Studies of mesenchymal and amoeboid migration on compliant substrates

THE INTERPLAY BETWEEN THE ENDOPLASMIC RETICULUM STRUCTURE AND THE CYTOSKELETON ORGANIZATION IN SCHIZOSACCHAROMYCES POMBE ZHANG DAN A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY TEMASEK LIFE SCIENCES LABORATORY NATIONAL UNIVERSITY OF SINGAPORE 2012 i DECLARATION I hereby declare that the thesis is my original work and it has been written by me in its entirety. I have duly acknowledged all the sources of information which have been used in the thesis. This thesis has also not been submitted for any degree in any university previously. Zhang Dan Dec 2012 ii ACKNOWLEDGEMENTS Firstly, I would like to express my heartfelt gratefulness to my supervisor Snezhka for her extraordinary guidance, continuous support and encouragement. Without her great scientific insights, emotional support and understanding, this thesis would not have been possible. I thank her for giving me opportunities and freedom to explore and experiment and for her great patience to my weakness. I am thankful to my co-supervisor Prof. Mohan Balasubramanian for his valuable advice and comments on my research work. I am also grateful to my thesis committee members Dr. Jedd Gregory, Dr. Wang Yue and Dr. Thirumaran Thanabalu for the time and efforts they put for my thesis work and their helpful suggestions on my studies. Several people have contributed to the completion of this work. I am very grateful to Aleksandar Vjestica, my great work partner and close friend, for his constant technical help and scientific discussion. I greatly enjoyed the collaboration with him as well as our friendship. I am very thankful to all my attachment students, Bhuvaneswari Shanmugam, Pooja Padmini, Indira Priyadarshini, Felicia Lim, Ranjay Jayadev, for their help in generating constructs and strains in this work. I thank the cell biology community, including the cell division group, Fungal Pathobiology group for sharing reagents, constructs and strains, and for their technical help and discussions. I thank Dr. Snezhka Oliferenko, Dr. Huang Yingyi, Dr. Maria Makarova who made critical comments and spent their valuable time to proof-read my thesis. I am thankful to TLL facilities and staff for general support, and Temasek Holdings, Singapore for the financial support. Many thanks to all my friends in and out of the lab, for wonderful moments spent together and their emotional supports throughout these years. Lastly, I owe my deepest gratitude and appreciation to my family, for their love, unfailing supports and understanding. iii TABLE OF CONTENTS TITLE PAGE i DECLARATION ii ACKNOWLEGEMENTS iii TABLE OF CONTENTS iv SUMMARY viii LIST OF FIGURES x ABBREVIATIONS xii PUBLICATIONS xiii CHAPTER I: Introduction ……………………………………….……………….1 1.1 Endoplasmic reticulum ………………………………………………………….2 1.1.1 Architecture of the endoplasmic reticulum …………………………….… .2 1.1.2 The ER shaping proteins ………………………………… …….………….3 1.1.3 Functions of the tubular ER …………………………………… .……… .5 1.1.4 Membrane contacts between the ER and other organelles ………….………6 1.1.5 The endoplasmic reticulum in yeast ……………………………………… 1.2 Actin cytoskeleton ………………………… .………………………… ……… 1.2.1 Actin structures …………………………………………………….………9 1.2.2 Actin cytoskeleton in S. pombe …………………………… …………… 10 1.2.2.1 Actin structures in interphase ………………………… ……… …10 1.2.2.2 Actomyosin ring assembly …………… .………………….………11 1.2.3 Actin cytoskeleton and endomembranes ……………….………….………14 1.3 Division site selection in S. pombe …………………………………… ……… 16 1.4 Closed mitosis ………………………………………………………… .………18 1.4.1 Spindle pole body ………………………………………………… .…… 18 1.4.2 The SPB anchorage and the NE remodeling during closed mitosis …… .19 1.4.3 Nuclear membrane expansion during closed mitosis ………… ………….20 1.5 Objectives …………………………………………………………… .… .……23 CHAPTER II: Materials and Methods …………………………………… ……26 2.1 Strains, reagents and genetic methods ……………………………… .…………26 2.1.1 Schizosaccharomyces pombe strains ……………… ……………… ……26 iv 2.1.2 Media and growth conditions …………… .………………………………34 2.1.3 Enzymes, antibodies and drugs ……………………………………………35 2.2 Molecular methods ……………………………………… ………………… …36 2.2.1 Recombinant DNA techniques .36 2.2.2 LiAc transformation of S. pombe .36 2.2.3 Extraction of S. pombe genomic DNA ……………………….…… .…….37 2.3 High-copy suppressor screening for cut11-6 …………………………………….37 2.4 Construction of S. pombe strains …………… .…………………………………38 2.4.1 Designs of artificial constructs …………… ……………………… .……38 2.4.2 Construction of knock out mutants …………… …………………… .….39 2.4.3 Epitope tagging of genes …………………… ………………………… 39 2.4.4 Generation of tts1 mutants …………………… ………………………….40 2.4.5 Generation of cells over-expressing Tts1 ……………………………… 40 2.5 Biochemistry ………………………………………………… ………… .……41 2.5.1 TCA protein precipitation ……………………………………… …… …41 2.5.2 Yeast total protein extraction …………………………… .………………41 2.5.3 Immunoprecipitation …………………………………… …………….….42 2.5.4 Western Blot ……………………………………………… .……….…….43 2.5.5 TAP affinity purification …………………………………… ……………44 2.6 Cell biology and microscopy …………………………… .…………….……….47 2.6.1 Generation of S. pombe spheroplasts …………………… ……….………47 2.6.2 Immunofluorescence staining ……………………………………….…… 47 2.6.3 Epifluorescent microscopy ………………… .……………………………48 2.6.4 Scanning confocal microscopy …………………………… .…………… 49 2.6.5 Time-lapse fluorescent microscopy ………………………………….…….49 2.7 Image analysis ………………………………………… .………………… … 50 2.7.1 3D rendering ……………………………………… .…………………… 50 2.7.2 Quantification of the cortical tubular ER domains ……… .……… .…….50 2.7.3 Mid1p-GFP intensity profiling ………………………… .……………… 51 2.7.4 Quantification of the specificity of Tts1 mutants in the tubular ER localization ………………………………………………………………………51 CHAPTER III: Results ……………………………….………………… .………52 3.1 Roles of the actin cytoskeleton in structuring the cortical ER in S. pombe 52 v 3.1.1 Extension of the cortical ER to the growing cell tips requires actin cables and type V myosins ……………………………………………………….…… 52 3.1.2 VAP proteins are required for attaching the cortical ER to the PM in the fission yeast ……………………………………………………………… .……57 3.1.3 The cortical ER is a compartmentalized network of sheets and tubules … 61 3.1.3.1 The tubular ER is accumulated at the mitotic cell equator … .……61 3.1.3.2 The equatorial accumulation of the tubular ER depends on the actomyosin ring and Mid1 during mitosis ………………… .…………….65 3.2 The ER-PM contacts necessitate the reticulated ER morphology ………………70 3.2.1 Tts1, Rtn1 and Yop1 function to maintain the reticular ER network … .…70 3.2.1.1 Tts1, Rtn1 and Yop1 form a complex at the tubular ER domains…70 3.2.1.2 Tts1, Rtn1 and Yop1 sustain the tubular ER domains …………….73 3.2.2 Cells defective in the cortical ER structure fail to position the division s…78 3.2.2.1 Cells lacking Tts1, Rtn1 and Yop1 have defects in division site positioning ……………………………… .……………………….………78 3.2.2.2 Mid1 spreads in mitotic cells lacking Tts1, Rtn1 and Yop1 ………82 3.2.3 The PM-attached ER shields the PM inner surface from cortical complexes…………………………………………… .…………………………87 3.2.3.1 Cortical Mid1 nodes localize in between the ER elements……… .87 3.2.3.2 Mid1 nodes are restricted at the equatorial cortex when the ER-PM contacts are abolished …………………………………………………… .91 3.2.3.3 The artificial ER-PM tethers restore the ER-PM contacts in cells lacking VAPs …………………………………………………… ……… 95 3.2.3.4 The cortical ER obstructs the PM recruitment of peripheral complexes .98 3.3 Functional analysis of the novel ER shaping protein Tts1 …………………….102 3.3.1 Identification of Tts1 …………………………………………………… 102 3.3.2 Tts1 assists Cut11 to anchor the mitotic SPB in the nuclear envelope … 102 3.3.3 Tts1 is required for structuring the mitotic nuclear envelope ……… …107 3.3.4 The NE expansion is necessary for closed mitosis ……………… … .…110 3.3.5 Domain analysis of Tts1 ………………………………………….………114 3.3.5.1 Conserved motifs of Tts1 are required for its tubular ER localization .114 3.3.5.2 Functional motifs responsible for two separate roles of Tts1 … 118 vi CHAPTER IV: Discussion ……………………………… .………………… .…122 4.1 The cortical ER expansion during cell growth …………………………………122 4.2 Roles of VAP proteins in S. pombe ……………………………………….… 123 4.3 The cortical ER-PM contacts and division site selection in fission yeast … ….124 4.4 Roles of Tts1 and reticulons in shaping the ER membranes in fission yeast … 126 4.5 The mitotic SPB insertion and the mitotic NE remodeling ……….……………127 4.6 Closed mitosis and the nuclear membrane expansion ………………………….129 4.7 Conclusions and perspectives ………………………………………………… 131 REFERECES 134 APPENDICES 150 vii SUMMARY The largest cellular organelle, the endoplasmic reticulum (ER), is shaped as an interconnected network of sheets and tubules. The functional significance of the characteristic shape of the ER remains unclear. The cytoskeletal systems, namely actin and microtubule, have been suggested to structure and distribute the ER membranes. To investigate the complex interplay between the ER and the cytoskeleton may shed light on the functionality of the specific ER morphologies. In the rod-shaped fission yeast Schizosaccharomyces pombe (S. pombe), the ER consists of the sheet-like nuclear envelope (NE) and the cortical ER that forms an intricate network tightly apposing to the plasma membrane (PM). In this work, I show that the type V myosins and actin cables effectively transport the cortical ER into the growing S. pombe cell tips. Moreover, the ER is tethered to the lateral cell cortex by the highly conserved vesicle-associated membrane protein-associated (VAP) proteins Scs2 and Scs22. I further demonstrate that the cortical ER network is maintained by a set of three membrane proteins: reticulon/Rtn1, DP1/Yop1 and a newly identified evolutionarily conserved protein Tts1. In the absence of the ER tubulating proteins, the ER network structure is lost. As a result, the large ER cisternae physically shield the PM preventing the recruitment of the key division site regulator Mid1 and actomyosin ring assembly at the equatorial cortex. Strikingly, the detachment of the ER from the PM alleviates the division site positioning defects in cells with impaired ER architecture. We thus propose that in cells with prominent ER-PM contacts, fine reticulation of the ER network allows to establish sufficient well-distributed plasma membrane surfaces accessible for binding of peripheral protein complexes. viii S. pombe undergoes a so-called “closed mitosis” where the NE–tethered spindle pole bodies (SPBs) organize the spindle assembly within an intact NE. The NE remodels to allow the insertion and extrusion of the mitotic SPBs in the NE, and increases its surface area by 30% during mitosis. Interestingly, when we restricted the membrane availability prior to mitosis, cells broke the elongating spindles and failed nuclear division. Therefore, it appears that the NE expansion is necessary for the closed mode of mitosis. From a genetic screen for modulators of the nucleoporin Cut11 function in the SPB/NE anchorage, I identified the novel ER shaping protein Tts1. I have shown that Tts1 also functions to assist Cut11 to anchor the mitotic SPBs in the NE and to sustain the structure of the dividing NE. I have generated a set of Tts1 mutant variants and found the motifs important for Tts1 functions in the ER shaping and the mitotic SPB anchorage. Taken together, I believe that my studies provide interesting insights into the interplay between the cortical ER and the actin, and between the NE and the spindle microtubules. Importantly, I have provided novel evidence that attributes a specific physiological function to the reticulated morphology of the cortical ER. Key words: S. pombe, endoplasmic reticulum, ER-PM contacts, VAPs, reticulon, DP1/Yop1, Tts1, Mid1, actomyosin ring, division site selection, closed mitosis, SPB ix LIST OF FIGURES Figure 3.1.1 Efficient recruitment of the cortical ER to interphase cell tips 56 is dependent on intact actin cables and type V myosins. Figure 3.1.2 The VAP proteins Scs2 and Scs22 link the ER to the lateral 60 cortex in S. pombe. Figure 3.1.3.1 Rtn1, Yop1 and Tts1 co-localize in the cortical ER and 64 accumulate at the mitotic cell equator. Figure 3.1.3.2 Accumulation of the tubular ER at the mitotic cell equator 69 depends on the actomyosin ring and Mid1. Figure 3.2.1.1 Rtn1, Yop1 and Tts1 are physically associated with each other. 72 Figure 3.2.1.2 Rtn1, Yop1 and Tts1 maintain the tubular structures in the 76 cortical ER. 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PLoS Biol 5, e170 149 APPENDICES Publications as listed on page xii. 150 [...]... recruitment of the ER tubules onto the segregated chromosomes, followed by the ER flattening and resealing (Anderson and Hetzer, 2007, 2008) However, the removal of reticulons and DP1/Yop1, and the resulting flattening of the ER membrane did not lead to large scale defects in the NE reformation (Anderson and Hetzer, 2008) Therefore, the data on roles of ER tubules in NE breakdown and reformation remain... segregation of the genetic material Interestingly, the actomyosin ring does not undergo constriction until the completion of nuclear division This is in contrast to the situation in animal cells where actomyosin furrows assemble and immediately constrict following chromosome segregation (Schroeder, 1990) Septation Initiation Network (SIN), a pathway of kinase cascade triggers contraction of the ring... model to study ER-cytoskeleton interactions Following sections provide an overview of the structure and function of the ER and the actin cytoskeleton The last two sections further review closed mitosis and cytokinesis in fission yeast where the interactions between the ER and the cytoskeletal structures appear to play a crucial role 1.1 Endoplasmic reticulum 1.1.1 Architecture of the endoplasmic reticulum... polarized cellular environment such as neuron cells Moreover, a recent observation that ER tubules contact the fission sites at the mitochondria may also implicate network morphology and remodeling of the tubular ER in regulating the mitochondrial division (Friedman et al., 2011) Hence another promising direction in functional study of the tubular ER might be focusing on its interaction with other organelles... in yeast is relatively simple and well-defined Furthermore, components that could contribute to the ER structure and the generation of the ER-associated membrane contacts are mostly conserved in evolution Therefore, yeast has been recognized as one of the popular unicellular models to study the ER architecture and function In particular, the pronounced ER-PM association makes yeast a great system to... elaborate architecture and multiple functions It consists of the sheet-like nuclear envelope, and the 2 peripheral ER which is morphologically divided into the flat cisternae and a polygonal network of membrane tubules (reviewed in Voeltz et al., 2002) These membrane sheets and tubules are interconnected and share a common continuous lumen (Terasaki et al., 1996) Based on its association with ribosomes,... regulation of ring assembly is based on the nuclear export of Mid1 and the nuclear positioning in preceding interphase Remarkably, Mid1 is loaded on the cortex as a band of a limited width, which has been proposed to be critical for ensuring assembly of a single ring (Vavylonis et al., 2008) What restricts the size of this region remains unclear The limited size could be a physical outcome of Mid1 diffusion... al., 1998) In fission yeast, simultaneous disruption of actin transport and the exocyst function leads to a complete loss of cell polarity (Bendezu and Martin, 2010) On the other hand, the actomyosin-based transport also participates in the polarization of the Trans-Golgi network (TGN) (Santiago-Tirado et al., 2011) and the exocyst components (Bendezu et al., 2012) which could contribute to the localized... insertion into the NE (Lau et al., 2004) Unlike in budding yeast, the fission yeast SPBs undergo a cycle of insertion and extrusion during mitosis, spending most of the cell cycle at outer side of the NE and settling into the NE briefly during mitosis (Ding et al., 1997) The NE fenestration and SPB insertion are well coordinated and a tight nucleocytoplasmic barrier is maintained throughout mitosis (Gonzalez... the depletion of Rtn1, Rtn2 and Yop1 causing the conversion of most 5 ER tubules into sheets (Voeltz et al., 2006), does not affect the normal cell growth Cumulatively, the physiological significance of the tubular morphology in the ER system remains unclear Cellular polarization is the basis underlying cell-cell communications and responses in multi-cellular organisms Indeed, depletion of tubulating . overview of the structure and function of the ER and the actin cytoskeleton. The last two sections further review closed mitosis and cytokinesis in fission yeast where the interactions between. recruitment of the ER tubules onto the segregated chromosomes, followed by the ER flattening and resealing (Anderson and Hetzer, 2007, 2008). However, the removal of reticulons and DP1/Yop1, and the. Yue and Dr. Thirumaran Thanabalu for the time and efforts they put for my thesis work and their helpful suggestions on my studies. Several people have contributed to the completion of this