanism that degrades mRNA bearing a

premature termination codon (PTC) (Wagner and Andersen, 2001; Ferraiuolo et al.,

2004). About 30% of inherited disorders including β-thalassemia, myotonia congenita

and retinal degeneration are the consequences of mutations that create a PTC

(Frischmeyer et al., 1999). The presence of eukaryotic translation initiation factors found

in the lysates is irritating at first glance. However reports exist describing that some

nuclear translation closely coupled to transcription takes place (Iborra et al., 2004). The

presence of translation initiation factors in the nucleus and how they act in the nucleus

has to be further elucidated.

More than twice the number of proteins contributing to cell architecture was detected in

mHaCaT cell lysates in comparison to HaCaT cell lysates. Proteins found only in the

Discussion

77

mHaCaT cell lysates participate mainly in the actin cytoskeleton. Many proteins with

known structural roles in the cytoskeleton are either localized in the nucleus or shuttle in

and out of the nucleus, supporting the existence of both cytoplasmic and nuclear

isoforms of key structural proteins. For actin it is reported that it shuttles between the

nucleus and the cytoplasm and assembles to nuclear-specific short polymers (Pederson

and Aebi, 2002; Pederson and Aebi 2005; McDonald et al., 2006). Diverse roles are

reported for nuclear actin including chromatin remodeling and transcription, processing

and export of mRNA (Bettinger et al., 2004, Olave et al., 2004). Included in the actin

network is α-actinin, Ras-GTPase activating like protein and myosin. Myoferlin is

required for myotube formation (Doherty et al., 2005). It has also been reported that

actin binds directly to LMNA/C at two actin binding sites in the tail region of LMNA and

at one region in LMNC (Simon et al., 2010). Therefore, actin and actin-related proteins

might be present in the detected protein pool due to interactions with lamin contained in

both lysates, and which interacts with SUN2.

In most cells tubulin resides only in the cytosol and not in the nucleus. The βII- isotype

of tubulin was recently found in the nuclei of several tumor cells but could not be

detected in biopsy samples of normal human tissues suggesting that the presence of

nuclear βII-tubulin may be correlated with the cancerous state of cells (Xu et al., 2002,

Yeh et al., 2004). However, the function of βII-tubulin in the nucleus is still unknown.

Microtubule polymers formed by α- and β-tubulin have been detected in both lysates.

Since only the N-terminus of SUN2 was used as bait, no indirect interactions with

microtubule through ONM-located nesprins can be responsible for the presence of

tubulin. Also, with regard to the reported findings of tubulin isoforms in tumor cell nuclei

HaCaT cells and not HeLa (human cervical cancer) cells have been used for pull down

assays. Therefore, the presence of tubulin might be due to microtubules that have

invaded the nuclear space upon nuclear envelope disassembly in prometaphase and

suggest a role for SUN2 in coupling the mitotic spindle microtubules to kinetochores or

to opposing microtubules (Loubery et al., 2008). Tubulin pools can be found in both

lysates because the normally grown HaCaT cells are not synchronized and therefore

some of the cells underwent mitosis when harvested for the assay.

Several subunits of coat protein complex I (COPI), namely COPα, β, γ, δ, could be

pulled down by the N-terminus of SUN2. Trafficking of many membrane proteins within

the Golgi and between Golgi and ER relies on the recognition of ER localization signals

by COPI. As described in 3.1 and shown in figure 4 and 5, four arginine residues at

Discussion

78

amino acid position 102-105 conserved in mammalian SUN2 but not in SUN1, provide a

ER localization signal as previously described for membrane proteins (Michelsen et al.,

2005). This 4R motif of mammalian SUN2 was recently shown to interact with COPI,

and mutations in this motif resulted in loss of the association (Turgay et al, 2010).

These observations underline not only the structural importance of SUN2 during the cell

cycle, but also point to SUN2 as a protein that scaffolds a variety of multi-protein

complexes at the inner nuclear membrane. The identity of the putative interactors

suggests that SUN2 might exhibit a network function at the inner nuclear membrane by

interlinking proteins with diverse functions. Also, the presence of several RNA-

processing proteins may point to a novel role for SUN2 in the processing of RNA, and

could extend the mode of operation for SUN2 in cellular processes. Therefore, SUN2

may not only be essential in the transmission of signals via the LINC complex, but may

also influence and mediate gene regulation at various levels and functions. Such

proposals are however still hypothetical as the interactions have not been verified by

different methods yet.

4.4 Direct interactions of LMNA/C with the N-terminus of SUN2 in vitro

A previous study (Crisp et al, 2006) reported the in vitro interaction of a GST-fusion

protein containing the first 165 amino acids of SUN2 with four lamin proteins: LMNB1,

LMNC, full-length LMNA, and mature LMNA, although the interaction with LMNB1 and C

appeared barely more than the background observed with GST as control alone. Since

LMNA/C was detected in the pull down experiments from HaCaT cell lysates, the

respective interacting domains of LMNA/C with SUN2 was mapped. Five GST-LMNC

fragments have been tested for interaction with the N-terminus of recombinant SUN2 by

GST pulldown assays: GST-LMNC N-term/LMNA (aa 1-127), GST-LMNC coil1B-∆ (aa

128-218), GST-LMNC coil2 (aa 243- 387), GST-LMNC tail (aa 384-566), GST-∆LMNC

(aa 128-572). The GST-fusion constructs LMNNt and coil1B∆ which are contained

within ∆LMNC could precipitate high amounts of SUN2Nt whereas decreased amounts

of SUN2Nt have been precipitated by ∆LMNC. These differences in the binding affinity

for ∆LMNC compared with LMNNt might be due to varying folding and dimerization

processes in the SUN2Nt protein as well as in the lamin-constructs. Both lamin

polypeptides (A- and B-types) harbor α-helical regions and form parallel coiled–coil

homodimers, which can in turn assemble into higher-order filamentous structures and

Discussion

79

therefore compete for binding with SUN2Nt. The data point to an extended interaction

area for SUN2 and LMNA/C. Multiple or extended interaction zones have also been

reported for c-Fos and LMNA/C (Maraldi et al., 2010).

In experiments in HeLa cells in which A-type lamins had been eliminated by RNA

interference, the NE localization of SUN2 was barely affected (Crisp et al, 2006).

Evidently, although A-type lamins can contribute to SUN2 localization, they are not the

only determinants. As recently published, three features are thought to jointly contribute

to the NE-localization of SUN2: 1) The N- terminal domain (aa38-52) recognized by

Importin α/β; 2) The 4R-motif serves as binding platform for coatomer I complex, and 3)

The C-terminal SUN domain establishing SUN-KASH interactions to stabilize the NE

localization (Turgay et al, 2010). Therefore, the direct interaction of SUN2 and LMNA/C

might be affected by mutations in LMNA and result in a defective interaction despite the

regular presence of both components.

Discussion

80

4.5 Characterization of fibroblast from Stiff skin syndrome (SSS), Duchenne

muscular dystrophy (DMD) and Emery-Dreifuss muscular dystrophy / Charcot-

Marie-Tooth syndrome (EDMD/CMT) patients

4.5.1 Stiff skin syndrome (SSS) patients

Stiff skin syndrome is characterized by hard, thick skin that limits joint mobility and

causes flexion contractures. Hypertrichosis, postural and thoracic wall abnormalities are

associated, and occasional findings include focal lipodystrophy and muscle weakness

(Liu et al., 2008). Although about forty cases have been described in literature, definitive

assignment of the inheritance pattern is precluded due to the lack of large multiplex

families. Thus, prior work concerning the pathogenesis of SSS has been mainly

observational with few mechanistic insights (Esterly et al., 1971; Amoric et al., 1991;

Bodemer et al., 1991; Fidzianska et al., 2000; Jablonska et al., 2004; Ferrari et al.,

2005; Geng et al., 2006; Pages et al, 2007). Findings include increased collagen

production with sclerotic collagen bundles in the deep reticular dermis and/or

subcutaneous septa. Also, increased levels of cytokines including TNFα, IL-6 and

TGFβ2 have been described (Jablonska et al., 2000; Loeys et al., 2010).

In this study, dermal fibroblast from a SSS patient are described which exhibit nuclear

alterations similar to laminopathy patient cells. Other findings revealed restricted growth,

decreased SUN2 transcript levels in senescent cells but overexpression of SUN2 in low

passages. Also, in comparison to control cells, cell adhesion and spreading was

remarkably decreased.

Ordered polymers of fibrillin1 (termed microfibrils) encoded by FBN1 initiate elastic fiber

assembly and bind to and regulate the activation of the profibrotic cytokine transforming

growth factor β (TGFβ) (Isogai et al., 2003). Excessive microfibrillar deposition is

accompanied by increased TGFβ concentration in stiff skin patients as described in a

recent report (Loeys et al., 2010). The study describes mutations in the only Arg-Gly-

Asp (RGD) sequence–encoding domain of fibrillin1 that mediates integrin binding as

causative for Stiff skin syndrome. Integrins that bind via this RGD-motif are integrin

αvβ3, αvβ6, and α5β1 which are also known to activate TGFβ (Neil et al., 2006; Galliher

et al., 2006).

Several studies have implicated aberrant integrin expression or function in fibrotic

phenotypes (Asano et al., 2005; Yang et al., 2007; Wipff et al., 2008). Integrins activate

a variety of adhesion-dependent signal cascades including FAK, MAPK and PI3K/PKB

Discussion

81

which regulate cell proliferation. Integrin β1 overexpression has been reported to inhibit

cell adhesion and accordingly, reduces PI3K/PKB pathway activity, subsequently

resulting in reduced cell proliferation through upregulation of the cyclin-dependent

kinase (CDC) inhibitor p21Kip1 (Fu et al, 2007).

Contrary, no disease relevant mutations could be found in the FBN1 gene for the patient

fibroblasts described in the present study. This is compatible with findings in further SSS

patients in which the FBN1 gene was also unaltered (personal communication Dr. P.

Robinson, Charité, Berlin). Thus, diminished growth and a remarkable decrease in cell

adhesion and spreading on the substratum observed in SSS fibroblasts described in this

study is most probably due to so far unknown mechanisms independent from FBN1

mutations. These results underline the hypothesis that microfibrills present in SSS

patients without mutations in FBN1 bind to and regulate the activation of TGFβ in a

concentration depending manner (Loeys et al., 2010). As a consequence of enhanced

nuclear signaling fibroblasts produce more collagen. Increased TGFβ concentration thus

results in increased signaling in the dermis, contributing to the disease phenotype.

Overexpression of microfibrillar bundles might also contribute to altered SUN2 transcript

levels observed in our SSS patient cells. Increased SUN2 protein levels at the NE might

strengthen links both to the nuclear lamina and, via the LINC complex, to the

cytoskeleton and therefore contribute to the cell’s stiffness. Consistent with this is the

observation of broadened rim staining for SUN2 indicating a thickened NE with

increased SUN2 accumulation. Similar observations were made for LMNA/C, LMNB1

and NESPRIN1. Therefore, presently unknown mutations might affect LINC complex

proteins resulting in a disruption of the LINC complexes potentially leading to an

enlargement of the perinuclear space between the ONM and the INM. In fact, previous

studies reported significant enlargement of the perinuclear space due to disruption of

LINC complexes (Crisp et al., 2006), supporting the involvement of LINC complex

proteins in the etiology of Stiff skin syndrome.

Discussion

82

4.5.2 Duchenne muscular dystrophy (DMD) and Emery-Dreifuss muscular

dystrophy / Charcot-Marie-Tooth syndrome (EDMD/CMT)

It has been reported that alterations in nuclear morphology correlate with chromatin

arrangement possibly involved in the control of gene expression. Defects in nuclear

architecture are associated with X-EDMD, including aberrant heterochromatin

distribution and leakage of amino acids into the cytoplasm, conceivably due to NE

fragility (Fidzianska et al, 2003; Muchir et al., 2004).

These changes include altered subnuclear targeting of transcription factors and/or

nuclear domains. Principal components of chromatin remodeling complexes include

actin and actin regulatory proteins. Between nuclear actin, LMNA/C and emerin a

molecular link has been suspected to exist (Fairly et al., 1999; Clements et al., 2000;

Vaughan et al., 2001). Previously reported and further elucidated in the study by Crisp

et al (2006), is a direct interaction of LMNA/C and SUN2. Loss or mutations that inhibit

the interaction between one of those proteins could result in an altered relationship

between the NE and chromatin. Subsequently, this is also true for any protein taking

part in the extended LINC-complex. Strikingly, putative interaction partners of SUN2

differ dramatically as far as architectural proteins are concerned in EDMD/CMT cell

lysates in comparison to control cell lysates. Actin and actin related proteins like α-

actinin, myosin and nesprin were only detected in control lysates. Since LMNA/C was

still detected in EDMD/CMT cell lysates, it is conceivable that the LMNA/C-SUN2

interaction is maintained in EDMD/CMT cells, and the interruption might be therefore up-

or downstream of the LMNA/C-SUN2 connection. The absence of the actin complex in

this patient cell lysate points to a possibly altered interaction with these proteins. The

NESPRIN2 peptide identified in control cells matches to a sequence in the last spectrin

repeat in all NESPRIN2 isoforms, namely NESPRIN2 Giant/NUANCE, NESPRIN2α2

and NESPRIN2α1. Spectrins are known to associate with actin (Nowak et al., 2009). In

a hypothetical scenario, mutated NESPRIN1α in EDMD/CMT patient might weaken the

actin complex interaction and by doing so, contribute to or be the crucial factor leading

to the disease phenotype. It is therefore conceivable that altered expression of LINC-

complex proteins and/or associated proteins which interact with a nuclear actin scaffold

may affect gene expression in repair and/or maintenance of muscle cells in laminopathy

patients. Further experiments assessing the interactions between LINC complex

proteins and actin complex proteins will shed light on these important questions.

Discussion

83

As already mentioned in 4.3, RNA processing is linked to several severe human

diseases. However, the putative proteome of SUN2NT in EDMD/CMT patient cell

lysates revealed no significant difference in RNA processing proteins compared to

control cell lysates. Still, further studies beyond pulldown assays are required to prove

SUN2 interactions with the RNA processing complex.

The diminished SUN2 transcript levels in senescent patient cells observed in this study

might be due to presently unknown mutations in LINC complex proteins abolishing or

weakening the interaction with SUN2 or through perturped mechanotransduction and

subsequent aberrant signaling pathways which can in turn adjust cellular and

extracellular structure. Since it is reported that protein-protein interactions stabilize the

NE localization of SUN2 (Turgay et al, 2010), it is conceivable that mutations in SUN2

interacting partners affect the efficiency of their recruitment to the NE, leading to an

enhanced degradation. Findings in this study underline the importance of SUN2 and its

interacting partners as part of the LINC complex in disease development. Interestingly,

the N-terminus of SUN2 is a serine rich region, which in general is suspected to be a

precondition for alternate protein interactions in a tissue specific way. The analysis of

phosphorylation sites and of tissue specific SUN2 interacting partners will give more

insights into the tissue specific manifestation and variable penetrance seen in the

diverse laminopathy phenotypes.

In muscular dystrophy disorders, there is a constant need for regeneration in recurrent

myofiber damage in the presence of even mild stress. Satellite cells play a major role in

postnatal muscle growth and repair. Clinical manifestations of severe muscle wasting

caused by fibrosis, calcium deposits and adipose accumulation supplant muscle tissue,

is postulated due to impaired function of muscle stem cells (Wilson et al., 2000; Sacco

et al., 2008; Sacco et al., 2010). Damaged muscle fibers require gene activation that

might be affected by chromatin arrangements occurring in laminopathy conditions. In

fact, in X-EDMD patients a certain percentage of muscle fibre nuclei show nuclear

lamina and chromatin alterations (Ognibene et al., 1999).

The mutation in the dystrophin gene found in patient 1 converts glutamic acid encoded

by GAA/GAG into a premature termination codon resulting subsequently in a truncated

protein. This loss of protein function disconnects the extra-cellular matrix from the

cytoskeleton. The dystrophin gene is highly complex, containing at least eight

independent, tissue-specific promoters and two polyA-addition sites. Dystrophin RNA is

Discussion

84

differentially spliced, producing a range of different transcripts encoding a large set of

protein isoforms. The dystrophin protein as encoded by the Dp427 transcript is a large,

rod-like cytoskeletal protein found at the inner surface of muscle fibers. Dystrophin is

part of the dystrophin-glycoprotein complex (DGC), which connects the extra-cellular

matrix with the inner cytoskeleton (F-actin) (NCBI RefSeq database; Soltanzadeh et al.,

2010). Although loss of the structural protein dystrophin is the primary defect in

Duchenne muscular dystrophy, the secondary molecular machinery based on the

hypothesis that mitochondrial dysfunction caused by alterations of the calcium signaling

system resulting in a deleterious amplification of stress-induced cytosolic calcium

signals and in an amplification of stress-induced ROS production, is not fully understood

(Shkryl et al, 2009).

Defects in dystrophin have been associated with reduced nitric oxide (NO) production,

chronic inflammation and tissue degeneration resulting in altered gene expression

profiles and deficient regeneration (Gucuyener et al, 2000; Kasai et al, 2004; Judge et

al, 2006). There is evidence that during the mechanical stretching or regeneration of

dystrophic muscles signal transduction pathways, including PI3K-AKT, are altered. AKT

is also involved in the regulation of the intracellular NO synthesis (Dimmeler et al, 1999;

Dogra et al, 2006; Peter et al., 2006). Protein phosphatase 2A activity is NO dependent,

and its reduced activity results in altered regulation of class IIa histone deacetylases

(HDACs) 4 and 5 which have been found altered in DMD patient muscle cells (Illi et al,

2008). HDAC activity is involved in the regulation of genes, including c-Fos

downregulation which is necessary for satellite cell conversion to myoblasts. In

comparison to healthy individuals, altered pattern of global histone modifications have

been found in DMD patient muscle cells (Cohen et al, 2007). Therefore, the connection

between NO signaling and the altered epigenetic profile described in muscle cells

deficient for dystrophin indicates a link between the dystrophin-activated NO signaling

and the remodeling of chromatin and therefore an epigenetic contribution to the

pathogenesis and progression of DMD (Colussi et al. 2009).

Through mechanotransduction mechanical forces are translated into biochemical

signals and activate diverse signaling pathways which can in turn adjust cellular and

extracellular structure. By this mechanosensitive feedback, cellular functions like

migration, proliferation, differentiation and apoptosis are modulatet. Multiple and

overlapping cellular signaling pathways are activated by mechanosensors that can be

activated by stretch even in the absence of ligands, including extracellular signal-

Discussion

85

regulated kinase 1/2 (ERK1/2) and the mitogen-activated protein kinase (MAPK)

signaling (Jaalouk and Lammerding, 2009). It is reported, that dystrophin deficiency

causes an aberrant mechanotransduction in muscle fibers and leads to deregulation of

only ERK1/2 among the MAP kinase signaling pathways (Kumar et al, 2004).

Additionally, the mutation found in NESPRIN1α2 in patient 1 and in NESPRIN1α1 in

patient 2 being without consequences in healthy individuals, might in these cases

contribute to the perturbed mechanotransduction and weaken LINC complex protein

interactions. In immunofluorescence analysis, diminished staining for NESPRIN1

detected in EDMD/CMT cells and cytoplasmic localization of NESPRIN1 in DMD

fibroblasts further support this hypothesis. The nucleus-centrosome distance maintained

by SUN-NESPRIN1/2 interactions might also be affected by these nesprin mutations.

However, a previous report identified emerin as a novel microtubule interacting protein

anchoring the centrosome to the nucleus (Salpingidou et al, 2007). These data are

further confirmed by findings that the centrosome-nucleus distance is increased in

emerin-null human dermal fibroblasts. Fibroblasts with similar nuclear morphological

defects but normal for emerin from a Greenberg dysplasia patient did not exhibit altered

nucleus-centrosome distance, suggesting that centrosome mislocalization is specific to

the loss of emerin from the NE (Hale et al., 2008; Hutchison et al., 2007). Therefore, the

so far unknown underlying mutations in the fibroblasts of the three patients described in

this study are conceivable to affect proteins that interact either directly or indirectly with

emerin. Since SUN2 binds directly to emerin and is diminished in senescence patient

cells, SUN2 might play an essential role. Also, presently unknown interactions of NE

proteins with microtubules are possible. Tubulinα and tubulinβ were pulled down by

SUN2Nt from control and EDMD/CMT patient cell lysates, thus SUN2 might be a

promising candidate for microtubule interactions.

LINC complex proteins and associated proteins are ubiquitously expressed; yet

mutations in one of these proteins affect predominantly cardiac and skeletal muscle

tissue (Cohen et al., 2001). The hypothesis that nuclear fragility, although present in all

cell types, is critical only for certain tissues does not explain arrhythmia heart defects

involving pace-making cells that mediate the conduction pathway, and is also not

sufficient to explain the pathogenesis of lipodystrophy since adipose tissue is not

subjected to critical mechanical stress levels (information obtained from Muscular

Dystrophy Association, http://www.mdausa.org). Tissue specific signaling pathways

mediated by tissue specific composition of LINC complex proteins and associated

Discussion

86

proteins might be an alternative model. In such a model, nuclear envelopathies might be

caused by defects in gene expression due to loss or mutations in proteins involved

either directly or indirectly in the maintenance of proper chromatin arrangement which is

crucial for tissue specific regulation of transcription. In fact, perturbed ERK and JNK

branches of the MAPK signaling cascade was found in hearts of a mouse model for

autosomal EDMD and X-linked EDMD (Muchir et al., 2007; Muchir and Wormann,

2007). Chronically increased ERK and JNK activation is deleterious for hearts and

treatment of EDMD mice with PD98059, an inhibitor of ERK signaling, prevents

development of cardiomyopathy (Muchir et al., 2009). These findings were consistent

with known alterations in MAPK signaling in cardiomypathy (Molkentin et al., 2004). This

model also includes that no single mechanism will account for the varying phenotypes

among laminopathies.

Also, the complexity of polynucleated skeletal muscle maintenance and recurrent repair

and differentiation processes in muscular dystrophies suggest that a large number of

molecular mechanisms might be involved in the pathogenic development. One complex

mechanism is proper nuclear positioning relative to the cell body which is important for

many cellular processes during mammalian development (Zhang et al., 2008,

Gundersen et al., 2011). In the syncytial skeletal muscle cells, more than 100 nuclei are

evenly distributed at the periphery of each cell, with 3–8 nuclei anchored beneath the

neuromuscular junction. These postsynaptic nuclei cluster together under the plasma

membrane at sites of neuronal contacts forming the neuromuscular junction and

synthesize the components of the neuromuscular junction that specify the overlying

membrane as the target site for innervation. Therefore, in another model, failure in

nuclear positioning due to disruption of the extended LINC complex leads to a failure of

correct anchoring and correct positioning of the nucleus within the cell, and

subsequently to failure in innervation and to the disease phenotype of muscular

dystrophy (Bruusgaard, 2003; Starr et al, 2005; Zhang et al, 2008). Taken together,

comparative studies on the three different laminopathy affected patient cells revealed

similar cellular phenotypes with regard to the experiments carried out in this study. This

emphasizes the hypothesis that interruption of mechanotransduction and subsequent

signaling pathways caused at multiple levels, leading to disease; schematical displayed

in figure 33. Identifying the proteins taking part in the extended linker complex and their

function in biochemical pathways will provide new possibilities of therapeutic

approaches for these diseases.

Discussion

87

Fig. 33: Scheme of the LINC complex and associated proteins involved in chromatin association

maintenance and transduction of cytoplasmic forces. Question marks indicate suggested but not proven

interactions. Black arrows show signal transduction from the cytoplasm or the cytoplasmic membrane into

the nucleus via the LINC complex. Gray dashed arrows crossed by red line illustrate interrupted signal

transduction from the cytoplasm or the cytoplasmic membrane into the nucleus via the LINC complex.

Gray brackets with red arrows point to potential mutations/interruptions of cytoskeletal/LINC complex

proteins leading ultimately to altert signaling and disturbed gene transcription.

assembled cytosceletal/LINC complex

gene silencing

disassembled cytosceletal/LINC complex

disturbed gene transcription:disease phenotype

nucleus

cytoplasm

MTOC

? during mitosis

ONM

INM

mechano transduction/

signaling

collagen/fibronectin

plasma membrane

actin-filaments/myosin emerin heterochromatin LMNA/C microtubules NESPRIN1/2 intermediate filaments repression complex SUN2 transcription complex

laminin

dystroglycan complex

dystrohin

integrin receptors

talin vinculin

Summary

88

Summary

LINC complexes serve in several cellular processes providing a connection between

cellular components and organelles. Here I have generated monoclonal antibodies

directed against the N-terminus of human SUN2 and used it for immunofluorescence

studies. I observed that during the cell cycle SUN2 is evenly distributed in a rim like

pattern localized to the inner nuclear membrane in inter- and prophase, and

associates with condensed chromatin during metaphase. During nuclear envelope

reassembly in anaphase, SUN2 again shows a rim like pattern and is enriched in

NPC-poor regions further supporting the proposed role of SUN2 in heterochromatin

maintenance. SUN2 colocalizes also with pericentrin at the centrosome.

An increased SUN2 presence at the NE was detected in human glioblastoma cells,

pointing to a putative role in tumorigenesis possibly due to aberrant recruitment of

interaction partners that are involved in the development of malignancy.

A first proteomic study of the N-terminus of SUN2 revealed interactions with proteins

of the chromatin remodeling complex, suggesting SUN2 might be involved in

maintaining gene silencing during the cell cycle by recruiting corepressor complexes

to the nuclear periphery. Also, the analysis of the putative proteome revealed the first

described interaction with the RNA processing complex, suggesting a novel role for

SUN2 in RNA processing and splicing.

Characterization of Duchenne muscular dystrophy (DMD), Emery-Dreifuss muscular

dystrophy/Charcot-Marie-Tooth syndrome (EDMD/CMT) and Stiff skin syndrome

(SSS) patient fibroblasts revealed various nuclear deformations, diminished cell

adhesion and cell spreading on the substratum. Furthermore, the nucleus-

centrosome distance was increased in all three patient cells.

In precipitation assays using the N-terminus of SUN2 as GST-fusion and carried out

with EDMD/CMT patient cell lysates several actin related proteins were not present

which have been detected in control cell lysates. This supports the hypothesis that

protein interactions that involve the extended LINC complex are weakened or

interrupted.

Taken together, the findings in this study are consistent with proposed disease

mechanisms involving altered cell stability and/or altered gene transcription and

underline the importance of SUN2 and its interactions as part of the LINC complex.

Furthermore, the analysis of tissue specific SUN2 interactions will give more insights

Summary

89

into the tissue specific manifestation and variable penetrance of the various

laminopathy phenotypes.

Zusammenfassung

90

Zusammenfassung

Der LINC-Komplex stellt eine Verbindung zwischen Zellkern und Zytoplasma dar und ist

an vielen zellulären Prozessen beteiligt. Ein in dieser Arbeit neu generierter

monoklonaler Antikörper gegen die N-terminale Domäne des humanen SUN2 Proteins

zeigte eine regelmäßige ringförmige Verteilung von SUN2 an der Kernmembran von

Inter- und Prophase-Kernen in HeLa Zellen. Während der Metaphase ko-lokalisiert

SUN2 mit den kondensierten Chromosomen. Außerdem konnte eine Kolokalisation mit

Pericentrin am Zentrosom gezeigt werden. Bei der Reassemblierung der Kernhülle in

der Anaphase ist SUN2 wieder im Bereich der Kernhülle detektierbar und ist besonders

in NPC-armen Bereichen angereichert. Dies ist ein weiterer Hinweis darauf, dass SUN2

an der Aufrechterhaltung von Heterochromatin beteiligt ist.

Die in Immunofluoreszenzanalysen beobachteten signifikant erhöhten SUN2 Mengen in

humanen Glioblastomzellen weisen auch auf eine mögliche Rolle von SUN2 bei der

Tumorentstehung hin. Dies könnte durch eine übermäßige Rekrutierung von

Interaktionspartnern erfolgen, die ihrerseits an der Zellentartung beteiligt sind.

In Präzipitationsversuchen mit dem N-Terminus von SUN2 und Lysaten von humanen

Keratinozytenzellen wurden putative SUN2 Interaktionspartner identifiziert. Dabei

konnte gezeigt werden, dass der nukleoplasmatisch lokalisierte N-Terminus nicht nur

direkt mit LaminA/C sondern auch mit einer Vielzahl von Komponenten des Chromatin-

Remodelierungskomplexes direkt oder indirekt interagiert. Dies lässt vermuten, dass

SUN2 durch die Rekrutierung von Ko-Repressoren an die Kernperipherie an der

Aufrechterhaltung von Heterochromatin beteiligt ist. Auch konnte durch die Analyse des

putativen Proteoms eine bisher nicht beschriebene Interaktion von SUN2 mit dem RNA-

prozessierendem Proteinkomplex gezeigt werden.

Bei der Charakterisierung von Duchenne Muskeldystrophie (DMD), Emery-Dreifuss

Muskeldystrophie /Charcot-Marie-Tooth Syndrom (EDMD/CMT) und Stiff skin Syndrom

(SSS) Patientenzellen konnten verschiedene Zellkerndeformationen und eine

wesentlich verminderte Zellsubstratanheftung und Zellausbreitung festgestellt werden.

Des Weiteren war das Expressionsprofil für SUN2 verändert und der Zentrosomen-

Nukleus Abstand vergrössert. Ein putatives SUN2-Proteom aus Lysaten von EMD/CMT

Patientenzellen gibt Hinweise auf eine mögliche Interaktionsstörung des LINC-Komplex

Proteins mit dem nukleären Aktin-Komplex.

Zusammenfassend unterstützen die in dieser Arbeit gewonnen Ergebnisse in der

Literatur beschriebene Pathomechanismen, die sowohl eine erhöhte

Zusammenfassung

91

Mechanosensitivität der Zellen als auch eine veränderte Transkriptionsregulation

fordern, und stellen die Bedeutung von Proteinen des LINC-Komplex und damit

assoziierter Proteinkomplexe heraus. Detaillierte Bindungsstudien werden einen

weiteren Aufschluss über die Pathomechanismen geben, die zu den phänotypisch

vielfältigen und gewebespezifischen Ausprägungen der Laminopathien führen.

References

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Preliminary Puplications

103

Preliminary Puplications:

• Poster presentation, Annual Conference of German Society of Cell Biology

(Deutsche Gesellschaft für Zellbiologie, DGZ) 2011: Interactions and subcellular

distribution of human SUN2

Puplications (submitted soon):

• Vaylann, E., 1, 2 Noegel, A.A. 1, 2 (2011): Subcellular distribution and interactions

of human SUN2.

1Institute for Biochemistry I, Medical Faculty, University of Cologne, Cologne,

Germany 2Center for Molecular Medicine Cologne (CMMC) and Cologne

Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases

(CECAD), Medical Faculty, University of Cologne, Cologne, Germany

• Taranum, S.,1,2#, Vaylann, E., 1,2# Abraham, S., 1,2 Karakesisoglou, I., 3, Wehnert,

M., 5, Noegel, A.A., 1,2 (2011): Characterization of primary fibroblast of Emery-

Dreifuss muscular dystrophy (DMD) and Emery-Dreifuss muscular dystrophy/

Charcot-Marie-Tooth syndrome (EDMD/CMT) patients.

1Institute for Biochemistry I, Medical Faculty, University of Cologne, Cologne,

Germany, 2Center for Molecular Medicine Cologne (CMMC) and Cologne

Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases

(CECAD), Medical Faculty, University of Cologne, Cologne, Germany,

3Department of Biological Sciences, The School of Biological and Biomedical

Sciences, The University of Durham, Durham, UK, 5Ernst-Moritz-Arndt-University,

Institute of Human Genetics, Greifswald, Germany

# both authors contributed equally to this work

104

Curriculum vitae

Personal details

Name: Eva Mawina Vaylann, nee Stürz

Date of Birth: 25.07.1978

Nationality: german

Doctoral studies, Professional experience, University studies

April 2009 – March 2011 February 2009 - April 2009 April 2008 –February 2009 September 2007 - March 2008 April 2007 - September 2007 November 2003 - April 2007 2003 - 2007

Doctoral studies, Institute for Biochemistry I, Medical Faculty, University of Cologne, Germany, Supervisor: Prof. Dr. Angelika A. Noegel, Title: Interactions and subcellular distribution of human SUN2 Research assistant, Kagando Hospital and Rural Development Center, Uganda, Africa Research assistant, Internal Medicine I, University Hospital/ Institute for Biochemistry I, Medical Faculty, University of Cologne temporary employment abroad in Africa (South Africa, Zimbabwe, Zambia and Mozambique), founding and supervision of a children’s care project „Children-in-need-africa“ in South Africa and Mozambique, Africa Research assistant, Institute for Biochemistry, Faculty of Mathematics and Natural Science, University of Cologne, Germany (Group Prof. Dr. Krämer, Dr. Marin) Student research assistant, Institute for Zoology (Prof. Dr. Plickert); Institute for Biochemistry (Group Prof. Dr. Krämer, PD. Dr. Niefind) Faculty of Mathematics and Natural Science, University of Cologne, Germany Diploma studies, major subjects: Biochemistry, Genetics, Pharmacology, Diploma thesis in Biochemistry, Institute for Biochemistry, Faculty of Mathematics and Natural Science, University of Cologne, Germany, Supervisor: Prof. Dr. Schomburg, Title: „Metabolom analysis during the diauxic shift in Saccharomyces cerevisiae“

105

Lebenslauf

Persönliche Daten:

Name, Vorname: Eva Mawina Vaylann, geb. Stürz

Geburtsdatum: 25.07.1978

Staatsangehörigkeit: deutsch

Promotion, Berufspraxis, Hochschulstudium April 2009 – März 2011 Februar 2009 – April 2009 April 2008 –Februar 2009 September 2007 - März 2008 April 2007 - Sebtember 2007 November 2003 - April 2007 2003 - 2007

Promotion am Institut für Biochemie I der Medizinischen Fakultät der Universität zu Köln, Universität zu Köln, Betreuerin: Prof. Dr. Angelika A. Noegel, Thema: Interaktionen und subzelluläre Verteilung von humanem SUN2 Wissenschaftliche Mitarbeiterin, Kagando Hospital und Entwicklungszenter, Kagando, Uganda, Afrika Tätigkeit als Wissenschaftliche Mitarbeiterin, Inneren Medizin I, Universitätsklinik/ Institut für Biochemie I, Medizinische Fakultät der Universität zu Köln Auslandsaufenthalt in Afrika ( Südafrika, Zimbabwe, Sambia und Mosambik), Gründung und Betreuung eines „Children-in- need-africa“ Hilfsprojektes in Südafrika und Mosambik, Afrika Tätigkeit als Wissenschaftliche Hilfskraft am Institut für Biochemie der Universität zu Köln, Mathematisch-Naturwissenschaftliche Fakultät (AG Prof. Dr. Krämer, Dr. Marin) Tätigkeit als Studentische Hilfskraft am Institut für Zoologie der Universität zu Köln, Molekulare Grundlagen von Entwicklungs- prozessen (Prof. Dr. Plickert); Institut für Biochemie der Universität zu Köln, Mathematisch-Naturwissenschaftliche Fakultät (AG Prof. Dr. Krämer, PD. Dr. Niefind) Biologiestudium an der Universität zu Köln, Mathematisch-Naturwissenschaftliche Fakultät, Hauptstudium: Biochemie, Genetik, Pharmakologie, Diplomarbeit am Institut für Biochemie, AG Prof. Dr. Schomburg: „Meta-bolomanalyse des diauxischen Wechsels bei Glucoselimitation in S. cerevisiae“

interactions and subcellular distribution of human sun2

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