!" #$"% "$&$$ ' () * ( +",-& . /01( ( /231( 4 /5 1 &.3 6 7&3 (3 7 3 3 ("& (583( & 583( " 835 4( &"9 ( " & " 3 :. ;( &< ( " &3 5 ( 4 " &35#5"# ( " &. " &. ( ( 4 & #$"% =>> && > ? @ == = = "A"B", 3-C%BC"%,ACD%%! 3-C%BC"%,ACD%B$ ! " ("$,C" 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. ii 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. !"!#$!%$"! iv 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 vi 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