Multifaceted Roles of the Transmembrane Nuclear Envelope Protein, Samp1

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Multifaceted roles of the transmembrane nuclear envelope protein, Samp1

Mohammed Hakim Jaffer Ali

Abstract

The eukaryotic nuclear envelope (NE), separates the nucleoplasm from cytoplasm and is made up of two concentric lipid membranes, the outer and the inner nuclear membranes (ONM and INM), the nuclear pore complexes (NPCs) and an underlying filamentous nuclear lamina. The INM contains hundreds of unique transmembrane proteins of which only a handful have been characterized. In this thesis, I aimed to understand the functional organization of proteins in the nuclear envelope and I focused on investigating the functions of a recently identified INM transmembrane protein, Samp1. We have developed a novel and robust approach, MCLIP, to identify specific protein- protein interactions taking place in live cells. Using MCLIP, we have shown that Samp1 interacts with proteins of the LINC complex, the nuclear lamina and components of the mitotic spindle. Samp1's specific interactions with a variety of binding partners, suggest that Samp1 plays important roles both in interphase and in mitosis. We have also shown that Samp1 can provide a binding site at the INM for the GTPase Ran, a master regulator of protein interactions in interphase and in mitosis. Furthermore, we have also investigated the role of Samp1 in cell differentiation using two independent model systems. In human iPSCs, ectopic expression of Samp1 promoted differentiation despite pluripotent culture conditions. In C2C12 myoblasts, depletion of Samp1 completely blocked differentiation into myotubes. The two studies complement each other and suggest that Samp1 has a strong differentiation promoting activity. Taken together, the findings in this thesis, give insights on the unexpected and unforeseen roles played by a transmembrane protein in different fundamental cellular process.

List of publications

This thesis is based on the following publications, referred to as Paper I, II, III, IV and V, in the text.

I. Jafferali, M., Vijayaraghavan, B., Figueroa, R., Crafoord, E., Gudise, S., Larsson, V., & Hallberg, E. (2014). MCLIP, an effective method to detect interactions of transmembrane proteins of the nuclear envelope in live cells. Biochimica Et Biophysica Acta (BBA) - Biomembranes, 1838(10), 2399- 2403.

II. Vijayaraghavan, B., Jafferali, M., Figueroa, R., & Hallberg, E. (2016). Samp1, a RanGTP binding transmembrane protein in the inner nuclear membrane. Nucleus, 7(4), 415-423.

III. Larsson, V., Jafferali, M., Vijayaraghavan, B., Figueroa, R., & Hallberg, E. Mitotic spindle stability and correct chromosome segregation is dependent on an integral nuclear membrane protein, Samp1 (Manuscript).

IV. Bergqvist, C*., Jafferali, M*., Gudise, S*., Markus, R., & Hallberg, E. An inner nuclear membrane protein induces rapid differentiation of human induced pluripotent stem cells. (Submitted Manuscript).

V. Jafferali, M., Hasan, M., Figueroa, R., & Hallberg, E. Spindle associated membrane protein 1 (Samp1) is required for the differentiation of muscle cells. (Submitted Manuscript).

*These authors contributed equally to the project

iii

Jafferali, M., Beckman, M., Kihlmark, M., & Hallberg, E. (2015). Nucleus and Nuclear Envelope: Methods for Preparation. Els, 1-4.

Jafferali, M., Figueroa, R., & Hallberg, E. (2016). MCLIP Detection of Novel Protein–Protein Interactions at the Nuclear Envelope. Methods In Enzymology, 503-515.

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Contents

Abstract ...... i

List of publications ...... iii

Abbreviations ...... vii

1 Introduction ...... 1 1.1 The nuclear envelope ...... 1 1.1.1 Outer nuclear membrane and inner nuclear membrane ...... 3 1.1.2 The nuclear lamina ...... 3 1.1.3 The LINC complex ...... 4 1.1.4 The LEM domain protein – emerin ...... 5 1.1.5 Samp1 ...... 5 1.1.6 The nuclear envelope and diseases ...... 7 1.2 The cell cycle ...... 9 1.3 GTPase Ran ...... 11 1.4 Differentiation ...... 12 1.4.1 Pluripotent stem cells ...... 13 1.4.2 Myogenesis ...... 14 1.4.3 NE proteins in muscle differentiation ...... 15 1.5 Methods to study protein-protein interactions ...... 15 1.6 Protein interaction studies in the nuclear envelope ...... 17

2 Aim ...... 19

3 Methodological considerations ...... 21 3.1 Cell cultures ...... 21 3.2 Short hairpin RNA (shRNA) ...... 22 3.3 Lentivirus like particle generation ...... 22

4 Results and discussion ...... 23 4.1 A novel approach to study NE protein interactions in live cells ...... 23

v 4.2 Molecular dissection of the interaction between Samp1 and Ran ...... 25 4.3 The role of transmembrane INM proteins in the mitotic spindle ...... 26 4.4 The role of Samp1 in differentiation ...... 27

5 Conclusion ...... 29

6 Future perspectives ...... 31

7 Populärvetenskaplig sammanfattning på svenska ...... 33

8 Acknowledgments ...... 35

9 References ...... 38

Table 1. Features of different methods to study protein-protein interactions...... 16 Table 2. Nuclear envelope protein-protein interaction studies...... 18

Figure 1. Schematic illustration of nuclear envelope proteins……………………………… 2 Figure 2. Schematic drawing of Human Samp1 vs. Ct-Samp1……………………………... 6 Figure 3. Schematic representation of diseases associated with NE proteins………………. 8 Figure 4. Schematic representation of the eukaryotic cell cycle……………………………..9 Figure 5. Structure of DSP………….……………………………………………………... 24

Abbreviations

APC/C Anaphase promoting complex/cyclosome BAF Barrier-to-autointegration factor CoIP Coimmunoprecipitation Ct Chaetomium thermophilum DSP Dithiobis succinimidyl propionate EDMD Emery-Dreifuss muscular dystrophy ER Endoplasmic reticulum ESC Embryonic stem cell GDP Guanosine diphosphate GTP Guanosine triphosphate ICM Inner cell mass INM Inner nuclear membrane iPSC induced pluripotent stem cell LEM (LAP2, emerin, MAN1) LINC Linker of nucleoskeleton and cytoskeleton MCLIP Membrane protein crosslink immunoprecipitation NE Nuclear envelope NEBD Nuclear envelope breakdown NET Nuclear envelope transmembrane protein NPC Nuclear pore complex ONM Outer nuclear membrane PNS Perinuclear space RCC1 Regulator of chromosome condensation 1 S.pombe Schizosaccharomyces pombe

vii SAF Spindle assembly factor SAMP1 Spindle associated membrane protein 1 TX-100 Triton X-100 VLP virus-like particle

viii 1 Introduction

1.1 The nuclear envelope

The hallmark of an eukaryotic cell is the nucleus, which is enclosed by a membranous, structure called nuclear envelope (NE). The NE is made up of two concentric phospholipid membrane bilayers (Fig. 1), the outer nuclear membrane (ONM) and the inner nuclear membrane (INM) (Gerace and Burke, 1988; Watson, 1955). The ONM is contiguous with the endoplasmic reticulum (ER). The ONM and INM are separated by a space called perinuclear space (PNS), which is continuous with the lumen of the ER. The ONM fuses with the INM at multiple points in the PNS forming specialized macromolecular circular apertures called nuclear pores, which harbour the nuclear pore complexes (NPCs). The molecular mass of the NPCs is 124 MDa and it contains approximately 30 different proteins in multiple copies. The NPCs provide a channel for bidirectional transport of RNA molecules, proteins and ribonucleoprotein particles (RNPs) between the nucleus and the cytoplasm (Gerace and Burke, 1988; Hetzer, 2010; Strambio-De-Castillia et al., 2010). Underneath the INM, a dense fibrillar network of proteins called the nuclear lamina, consisting of lamin proteins and lamin associated proteins. The nuclear lamina functions as an anchor for chromatin, chromatin associated proteins, transmembrane NE proteins and the NPCs (Georgatos et al., 1994; Simon and Wilson, 2011). Until the discovery of envelopathies (laminopathies) - diseases linked to mutation in the nuclear lamina and NE proteins (Burke et al., 2001; Worman et al., 2010) - the NE was merely considered as a membrane barrier.

1.1.1 Outer nuclear membrane and inner nuclear membrane

Until the previous decade, the ONM was considered to be simply a membrane extension of ER containing the same pool of proteins. Both the ONM and ER are studded with ribosomes engaged in protein synthesis. In the 2000s, it was shown that at least one protein family is distinct to the ONM – the nesprins (Zhang et al., 2001; Zhen et al., 2002). Nesprins are giant spectrin repeat containing transmembrane proteins that connect the cytoskeleton to the nucleus.

On the other hand, the INM was known to have, a unique protein composition long before the ONM. Several transmembrane INM proteins such as Lamin B receptor (Worman et al., 1988), LAP1 and LAP2 (Senior and Gerace, 1988), gp210 (Greber et al., 1990; Wozniak et al., 1989), POM121 (Hallberg et al., 1993), emerin (Manilal et al., 1996), SUN1 (Malone et al., 1999), SUN2 (Hoffenberg et al., 2000) and MAN1 (Liu et al., 2003) had been discovered and characterized. Recently, subtractive proteome analysis of the NE of rat liver, rat muscle and human leukocytes reported several hundreds of unique putative transmembrane NE proteins (Korfali et al., 2012; Schirmer et al., 2003), among them only a few have been characterized so far.

1.1.2 The nuclear lamina

Commonly referred to as ‘detergent-high-salt-resistant-proteins’, the nuclear lamins (Aaronson and Blobel, 1975; Dwyer and Blobel, 1976), include three major lamin proteins, termed lamin A, B, and C (Gerace et al., 1978; Gerace et al., 1984). In the majority of cells, A-type lamins include Lamin A and Lamin C, which are alternatively spliced products of the gene, LMNA. A- type lamins are developmentally regulated and expressed in most differentiated cells but absent in embryonic cells (Harborth et al., 2001). B-type lamins are ubiquitously expressed in all embryonic and adult cells (Broers et al., 1997). B-type lamins are products of two separate genes - the LMNB1 encoding Lamin B1 (Lin and Worman, 1995), or the LMNB2, encoding Lamin B2 and Lamin B3 (Broers et al., 2006). Lamins are known to bind to many INM proteins, including Samp1, emerin, SUN1, SUN2, MAN1, LBR, LAP1 and 3 LAP2 (see table 2). Lamins also binds to chromatin proteins, histone H2A, barrier-to-autointegration factor (BAF) and DNA-replication complexes (Gruenbaum et al., 2003; Zastrow et al., 2004). Studies on somatic cells and laminopathy patients cells have shown that nuclear lamins are essential in a wide range of nuclear functions, including DNA repair, transcription, cell proliferation and cell differentiation (Dechat et al., 2008). Furthermore, advanced microscopic studies on mammalian cells have shown that A- and B- type lamins form distinct compartments in the NE and suggest that each Lamin network may have specific roles in gene expression and chromatin organization. This could be the reason why mutations in the LMNA gene causes a wide range of human diseases (Shimi et al., 2008)

1.1.3 The LINC complex

An evolutionarily conserved system of intracellular communication was reported in 2006, the LINC (linker of nucleoskeleton and cytoskeleton) complex. The INM SUN domain proteins interact with the ONM KASH domain proteins in the lumen of the NE and physically connect the nuclear interior with the cytoskeleton (Crisp et al., 2006; Haque et al., 2006; Razafsky and Hodzic, 2009). SUN domain proteins are type II integral proteins typically located in the INM of the NE. In mammals, two major SUN domain proteins are expressed, SUN1 and SUN2 (Crisp et al., 2006; Padmakumar et al., 2005). Mutation studies of LINC complex proteins and wound-healing assays have shown how LINC complexes are indispensable for cell polarization, cell migration, nuclear positioning, centrosome-nucleus connection, signal transduction and repositioning of chromosomes within the nucleus (Burke and Roux, 2009; Fridolfsson and Starr, 2010). Furthermore, LINC complexes have been shown to be necessary for communication of mechanochemical signaling to the nucleus (Lombardi et al., 2011). LINC complex association with the nuclear lamina and INM proteins emerin and Samp1, suggests that these interactions are required to strengthen the LINC complex anchorage during nuclear movement and to resist the high mechanical force imposed on them (Borrego-Pinto et al., 2012; Chang et al., 2015; Gudise et al., 2011).

4 1.1.4 The LEM domain protein – emerin

Emerin, a dynamic nuclear envelope protein encoded by the EMD gene (Emery, 2000), is linked to EDMD disease (see section 1.1.6). Emerin is a type II integral protein and consists of 254 amino acids (Manilal et al., 1996; Nagano et al., 1996). The N-terminal domain of emerin is exposed to the nucleoplasm and the C-terminal domain in the PNS. Emerin, being ubiquitously expressed in many cell types has been shown to be involved in a diverse array of functions such as regulation of transcription factors, nuclear structure, and chromatin architecture (Berk et al., 2013). Emerin, together with MAN1 and LAP2, contains a LEM domain, a ~40-residue long structurally conserved sequence (Cohen et al., 2001; Dechat et al., 2000b; Lin et al., 2000), which binds directly to a conserved chromatin protein called BAF (Berk et al., 2013; Holaska and Wilson, 2006). Emerin has been shown to be involved in chromatin organization and tethering at the NE by interacting directly with the chromatin-silencing protein HDAC3 and associating with A-type and B-type lamins (Holaska and Wilson, 2006). Studies on null EDMD patient cells and mouse models have shown that loss of emerin affects muscle and heart related genes regulated by Rb (retinoblastoma protein) and MyoD. Furthermore loss of emerin also perturbs genes regulated by JNK, MAPK and the Wnt signalling pathways (Berk et al., 2013).

1.1.5 Samp1

Samp1 is a recently identified and characterized transmembrane protein specifically located in INM of the NE (Buch et al., 2009). Samp1 is encoded by the gene TMEM201 and is also referred to as NET5, since it was initially indicated as a putative transmembrane NE protein in rat liver (Schirmer and Gerace, 2005). Human cells have three predicted isoforms of Samp1 (Samp1- a, b, c) and these isoforms share a similar N-terminal portion but differ at their C-termini (Borrego-Pinto et al., 2012). Hallberg’s group characterized the short isoform of Samp1 (Samp1a) and reported it as a type II integral protein specifically located in the INM (Buch et al., 2009). Samp1 is conserved from Schizosaccharomyces pombe (S.pombe) to human (Buch et al., 2009; Gudise et 5

A well-known feature during mitosis is, that integral proteins of the nuclear membrane become dispersed and diffused throughout the ER (Ellenberg et al., 1997) when the mitotic spindle forms. However, a subpopulation of Samp1 (Buch et al., 2009) and two other proteins, WFS1 and TMEM214 (Wilkie et al., 2011) were found to be concentrated along the spindle microtubules during mitosis. The functional role of Samp1 in the mitotic spindle is yet to be described (see Paper III). Genome wide siRNA screens in C. elegans (Sonnichsen et al., 2005) and HeLa cells (Neumann et al., 2010) have indeed shown that Samp1 depletion gives rise to phenotypes associated with mitotic defects, suggesting that Samp1 might play an important role in mitosis.

1.1.6 The nuclear envelope and diseases

Mutations in NE proteins, give rise to a broad spectrum of diseases commonly called, nuclear envelopathies or laminopathies (Burke et al., 2001; Worman et al., 2010). There are at least 15 heritable human diseases associated with mutations in genes encoding NE proteins (Jacob and Garg, 2006). Some of them are, EMD, LMNA, LMNB1, LMNB2, LEMD3, LBR, MAN1, and SYNE1 (Worman and Bonne, 2007). In 1994, EMD gene was identified as causing X-linked Emery-Dreifuss muscular dystrophy (X-EDMD). Its gene product was called emerin. Later, studies have shown autosomal dominant Emery-Dreifuss muscular dystrophy (AD-EMD), a disease that causes the same phenotype as X-EDMD, can also be caused by mutations in the LMNA gene. Mutations in genes encoding emerin and A-type lamins account for only two-thirds of the EDMD diseases (Pillers and Von Bergen, 2016), which make it possible for other NE proteins to be involved in the disease mechanism. Several other diseases (Fig. 3) caused by mutations in NE proteins include lipodystrophy, neuropathy, dermopathy and premature ageing syndromes (reviewed in Worman and Bonne, 2007; Worman et al., 2010).

The following five phases take place sequentially during mitosis

• Prophase – Duplicated chromosomes condense. Duplicated centrosomes with microtubules move towards different poles of the nucleus. • Prometaphase – NEBD takes place, the INM proteins are dispersed throughout the ER (Ellenberg et al., 1997). The nuclear lamina and the NPCs are disassembled. Condensed chromosomes are accessed by the kinetochore microtubules and the mitotic spindle begins to assemble. • Metaphase –The push force by the kinetochores microtubules and the pull force by the astral microtubules keep the chromosome in continuous oscillatory movement at the metaphase plate. Mitotic cells normally spend half of their M phase in metaphase with the chromosomes aligned at the metaphase plate, awaiting for the checkpoint proteins to initiate separation of the sister chromatids. • Anaphase – All of a sudden anaphase starts with the release of cohesin, a protein complex that holds sister chromatids at the metaphase plate. The anaphase-promoting complex (APC) is activated, which activates a set of downstream proteins to finally activate separase, a protease. Separase cleaves cohesin and unglues the sister chromatids, which immediately separate and form daughter chromosomes. The kinetochore microtubules start to disassemble and NE begins to reassemble. • Telophase – Segregated chromosomes decondense, the NE with the NPCs is reformed around each of the two new daughter nuclei marking the completion of mitosis.

Cytokinesis follows immediately after mitosis. The cytoplasm divides into two by a contractile ring of actin and myosin filaments that pinch off the cell membrane to create two daughter cells, with each containing one nucleus (Alberts et al., 2008).

The cell cycle checkpoint proteins are the controllers and regulators of the eukaryotic cell cycle. These checkpoint proteins ensure that each process is completed accurately before progressing into the next phase. Cyclin-dependent

10 protein kinase (Cdk) complexes with cyclins such as G1/S-cyclins, S-cyclins and M-cyclins control the eukaryotic cell cycle (Alberts et al., 2008).

Proper segregation of sister chromatids during anaphase is one of the most crucial steps in mitosis. Uneven segregation of chromosomes results in severe mitotic defects and if this happens over consecutive cell cycles, it leads to chromosome instability (Rajagopalan and Lengauer, 2004). The segregation of sister chromatids is controlled by the APC/C (anaphase promoting complex or cyclosome) (Alberts et al., 2008). Some of the proteins that play an important role in spindle assembly are the spindle assembly factors (SAFs such as augmin, γ-tubulin, NuMA and TPX2), the transport receptor importin β and the small GTPase Ran (Alberts et al., 2008; Forbes et al., 2015; Meunier and Vernos, 2012).

1.3 GTPase Ran Ran is a G protein that is involved in nuclear transport during interphase (Harel and Forbes, 2004), whereas in mitosis it plays a major role in spindle assembly and post-mitotic NE reformation (Melchior, 2001). Ran acts as a ‘molecular switch’ and exists either as RanGDP or RanGTP. RanGDP takes the help of nuclear transport factor 2 (NTF2) to enter into the nucleus (Fried and Kutay, 2003). The guanine nucleotide exchange factor (GEF) for Ran is a nuclear protein, RCC1 (regulator of chromosome condensation 1), which is bound to the chromatin. RCC1 promotes release of GDP from RanGDP and creates a situation for Ran to bind either to GTP or GDP. Since the levels of GTP are higher than GDP inside the nucleus, RanGTP is most likely to form (Moore, 1998). The Ran GTPase activating protein (RanGAP), located along the cytoplasmic filaments of NPCs, stimulates the GTPase activity of Ran, so that RanGTP can be converted to RanGDP (Harel and Forbes, 2004).

During interphase, Ran is mainly involved in the nucleocytoplasmic transport by triggering assembly and disassembly of transport complexes. RanGTP’s high affinity towards importin β and exportin are lost once the γ- phosphate of the GTP is hydrolysed to form RanGDP (Guttler and Gorlich, 2011; Harel and Forbes, 2004). The binding of RanGTP to the import-cargo

11 complex and the hydrolysis of GTP in Ran of the export-cargo complex aids the release of cargo into the nucleus or the cytoplasm, respectively (Harel and Forbes, 2004). Apart from the nucleocytoplasmic transport, other functions of Ran in interphase are yet to be described (see results section 4.2).

The major components of the mitotic spindle are, the spindle poles, the microtubules and the chromosomes. The mitotic spindle formation is controlled by RanGTP and SAFs (Meunier and Vernos, 2012). Ectopic expression or silencing of SAFs, leads to severe mitotic defects and sometimes even lethality (Ban et al., 2007; Bruning-Richardson et al., 2012; Meunier and Vernos, 2012; Radulescu and Cleveland, 2010). To build a bipolar mitotic spindle, the SAFs have to be activated locally around chromosomes and not elsewhere in the mitotic cytoplasm. This is accomplished by the RanGTP / importin β system. Importin β binds to different SAFs and keeps them in an inhibitory complex. The high concentration of RanGTP around the chromosomes binds to importin β and, subsequently releases the SAFs from the importin inhibitory complexes and allows spindle assembly to take place in the vicinity of the chromosomes (Harel and Forbes, 2004; Meunier and Vernos, 2012). A recent study demonstrated the discovery of microtubule branching mechanism in the mitotic spindle. This new branching is created on the sides of the existing microtubules with the help of the microtubule nucleating protein augmin, γ-tubulin, TPX2 and RanGTP. The exact mechanism of their roles remains unknown (but see 4.3). Branching of the mitotic spindle microtubules enhances the spindle stability and is thus important for correct chromosome segregations (Forbes et al., 2015)

1.4 Differentiation Differentiation is a process in which a less specialized cell matures or develops to become a cell with more distinct functions. Specialized cells in tissues and organs are the products of differentiation. During early embryogenesis, a mass of cells (also called as inner cell mass (ICM)) inside the primordial embryo develops into an embryoblast. The ICM is then rearranged to form three germ layers: an outer ectoderm, giving rise to the central nervous system, sensory epithelia of eye, ear and nose, and skin. The middle mesoderm 12 develops into connective tissues, cartilage, bone, kidneys, spleen, heart, striated and smooth muscles. The inner endoderm develops into lungs, liver and gastrointestinal tract (Pansky, 1982). There are two broad categories of stem cells: Embryonic Stem Cells (ESCs) and adult stem cells. Adult stem cells are present at specific sites in the body and are able to divide to regenerate damaged tissues or to replace dying cells. Unlike ESCs, adult stems cells are often restricted to differentiate into certain lineages. Adult stem cells also possess the ability of transdifferentiation, i.e. cell of one lineage to become a cell of another lineage (Eisenberg and Eisenberg, 2003).

1.4.1 Pluripotent stem cells Embryonic stem cells (ESCs) are pluripotent cells derived from the ICM of the blastocyst during gastrulation (Donovan and Gearhart, 2001). By arresting the embryonic progression and isolating from blastocyst, the ICM can be developed into ES cell lines. Culturing of these cells, gives rise to embryoid bodies through which embryonic lineage can be obtained (Sylvester and Longaker, 2004) and these cells have unlimited self-renewal and differentiation potential (Spradling et al., 2001). Isolation of human ESCs offers great potential to detect genetic diseases at very early stages, even before implantation of the embryo (Sermon et al., 2009). Immune rejections along with ethical concerns have diminished the progress of human ESCs in regenerative medicine.

Knowledge from somatic cell nuclear transfer (Gurdon, 1962), the discovery of the “master” transcription factors (Davis et al., 1987) and the generation of human and mouse ESCs (Evans and Kaufman, 1981; Smith et al., 1988) over a period of four decades led Yamanaka and his colleague to generate induced pluripotent stem cells (iPSCs). In 2006, Yamanaka and Takahashi demonstrated that four defined factors (Oct3/4, Sox2, c-Myc and klf4) were sufficient to reprogram mature fibroblast cells into pluripotent stem cells (Takahashi and Yamanaka, 2006). These human iPSCs were similar to human ESCs in their morphology, gene expression, and proliferation (Takahashi et al., 2007). Upon injection into animals, the iPSCs develop into a teratoma that contains all three germ layers (Takahashi et al., 2007; Takahashi 13 and Yamanaka, 2006). Application of iPSCs led to progress in regenerative medicine (Kriks et al., 2011; Tsuji et al., 2010), disease modelling and drug screening (Park et al., 2008). Directed differentiation is another approach where the iPSCs are forced to differentiate into a particular somatic lineage with a single or combination of factors. In most cases it is challenging to find a specific protocol that can generate enough cells of interest. Protocols for generation of iPSCs that can differentiate into a cell type of choice, for reintroduction into patient are complex and time-consuming (Hirschi et al., 2014).

1.4.2 Myogenesis Myogenesis is an event in embryonic development during which muscular tissue is formed. In the adults, myogenesis often takes place to replenish dying muscle cells or to regenerate damaged tissues. During adult myogenesis, mitotically quiescent satellite cells that are residing near associated muscle fibers (for example skeletal muscle), can be activated to proliferate upon inducing stress or trauma, such as injuries. The activated satellite cells then give rise to skeletal myoblasts that undergo multiple rounds of proliferation to regenerate the damaged tissue (Le Grand and Rudnicki, 2007). In developing embryo, myoblasts can either proliferate or differentiate. The factors that control this choice are not clearly defined. In cell culture, after withdrawal of growth factors, the myoblasts cease to undergo cell division and proceed to terminal differentiation and to form myotubes (Tapscott et al., 1988). Myoblast differentiation takes place through three succeeding steps – First, cell cycle exit and expression of certain muscle specific transcription factors and genes. Second, migration, recognition and adhesion of myoblasts to each other. Third, plasma membrane fusion of the adhering myoblasts. The above steps are repeated iteratively until the final muscle tissue is formed (Rochlin et al., 2010). In mammals the following myogenic factors from the MyoD family, myf-5, MyoD, myogenin and MRF4, are expressed during skeletal muscle formation and maturation. These members belong to the helix- loop-helix class of transcription factors, that are involved in activating muscle specific genes (Buckingham, 1992). MyoD and myf-5 are expressed both in

14 myoblasts and myotubes, whereas myogenin starts to be expressed just before myoblasts differentiation and MRF4 expression is observed only in myotubes (Buckingham, 1994). Knockout studies of the four myogenic factors in mice suggest that expression of either MyoD or myf-5 is necessary for the formation and/or propagation and maintenance of precursor myoblasts. Myogenin and MRF4, appear at the level of myoblast differentiation (Buckingham, 1994).

1.4.3 NE proteins in muscle differentiation Little is known about the role of NE transmembrane proteins in cell differentiation. Mutations in genes encoding emerin and A-type lamins have been reported for several muscle degenerative diseases (Maraldi et al., 2011; Mounkes and Stewart, 2004; Worman and Courvalin, 2005). To understand how NE transmembrane proteins are involved in such diseases, researchers investigated their role in myogenesis. The expression profiles of some nuclear envelope transmembrane proteins (NETs) differ widely in different tissues but are high in muscles (Korfali et al., 2012). During skeletal myoblast differentiation, NETs were shown to be either up regulated or down regulated (Chen et al., 2006). A recent study has shown that some NETs are involved in repositioning muscle specific genes from centre to the nuclear periphery (Robson et al., 2016). The same study showed that combined knockdown of three muscle specific NETs, NET39, Tmem38a and WFS1 repressed myogenesis more efficiently than when knocked down individually. This suggests that some NETs may work together during myogenesis, however this condition is not true in all cases (see section 4.4).

1.5 Methods to study protein-protein interactions

Different biochemical and genetical approaches have been developed over the years to study protein-protein interactions. Protein interactions are often transient or weak making it a challenge to study them. Some of the commonly used methods to study interacting proteins are, coimmunoprecipitation (CoIP), the yeast two-hybrid system (Y2H), pull down assay, fluorescence resonance energy transfer (FRET), surface plasmon resonance (SPR), blot overlay assay, MST (microscale thermophoresis), BioID 15 propionate, DSP) reacts with primary amines on proteins and forms stable amide bonds (Lomant and Fairbanks, 1976). Membrane permeability and cleavability of the crosslinker are extremely advantageous for identifying specific protein interactions taking place in live cells (Berggard et al., 2007; Kluger and Alagic, 2004). Combining with IP and mass spectrometry, chemical crosslinking is a powerful tool to study protein-protein interactions (Percipalle et al., 2002; Smith et al., 2011) (Paper 1).

1.6 Protein interaction studies in the nuclear envelope Identifying protein-protein interactions in the NE helps to understand and describe the functional diversity of transmembrane proteins. Pull down experiments using recombinant proteins is a good way to map interaction sites, but it does not conclude whether such interactions always happen in live cells or cell extracts, where competing binding proteins are present. Some NE transmembrane proteins that are tightly associated with the nuclear lamina, present a big challenge to researchers to solubilize them (Dwyer and Blobel, 1976; Radu et al., 1993; Snow et al., 1987) without disrupting their protein- protein interactions. Therefore employing classical CoIP to identify interacting partners of nuclear lamina and NE proteins can be an unattractive approach. An overview of different NE proteins and their binding partners are represented in table 2. The most studied among them such as A-type and B- type lamins and emerin have a variety of binding partners consistent with their functional diversity. One can see from table 2, that most of the studies were performed either by CoIP or pull down experiments, which is not always the best choice, especially when solubilization properties vary among the NE proteins. This highlighted the need to develop a novel, specific and robust approach, which will be necessary in the future to unravel the dynamic interactome of the NE.

2 Aim

This thesis is focused on gaining a deeper understanding of the functional organization of the nuclear envelope and its proteins. A broad knowledge in NE proteins would help us to understand why such a diverse repertoire of transmembrane proteins is present in the nuclear envelope and their functional roles apart from anchoring the nuclear lamina and chromatin associated proteins to the nuclear periphery.

Specific aims:

• to improve methods to detect interactions of extraction resistant transmembrane proteins in live cells and to investigate the functional significance of these interactions.

• to elucidate the functional significance of the interaction between the RanGTPase and Samp1.

• to elucidate the possible role of the transmembrane protein Samp1 in the mitotic process.

• to explore the role of Samp1 in the cell differentiation.

3 Methodological considerations 3.1 Cell cultures iPSCs (ATCC ACS 1011) used in paper IV are retroviral reprogrammed cells derived from human foreskin fibroblasts. These cells are similar to ESCs in their morphology and express the same markers as ESCs. This commercial iPSC line was characterized for expression of pluripotent markers and their ability to differentiate into the three germ layers. The iPSCs were cultured on matrigel, under pluripotent conditions. We noticed that these cells were very sensitive to matrigel concentrations and mechanical vibrations, which had to be optimized before starting the study to prevent spontaneous differentiation.

C2C12 myoblasts (ATCC) used in paper V are immortalized cell lines derived from mouse skeletal muscles which behave similar to that of progenitor lineage. These cells readily proliferate in growth medium containing high serum. In low serum medium the cells differentiate and express characteristic muscle proteins. The myoblast cell cultures must not be allowed to become confluent, as this will deplete the myoblast population in the cell culture. The cells were thus always maintained at less than 60% confluence. These cells are also sensitive to the surface on which they grow, so it must be optimized before starting the study, to prevent detachment from the surface. We noticed difficulties in growing myotubes on glass coverslips as the cells started to detach from the surface upon inducing differentiation. For imaging myotubes, cells were grown on glass-bottom dishes and handled carefully during the process of immunostaining.

21 3.2 Short hairpin RNA (shRNA) ShRNA is a vector based artificial RNA molecule that can be used to specifically silence targeted gene expression through RNA interference (RNAi) (Tuschl, 2001). In paper V, we have used two independent shRNA vectors targeting murine Samp1 to generate Samp1 knockdown C2C12 myoblast cell lines. Stable integration of the shRNA into the host genome gives continuous expression of the shRNA vectors and thereby extends the silencing of the target gene.

3.3 Lentivirus like particle generation Lentivirus based virus-like particles (VLPs), stemming from a subset of retroviruses, are the most common type of VLPs used for transduction of “hard-to-transfect” cells types e.g. C2C12. The genetic material of VLPs integrates stably into the host genome enabling long-term transgenic expression. In the lentiviral system used in Paper V, the component required to produce infectious VLPs is divided into multiple plasmids. It includes transfer plasmid encoding insert of interest (cDNA or shRNA), packaging plasmid and envelope plasmid. The above plasmids were transfected into HEK293T cells to produce VLPs. After post transfection medium change, the cell culture medium is collected between 36-48h and the VLPs are harvested by centrifugation. The harvested lentiviral suspension can be delivered directly to proliferating cells without any additional agents (e.g. Lipofectamine).

4 Results and discussion

Subtractive proteomic studies have shown that there are several hundreds of unique transmembrane proteins in the NE (Wilkie et al., 2011), but only a few of them have been characterized so far. The overall aim of this thesis is to understand the functional organization of the NE. In order to achieve this, it is important to identify the protein-protein interactions in the NE. I have focused on unravelling the functional role of the recently identified INM transmembrane protein, Samp1 (Buch et al., 2009; Gudise et al., 2011), in interphase and mitosis. Identification of interaction partners required development of novel methodology.

4.1 A novel approach to study NE protein interactions in live cells

Several different biochemical and genetic approaches are available to study protein-protein interactions (Table 1). The three approaches that can be used in combination with mass spectrometry (MS) for detecting protein interactions in mammalian cells are: CoIP, BioID and chemical cross-linking. The most commonly used approach, CoIP, has limitations for proteins with poor solubility. Since INM proteins are strongly associated with ‘detergent- high-salt-resistant-nuclear lamins’ (Aaronson and Blobel, 1975; Dwyer and Blobel, 1976) it is often impossible to solubilize them under non-denaturing conditions. For example, the LINC complex protein SUN1 resists solubilization even at 1M NaCl and 1% TX-100 (Paper I and (Haque et al., 2006). The recently invented BioID method (Roux et al., 2012), overcomes this problem and also detects protein interactions in live cells. However, it requires relatively long labelling hours (16h) making it unfeasible to study dynamic protein interactions that occur over short time periods, e.g., mitosis. 23

4.2 Molecular dissection of the interaction between Samp1 and Ran

In a protein interactome database Samp1 was predicted to interact with the small monomeric GTPase Ran (Alexeyenko and Sonnhammer, 2009). Since Ran is a soluble protein playing a key role in many nuclear assembly processes and previously not shown to interact with any transmembrane protein, we wanted to investigate whether this predicted interaction could happen in reality. To investigate this we performed MCLIP on live cells and in vitro pull down experiments on recombinantly expressed proteins. Since we had problems to achieve human recombinant Samp1 in soluble form, we took advantage of the Samp1 homologue, Ct-Samp1, in a thermophilic fungus, (see section 1.1.5) for the in vitro pull down studies. The nucleoplasmic N-terminal domain of Ct-Samp1 shares a high homology and functionality with human Samp1 with its four conserved -CXXC- motifs and also binds to the INM protein emerin (Paper I) as human Samp1 does (Gudise et al., 2011).

Results from pull down experiments with recombinantly expressed proteins demonstrated that Samp1 and Ran are indeed able to bind to each other directly. Furthermore, we also identified the Ran binding domain to amino acids 75-135 of Ct-Samp1. This region of Samp1 has a unique sequence that was not described previously in any of the known Ran binding proteins (Paper II). Despite the conserved CXXC motif in Samp1, the binding to Ran was not dependent on zinc. Thus Samp1 is the first transmembrane protein discovered to bind to Ran and possesses a unique Ran binding domain (Paper II).

Ran, a monomeric G protein is known to exist in two forms, inactive RanGDP or active RanGTP (Forbes et al., 2015; Harel and Forbes, 2004). The two forms act like a molecular switch to regulate protein interactions in key events such as, nucleocytotplasmic transport (Moore and Blobel, 1993), assembly of the mitotic spindle (Nachury et al., 2001) and postmitotic nuclear assembly (Harel et al., 2003; Walther et al., 2003). Using a temperature sensitive RCC1 mutation in tsBN2 cells as a model, we were able to turn off 25 the accumulation of RanGTP in favor of RanGDP in live cells. By performing MCLIP experiments in tsBN2 cells at two temperatures, we found that Samp1 preferred to interact with RanGTP over RanGDP. Results from pull down experiments with recombinantly expressed proteins also showed a binding preference of Samp1 to RanGTP. Furthermore, Samp1 was also able to recruit Ran to the nuclear periphery (Paper II). This suggests that Samp1 could act as local binding site for RanGTP to carry out RanGTP triggered processes at specific positions close to the INM (Paper II).

4.3 The role of transmembrane INM proteins in the mitotic spindle

The nuclear membrane is continuous with the rough ER. At NEBD, INM proteins are generally dispersed out in the mitotic ER, and after chromosome segregation recruited back to the INM of the reforming daughter nuclei. In 2009, Hallberg’s group demonstrated distribution of the transmembrane protein, Samp1 in the mitotic spindle (Buch et al., 2009). Later two more NETs (WFS1 and TMEM214) were also reported to be distributed in the mitotic spindle (Wilkie et al., 2011). Another research group have also shown low levels of transmembrane ER proteins in the mitotic spindle (Lu et al., 2009). Samp1 depleted cells showed a significant increase in cells with unevenly segregated chromosomes, indicating mitotic defects (Paper III). This implicates that Samp1 plays an important role in the mitotic machinery. Time- lapse microscopy of Samp1 depleted cells revealed, a significant, 6-fold prolongation specifically of the metaphase, indicating destabilization of the mitotic spindle (Paper III).

To understand why, we searched for binding partners of Samp1 in HeLa cells that were synchronized at metaphase. We already knew that Samp1 interacts with Ran (Paper I & II), a master regulator of the mitotic spindle. Using MCLIP, we identified augmin and γ-tubulin as other interaction partners of Samp1 during metaphase. A significant increase in spindle length and decreased recruitment of augmin and γ-tubulin was observed in Samp1 depleted cells (Paper III), supporting the idea of destabilization of the mitotic

26 spindle as suggested by the specific prolongation of metaphase. A recent discovery has shown branching of microtubules in the mitotic spindle. This branching is suggested to provide increased density to bundles of the spindle microtubules and stabilize the mitotic spindle for faithful chromosome segregation (Forbes et al., 2015; Kamasaki et al., 2013). Our data in paper III suggests that Samp1 could possibly provide anchoring points for augmin and γ-tubulin to promote microtubule branching in the mitotic spindle.

4.4 The role of Samp1 in differentiation

Samp1 is highly expressed in nerve and muscle cells (Thanisch et al., 2017). Recent studies have shown that certain NETs, that are highly expressed in muscle are involved in myogenesis (Korfali et al., 2012; Wilkie et al., 2011). The mechanism behind this is yet to be fully understood. iPSCs (Paper IV) and C2C12 cells (Paper V) are two independent model systems that we investigated for the possible role of Samp1 in differentiation. Samp1 expression levels increased during differentiation of iPSCs (Paper IV) and muscle cells (Paper V).

We started the study by classifying undifferentiated and differentiated iPSCs or C2C12 myoblasts based on their established morphological characteristics and the expression of well known differentiation markers, Lamin A/C (for iPSCs studies) and myosin heavy chain (MyHC) (for myogenesis). In both studies, Samp1 displayed more pronounced rim staining in the nuclear periphery of the differentiated cells than the undifferentiated ones. The increase of Samp1 protein levels was more pronounced in differentiating myoblasts (Paper V) compared to iPSCs (Paper IV).

Ectopic expression of Samp1 in a few iPSCs drastically modified colony morphology, and caused the entire cell mass to differentiate despite the fact that cells were still maintained under pluripotent conditions. Further studies showed that some of the cells differentiated by Samp1 over expression expressed βIII tubulin, a neuronal marker (Paper IV). In a different experimental setup, we investigated the fate of C2C12 myoblast differentiation after knockdown of Samp1. For this, we generated stable Samp1 knockdown 27 cell lines and induced differentiation by change to differentiation medium. Samp1 depleted cells failed to differentiate and also did not express the myogenic markers, myogenin or MyHC upon induction of differentiation (Paper V). This suggests that Samp1 is essential for myoblast differentiation. Ectopic expression of human Samp1 in Samp1 knockdown cell lines completely rescued differentiation of the myoblasts. This was clearly evident from the formation of myotubes and restoration of the myogenic index in rescue experiments (Paper V). The mechanism behind how Samp1 is involved in differentiation is not known at the moment and needs further investigations.

A recent study by Solovei et al. (2013) has shown that cells tether chromatin to the nuclear periphery by a so called A-tether (Lamin A/C dependent) and/or B-tether (Lamin B receptor dependent) mediated mechanism. The A-tether includes A-type lamins, LEM domain proteins and yet unidentified INM proteins. The B-tether includes Lamin B receptor (LBR) and B-type lamins. During cell differentiation, LBR is expressed initially followed by Lamin A/C expression. The difference in expression sequence of these two proteins in different cell types suggests that peripheral chromatin tethering might regulate cell differentiation (Solovei et al., 2013). Samp1’s interaction with emerin (Paper I), Lamin A/C (Borrego-Pinto et al., 2012) and results from differentiation studies (Paper IV & Paper V) suggest that Samp1 might promote differentiation by interacting with chromatin binding proteins and A-tether component.

5 Conclusion

We have developed a novel, robust and accurate method, MCLIP that detects specific protein-protein interactions of notoriously “difficult to solubilize” proteins in live cells. By applying MCLIP, we were able to identify specific interacting partners of Samp1, both in the NE and in the mitotic spindle (Paper I & III). Some of the interactions identified by MCLIP are now in fact proved to be direct binding interactions (Paper I, II & III). The identification of different interacting partners of Samp1 during interphase has thrown new light on the diverse functionality of the NE protein in different cellular processes. For example, by interacting with the G-protein Ran, Samp1 can provide a binding site at the INM for Ran to carryout RanGTP activated processes in the nuclear periphery (Paper II). Similarly, in mitosis, we propose that Samp1 may act as an anchoring point for augmin and γ-tubulin in the mitotic spindle and promote microtubule branching, which stabilizes the mitotic spindle (Paper III). Results from two independent differentiation model systems (Paper IV and Paper V) suggest that Samp1 has differentiation promoting activity in a tissue-wide perspective.

6 Future perspectives

Our recently developed MCLIP method can be combined with mass spectrometry (MS) to continue identifying new interacting partners of Samp1 both in interphase and mitosis (Paper I and III). Among several hundreds of putative transmembrane proteins reported in the NE, only a few have been characterized and far less is known about their interactions. MCLIP / MS studies on NE protein interactions will guide our understanding of the functional organization of the NE. In the future, the existing MCLIP protocol can be optimized with crosslinkers that differ in the specificity of functional groups to capture new interaction partners.

Until the discovery of Samp1 (Buch et al., 2009), the mitotic spindle was believed to be devoid of NE transmembrane proteins. Recent studies made by us and others (see section 4.4.3) provide emerging proofs that NE transmembrane proteins do exist in the mitotic spindle and that Samp1 has a functional role in mitosis (Paper III). The fact that this phenomenon has not been explored previously highlights the need for new methods to identify protein-protein interactions during such a short and dynamic process as mitosis. MCLIP / MS studies on cells synchronized in mitosis in order to identify other interacting partners of Samp1 could help us to shed light on mitotic defects and chromosome instability involved in cancer development.

It would also be interesting to continue studying the functional significance of the interaction between Samp1 and Ran (Paper II). For this, it would be useful to map the amino acids in Samp1 that are needed for this binding. Expression of recombinant deletion mutants of Ct-Samp1or competing peptides to interfere with Samp1-Ran binding would be useful in

31 studies of proteins assembly in the nuclear periphery. For this, in vitro nuclear assembly models using Xenopus eggs can be used.

Samp1 strongly promotes differentiation in two independent systems (Paper IV and Paper V) suggesting that Samp1 may have a general role in cell differentiation. To describe the mechanism behind this, MCLIP / MS studies on differentiating myoblasts can be useful. This may give us a list of interaction partners, including transcription factors, chromatin binding proteins, or other NE proteins, which may help us to answer whether Samp1 follows a similar mechanisms in the two differentiation models. If Samp1 interacts with chromatin binding proteins and affects expression of genes required for differentiation, we can extend our studies with fluorescent in situ hybridization (FISH) studies of gene positioning in myoblast cell lines after knockout of selected NE proteins.

In iPSCs, ectopic expression of Samp1 induced direct differentiation of iPSCs into cells expressing β-III tubulin (Paper IV). The next step is to investigate the extent to which the differentiated cells are expressing other neuronal markers to determine their specific cell type and functional properties (i.e. electrophysiology studies). If Samp1 induces development of functional neurons, it may open new possibilities to use Samp1 as a reprogramming factor in regenerative medicine.

The studies suggested will contribute to the emerging view that the NE is not just a physical barrier but a network of micro domains with unique set of transmembrane proteins carrying out fundamental cellular processes.

7 Populärvetenskaplig sammanfattning på svenska

Det genetiska materialet i den mänskliga cellen, genomet, är innesluten i en cellkärna. Kärnan är omgiven av ett membran som kallas kärnmembran. Kärnmembran har en yttre och ett inre skikt. Kärnmembran var oftast betraktat som enkel membranbarriär, att fysiskt skydda och separera DNA från biomolekyler och andra proteiner i cellen. Nyligen genomförda studier på kärnmembran har dock visat att det innehåller hundratals unika transmembranproteiner, varav endast en handfull har studerats hittills. Min avhandling är främst fokuserad på att ta reda på de funktionella roller som kärnmembran och dess transmembranproteiner har.

Kärndelning eller mitos är en viktig händelse i cellen, där de två identiska kopiorna av genomet delas upp av den mitotiska maskinen, med en kopia i vardera ny dotterkärna. Misstag som sker under denna process är mycket allvarligt och kan leda till utvecklingen av cancer. Cellen delar sig via cytokines och bildar två identiska celler med vardera ny cellkärna. Under 2009 visade Hallberg’s grupp för första gången att kärnmembran transmembranproteiner existerar i den mitotiska spindeln. Proteinet de upptäckte döptes till Samp1 (spindle associated membrane protein 1)

Vi var intresserade i att identifiera de funktionella roller Samp1 har mellan celldelningarna (interfas) och under mitos. För detta bestämde jag mig för att först identifiera bindnings partner till Samp1 i levande celler under interfas och mitos. Eftersom proteiner i kärnmembran är särskilt utmanande att studera därför att starkt förankrade i kärnlamina och interagerar även med andra proteiner. Jag har utvecklat en ny metod som möjliggör studier av bidning mellan proteiner (MCLIP, Membrane protein crosslinking immunoprecipitation) i leverande celler.

33 MCLIP kan kombineras med masspektrometri för att identifiera alla möjliga interagerade proteiner av intresse. Dena lista över bindnings partner som identifieras med hjälp av MCLIP kan sedan leda oss vidare i utredningen om Samp1´s funktion. Detta genom att tysta ner Samp1 proteinuttryck och sedan titta på vad som händer med de identifierade interaktionspartnerna. Genom att använda MCLIP, kunde vi identifiera specifika interaktionspartners till Samp1 under interfas och mitos. Detta hjälpte oss att demonstrera att (i) Samp1 binder till kärnmembran proteiner, LINC complex proteiner och proteiner från den mitotiska spindeln (ii) Samp1 spelar en viktig roll i att upprätthålla stabiliteten i det mitotiska maskineriet under delningen av genomet.

I andra halvan av min avhandling, undersökte jag rollen av kärnmembran proteiner i celldifferentiering. Differentiering är en process där en mindre specialiserad cell utvecklas till att bli en cell med mer distinkta funktioner. För denna studie användes mänskliga inducerbara pluripotenta stamceller (iPSCs) och muskelstamceller från mus, liknande C2C12 myoblastceller. iPSCs är multipotenta celler som har möjlighet att differentiera till alla typer av specialiserade celler medan C2C12 är celler som endast kan differentiera till muskelceller myotuber och myofibrer. I dessa två modellsystem, observerade vi att Samp1 främjar celldifferentiering. Överuttryck av Samp1 i iPSCs celler gav upphov till neuronala celler inom sex dagar. Dessutom, i myoblast muskelceller där Samp1 helt tystats ner slutade myoblaster att differentieras.

De viktigaste resultaten i denna avhandling är att ge insikt i de olika och oväntade rollerna som ett transmembranprotein han ha i olika cellulära processer. Detta visar tydligt att kärnmembran inte bara är en fysisk barriär utan också ett funktionellt membran fyllt med transmembranproteiner som utför viktiga nyckelfunktioner i cellen.

8 Acknowledgments

Alhamdullilah

I would like to express my sincere thanks to my supervisor Einar Hallberg for giving me an opportunity to work in many interesting projects. He has been an excellent teacher, mentor and helped me through out my PhD and in writing this thesis. I would like to thank you for encouraging my ideas and allowing me to grow as an independent researcher. This would also be a good time to thank you for letting me to experience sailing.

In this occasion, I would like to remember my co-supervisor Kerstin Iverfeldt. She introduced me to the world of recombinant protein studies during my masters and has been a good critic of my research. She nurtured my scientific discussions in the departmental seminars and held out a lending hand in infrastructural support. Your memory and contributions to science are eternal.

All the present members of the EHA group: Ricardo, Mehedi, Veronica, Balaje, Cissi and Frida - without your help, support and intelligent comments, my work would not have been as fruitful as it is. Ricardo, thank you for introducing me to microscopy and showing me the ethical ways of imaging. Mehedi, thanks for your effort in cloning challenging vectors. Veronica, thank you for all the late nights you put into synchronizing cells and your persistent efforts in the mitosis project. Thank you Cissi, for teaching me how to handle stem cells and reading the mind of the micromanipulator! You and Veronica exposed me the Swedish way of doing things both in the lab and outside, which made me feel at home. Balaje, thanks for all your efforts in the different projects and your smart thoughts on cloning strategies. Frida, thanks for 35 modernizing our weekly group meeting. Good luck with your studies. I would also like to acknowledge the previous members of the EHA group Marie, Ellinor, Santhosh and Robert for all the good times.

I am very much obliged to all the wonderful teachers at the department- Anders Unden, Anna-Lena Ström, Anna Forsby, Ülo Langel, Bengt Mannervik and Henrietta Nielsen for your insightful comments and thought provoking discussions at Tuesday seminars and at PhD student - Staff meetings.

I would also like to thank the lab assistants of the recombinant course, Elena, Jonas, Anna and Preeti. We conducted the course with unexpected surprises in every year. Kristina, thanks for your wise thoughts about the Swedish health care system and for smooth functioning of the PhD meetings. Carmine, thank you for arranging barbecues every summer and making sure that mingling happens now and then. Thank you Ying, for the cheerful positivity that you bring. Without your notes, I couldn't have gotten through the final exam. Totally indebted to you. Maxime, thanks for the nice company during the Swedish courses, we should have attended a few more! Niina, thank you for the generous support with chemicals and reagents. Tönis, the PS3 in the conference was epic. I appreciate that episode! Helena & Birgitta - discussions on the cultural differences in Stockholm have always been fun, thank you! Luís Daniel, thanks for nice discussion ‘man’ and for showing me where to find pastie de nata. Jessica, thanks for lending the balances. Moataz, thanks for the nice discussions.

I would also like to thank the recently graduated students Andrés, gracias for exposing me to the hilarious existence of cultural versions of spiderman and rambo. Those memories still send me into splits! Henrik and Jackob, I appreciate your tips to pass the final exam, thank you! Karim, thanks for being supportive and helpful during my D-course project. Ajayi and Xin, thanks for the lots of technical and non-technical discussion. Chats with you guys have been enjoyable and motivating.

36 Thanks Marie-Louise and Sylvia for all the help and patience with all my questions and making the paper work easy in my times of difficulty. You both are the backbone of the Neurokemi department! Project students- Mahmud, Paulo, Fausto, Hanna, and Martin - you were all very curious students and made my job easy. I learnt a lot from all of you. Thanks to everyone in the department for your presence and support at all times. I really appreciate the nice discussion and fun times during PhD meetings, Christmas dinner, barbecues, kubb and boule that we all enjoyed together, thank you all.

I would also like to thank my friends Imran, Nayeem, Ahamed, Hani and Hand Nasser from DBB and Organic chemistry. Meeting you guys frequently did take off some stress from daily ordeals. I would also like to thanks my friends outside work- Khan, Sadeak, Humayun and their families who understood my busy schedules and constantly encouraged me. I am indebted to their prayers, support and for ever being there for me.

Finally my family members whose immense love and support helped me to reach where I am today.

Thanks Yasar for all the love and support. Shaheen, thanks for your genuine effort in trying to undertand my research and refilling our mystery box. I appreciate your visit to Stockholm, for making wonderful scandinavian memories, and for the good times in Chennai. Hasina, words will never do justice to describe what you are to me, you are my best friend and critic. I am lucky that you are part of my life.

9 References

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