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. Figure 3.2.2.1 Cells lacking Rtn1, Yop1 or Tts1 fail to position the division 81 sites in S. pombe. Figure 3.2.2.2 Mid1 disperses along the lateral cortex of cells lacking Tts1, 85 Rtn1 and Yop1. Figure 3.2.3.1 Mid1 nodes localize in between the ER elements during the 90 actomyosin ring compaction. Figure 3.2.3.2 Mid1 nodes are restricted at the equatorial cortex when the 94 ER-PM contacts are abolished. Figure 3.2.3.3 The artificial ER-PM tethers restore the cortical ER contacts 97 in cells lacking VAPs. x Bahler, J., and Pringle, J.R. (1998). Pom1p, a fission yeast protein kinase that provides positional information for both polarized growth and cytokinesis. Genes Dev 12, 1356-1370. Bahler, J., Steever, A.B., Wheatley, S., Wang, Y., Pringle, J.R., Gould, K.L., and McCollum, D. (1998). Role of polo kinase and Mid1p in determining the site of cell division in fission yeast. The Journal of cell biology 143, 1603-1616. Bendezu, F.O., and Martin, S.G. (2010). Actin cables and the exocyst form two independent morphogenesis pathways in the fission yeast. Mol Biol Cell 22, 44-53. Bendezu, F.O., Vincenzetti, V., and Martin, S.G. (2012). Fission yeast Sec3 and Exo70 are transported on actin cables and localize the exocyst complex to cell poles. PLoS One 7, e40248. Blanchette-Mackie, E.J., Dwyer, N.K., Barber, T., Coxey, R.A., Takeda, T., Rondinone, C.M., Theodorakis, J.L., Greenberg, A.S., and Londos, C. (1995). Perilipin is located on the surface layer of intracellular lipid droplets in adipocytes. J Lipid Res 36, 1211-1226. Boettcher, B., Marquez-Lago, T.T., Bayer, M., Weiss, E.L., and Barral, Y. (2012). Nuclear envelope morphology constrains diffusion and promotes asymmetric protein segregation in closed mitosis. The Journal of cell biology 197, 921-937. Bridge, A.J., Morphew, M., Bartlett, R., and Hagan, I.M. (1998). The fission yeast SPB component Cut12 links bipolar spindle formation to mitotic control. Genes Dev 12, 927-942. Bullitt, E., Rout, M.P., Kilmartin, J.V., and Akey, C.W. (1997). The yeast spindle pole body is assembled around a central crystal of Spc42p. Cell 89, 1077-1086. Byers, B., and Goetsch, L. (1975). Behavior of spindles and spindle plaques in the cell cycle and conjugation of Saccharomyces cerevisiae. J Bacteriol 124, 511-523. Campbell, J.L., Lorenz, A., Witkin, K.L., Hays, T., Loidl, J., and Cohen-Fix, O. (2006). Yeast nuclear envelope subdomains with distinct abilities to resist membrane expansion. Molecular biology of the cell 17, 1768-1778. Carman, G.M., and Han, G.S. (2006). Roles of phosphatidate phosphatase enzymes in lipid metabolism. Trends Biochem Sci 31, 694-699. Carnahan, R.H., and Gould, K.L. (2003). The PCH family protein, Cdc15p, recruits two F-actin nucleation pathways to coordinate cytokinetic actin ring formation in Schizosaccharomyces pombe. J Cell Biol 162, 851-862. 135 Castagnetti, S., Oliferenko, S., and Nurse, P. (2010). Fission yeast cells undergo nuclear division in the absence of spindle microtubules. PLoS biology 8, e1000512. Celton-Morizur, S., Bordes, N., Fraisier, V., Tran, P.T., and Paoletti, A. (2004). C-terminal anchoring of mid1p to membranes stabilizes cytokinetic ring position in early mitosis in fission yeast. Molecular and cellular biology 24, 10621-10635. Celton-Morizur, S., Racine, V., Sibarita, J.B., and Paoletti, A. (2006). Pom1 kinase links division plane position to cell polarity by regulating Mid1p cortical distribution. Journal of cell science 119, 4710-4718. Chang, F., Drubin, D., and Nurse, P. (1997). cdc12p, a protein required for cytokinesis in fission yeast, is a component of the cell division ring and interacts with profilin. J Cell Biol 137, 169-182. Chang, F., Woollard, A., and Nurse, P. (1996). Isolation and characterization of fission yeast mutants defective in the assembly and placement of the contractile actin ring. J Cell Sci 109 ( Pt 1), 131-142. Chernomordik, L.V., and Kozlov, M.M. (2008). Mechanics of membrane fusion. Nat Struct Mol Biol 15, 675-683. Choi, H.S., Su, W.M., Morgan, J.M., Han, G.S., Xu, Z., Karanasios, E., Siniossoglou, S., and Carman, G.M. (2011). Phosphorylation of phosphatidate phosphatase regulates its membrane association and physiological functions in Saccharomyces cerevisiae: identification of SER(602), THR(723), AND SER(744) as the sites phosphorylated by CDC28 (CDK1)-encoded cyclin-dependent kinase. The Journal of biological chemistry 286, 1486-1498. Cui, Z., Vance, J.E., Chen, M.H., Voelker, D.R., and Vance, D.E. (1993). Cloning and expression of a novel phosphatidylethanolamine N-methyltransferase. A specific biochemical and cytological marker for a unique membrane fraction in rat liver. J Biol Chem 268, 16655-16663. D'Souza V, M., Naqvi, N.I., Wang, H., and Balasubramanian, M.K. (2001). Interactions of Cdc4p, a myosin light chain, with IQ-domain containing proteins in Schizosaccharomyces pombe. Cell Struct Funct 26, 555-565. Daga, R.R., and Chang, F. (2005). Dynamic positioning of the fission yeast cell division plane. Proc Natl Acad Sci U S A 102, 8228-8232. Dawson, T.R., Lazarus, M.D., Hetzer, M.W., and Wente, S.R. (2009). ER membrane-bending proteins are necessary for de novo nuclear pore formation. J Cell 136 Biol 184, 659-675. de Brito, O.M., and Scorrano, L. (2008). Mitofusin tethers endoplasmic reticulum to mitochondria. Nature 456, 605-610. De Craene, J.O., Coleman, J., Estrada de Martin, P., Pypaert, M., Anderson, S., Yates, J.R., 3rd, Ferro-Novick, S., and Novick, P. (2006). Rtn1p is involved in structuring the cortical endoplasmic reticulum. Mol Biol Cell 17, 3009-3020. Decottignies, A., Zarzov, P., and Nurse, P. (2001). In vivo localisation of fission yeast cyclin-dependent kinase cdc2p and cyclin B cdc13p during mitosis and meiosis. Journal of cell science 114, 2627-2640. Ding, R., West, R.R., Morphew, D.M., Oakley, B.R., and McIntosh, J.R. (1997). The spindle pole body of Schizosaccharomyces pombe enters and leaves the nuclear envelope as the cell cycle proceeds. Molecular biology of the cell 8, 1461-1479. Eng, K., Naqvi, N.I., Wong, K.C., and Balasubramanian, M.K. (1998). Rng2p, a protein required for cytokinesis in fission yeast, is a component of the actomyosin ring and the spindle pole body. Curr Biol 8, 611-621. Ercan, E., Momburg, F., Engel, U., Temmerman, K., Nickel, W., and Seedorf, M. (2009). A conserved, lipid-mediated sorting mechanism of yeast Ist2 and mammalian STIM proteins to the peripheral ER. Traffic 10, 1802-1818. Estrada, P., Kim, J., Coleman, J., Walker, L., Dunn, B., Takizawa, P., Novick, P., and Ferro-Novick, S. (2003). Myo4p and She3p are required for cortical ER inheritance in Saccharomyces cerevisiae. J Cell Biol 163, 1255-1266. Fankhauser, C., Reymond, A., Cerutti, L., Utzig, S., Hofmann, K., and Simanis, V. (1995). The S. pombe cdc15 gene is a key element in the reorganization of F-actin at mitosis. Cell 82, 435-444. Fawcett, D.W. (1981). The Cell, 2nd Edn (Philadelphia, W.B. Saunders Company). Feierbach, B., and Chang, F. (2001). Roles of the fission yeast formin for3p in cell polarity, actin cable formation and symmetric cell division. Curr Biol 11, 1656-1665. Fischer, R., Zekert, N., and Takeshita, N. (2008). Polarized growth in fungi--interplay between the cytoskeleton, positional markers and membrane domains. Mol Microbiol 68, 813-826. Flory, M.R., Morphew, M., Joseph, J.D., Means, A.R., and Davis, T.N. (2002). Pcp1p, an Spc110p-related calmodulin target at the centrosome of the fission yeast 137 Schizosaccharomyces pombe. Cell Growth Differ 13, 47-58. Franke, W.W., and Kartenbeck, J. (1971). Outer mitochondrial membrane continuous with endoplasmic reticulum. Protoplasma 73, 35-41. Friederichs, J.M., Ghosh, S., Smoyer, C.J., McCroskey, S., Miller, B.D., Weaver, K.J., Delventhal, K.M., Unruh, J., Slaughter, B.D., and Jaspersen, S.L. (2011). The SUN protein Mps3 is required for spindle pole body insertion into the nuclear membrane and nuclear envelope homeostasis. PLoS Genet 7, e1002365. Friedman, J.R., Lackner, L.L., West, M., DiBenedetto, J.R., Nunnari, J., and Voeltz, G.K. (2011). ER tubules mark sites of mitochondrial division. Science 334, 358-362. Gachet, Y., and Hyams, J.S. (2005). Endocytosis in fission yeast is spatially associated with the actin cytoskeleton during polarised cell growth and cytokinesis. J Cell Sci 118, 4231-4242. Galletta, B.J., and Cooper, J.A. (2009). Actin and endocytosis: mechanisms and phylogeny. Curr Opin Cell Biol 21, 20-27. Gautier, R., Douguet, D., Antonny, B., and Drin, G. (2008). HELIQUEST: a web server to screen sequences with specific alpha-helical properties. Bioinformatics 24, 2101-2102. Gehlen, L.R., Nagai, S., Shimada, K., Meister, P., Taddei, A., and Gasser, S.M. (2011). Nuclear geometry and rapid mitosis ensure asymmetric episome segregation in yeast. Current biology : CB 21, 25-33. Gonzalez, Y., Meerbrey, K., Chong, J., Torii, Y., Padte, N.N., and Sazer, S. (2009). Nuclear shape, growth and integrity in the closed mitosis of fission yeast depend on the Ran-GTPase system, the spindle pole body and the endoplasmic reticulum. Journal of cell science 122, 2464-2472. Grallert, A., Martin-Garcia, R., Bagley, S., and Mulvihill, D.P. (2007). In vivo movement of the type V myosin Myo52 requires dimerisation but is independent of the neck domain. J Cell Sci 120, 4093-4098. Hachet, O., and Simanis, V. (2008). Mid1p/anillin and the septation initiation network orchestrate contractile ring assembly for cytokinesis. Genes Dev 22, 3205-3216. Hagan, I.M. (2008). The spindle pole body plays a key role in controlling mitotic commitment in the fission yeast Schizosaccharomyces pombe. Biochemical Society transactions 36, 1097-1101. 138 Han, G.S., Wu, W.I., and Carman, G.M. (2006). The Saccharomyces cerevisiae Lipin homolog is a Mg2+-dependent phosphatidate phosphatase enzyme. The Journal of biological chemistry 281, 9210-9218. He, W., Lu, Y., Qahwash, I., Hu, X.Y., Chang, A., and Yan, R. (2004). Reticulon family members modulate BACE1 activity and amyloid-beta peptide generation. Nat Med 10, 959-965. Hodge, C.A., Choudhary, V., Wolyniak, M.J., Scarcelli, J.J., Schneiter, R., and Cole, C.N. (2010). Integral membrane proteins Brr6 and Apq12 link assembly of the nuclear pore complex to lipid homeostasis in the endoplasmic reticulum. Journal of cell science 123, 141-151. Hu, J., Shibata, Y., Voss, C., Shemesh, T., Li, Z., Coughlin, M., Kozlov, M.M., Rapoport, T.A., and Prinz, W.A. (2008). Membrane proteins of the endoplasmic reticulum induce high-curvature tubules. Science (New York, NY 319, 1247-1250. Hu, J., Shibata, Y., Zhu, P.P., Voss, C., Rismanchi, N., Prinz, W.A., Rapoport, T.A., and Blackstone, C. (2009). A class of dynamin-like GTPases involved in the generation of the tubular ER network. Cell 138, 549-561. Huang, Y., Chew, T.G., Ge, W., and Balasubramanian, M.K. (2007). Polarity determinants Tea1p, Tea4p, and Pom1p inhibit division-septum assembly at cell ends in fission yeast. Dev Cell 12, 987-996. Huang, Y., Yan, H., and Balasubramanian, M.K. (2008). Assembly of normal actomyosin rings in the absence of Mid1p and cortical nodes in fission yeast. J Cell Biol 183, 979-988. Jacquier, N., Choudhary, V., Mari, M., Toulmay, A., Reggiori, F., and Schneiter, R. (2011). Lipid droplets are functionally connected to the endoplasmic reticulum in Saccharomyces cerevisiae. J Cell Sci 124, 2424-2437. Jaspersen, S.L., and Ghosh, S. (2012). Nuclear envelope insertion of spindle pole bodies and nuclear pore complexes. Nucleus 3, 226-236. Kagiwada, S., and Hashimoto, M. (2007). The yeast VAP homolog Scs2p has a phosphoinositide-binding ability that is correlated with its activity. Biochem Biophys Res Commun 364, 870-876. Kagiwada, S., and Zen, R. (2003). Role of the yeast VAP homolog, Scs2p, in INO1 expression and phospholipid metabolism. J Biochem 133, 515-522. Kaiser, S.E., Brickner, J.H., Reilein, A.R., Fenn, T.D., Walter, P., and Brunger, A.T. 139 (2005). Structural basis of FFAT motif-mediated ER targeting. Structure 13, 1035-1045. Kaksonen, M., Sun, Y., and Drubin, D.G. (2003). A pathway for association of receptors, adaptors, and actin during endocytic internalization. Cell 115, 475-487. Kamasaki, T., Osumi, M., and Mabuchi, I. (2007). Three-dimensional arrangement of F-actin in the contractile ring of fission yeast. J Cell Biol 178, 765-771. Karanasios, E., Han, G.S., Xu, Z., Carman, G.M., and Siniossoglou, S. (2010). A phosphorylation-regulated amphipathic helix controls the membrane translocation and function of the yeast phosphatidate phosphatase. Proceedings of the National Academy of Sciences of the United States of America 107, 17539-17544. Keeney, J.B., and Boeke, J.D. (1994). Efficient targeted integration at leu1-32 and ura4-294 in Schizosaccharomyces pombe. Genetics 136, 849-856. Kim, Y., Gentry, M.S., Harris, T.E., Wiley, S.E., Lawrence, J.C., Jr., and Dixon, J.E. (2007). A conserved phosphatase cascade that regulates nuclear membrane biogenesis. Proc Natl Acad Sci U S A 104, 6596-6601. Kitayama, C., Sugimoto, A., and Yamamoto, M. (1997). Type II myosin heavy chain encoded by the myo2 gene composes the contractile ring during cytokinesis in Schizosaccharomyces pombe. J Cell Biol 137, 1309-1319. Kornmann, B., Currie, E., Collins, S.R., Schuldiner, M., Nunnari, J., Weissman, J.S., and Walter, P. (2009). An ER-mitochondria tethering complex revealed by a synthetic biology screen. Science 325, 477-481. Krapp, A., Gulli, M.P., and Simanis, V. (2004). SIN and the art of splitting the fission yeast cell. Curr Biol 14, R722-730. Kuerschner, L., Moessinger, C., and Thiele, C. (2008). Imaging of lipid biosynthesis: how a neutral lipid enters lipid droplets. Traffic 9, 338-352. Kupke, T., Di Cecco, L., Muller, H.M., Neuner, A., Adolf, F., Wieland, F., Nickel, W., and Schiebel, E. (2011). Targeting of Nbp1 to the inner nuclear membrane is essential for spindle pole body duplication. The EMBO journal 30, 3337-3352. Lau, C.K., Giddings, T.H., Jr., and Winey, M. (2004). A novel allele of Saccharomyces cerevisiae NDC1 reveals a potential role for the spindle pole body component Ndc1p in nuclear pore assembly. Eukaryotic cell 3, 447-458. Lee, I.J., Coffman, V.C., and Wu, J.Q. (2012). Contractile-ring assembly in fission 140 yeast cytokinesis: Recent advances and new perspectives. Cytoskeleton (Hoboken) 69, 751-76. Le Goff, X., Motegi, F., Salimova, E., Mabuchi, I., and Simanis, V. (2000). The S. pombe rlc1 gene encodes a putative myosin regulatory light chain that binds the type II myosins myo3p and myo2p. J Cell Sci 113 Pt 23, 4157-4163. Lev, S., Ben Halevy, D., Peretti, D., and Dahan, N. (2008). The VAP protein family: from cellular functions to motor neuron disease. Trends Cell Biol 18, 282-290. Levine, T.P., and Munro, S. (2001). Dual targeting of Osh1p, a yeast homologue of oxysterol-binding protein, to both the Golgi and the nucleus-vacuole junction. Mol Biol Cell 12, 1633-1644. Lim, H.W.G., Huber, G., Torii, Y., Hirata, A., Miller, J., and Sazer, S. (2007). Vesicle-like biomechanics governs important aspects of nuclear geometry in fission yeast. PLoS One 2, e948. Lipschutz, J.H., and Mostov, K.E. (2002). Exocytosis: the many masters of the exocyst. Curr Biol 12, R212-214. Loewen, C.J., Gaspar, M.L., Jesch, S.A., Delon, C., Ktistakis, N.T., Henry, S.A., and Levine, T.P. (2004). Phospholipid metabolism regulated by a transcription factor sensing phosphatidic acid. Science 304, 1644-1647. Loewen, C.J., Roy, A., and Levine, T.P. (2003). A conserved ER targeting motif in three families of lipid binding proteins and in Opi1p binds VAP. EMBO J 22, 2025-2035. Loewen, C.J., Young, B.P., Tavassoli, S., and Levine, T.P. (2007). Inheritance of cortical ER in yeast is required for normal septin organization. J Cell Biol 179, 467-483. Lord, M., and Pollard, T.D. (2004). UCS protein Rng3p activates actin filament gliding by fission yeast myosin-II. J Cell Biol 167, 315-325. Marks, J., Hagan, I.M., and Hyams, J.S. (1986). Growth polarity and cytokinesis in fission yeast: the role of the cytoskeleton. J Cell Sci Suppl 5, 229-241. Martin, S.G., and Berthelot-Grosjean, M. (2009). Polar gradients of the DYRK-family kinase Pom1 couple cell length with the cell cycle. Nature 459, 852-856. Martin, S.G., and Chang, F. (2005). New end take off: regulating cell polarity during the fission yeast cell cycle. Cell Cycle 4, 1046-1049. 141 Martin, S.G., and Chang, F. (2006). Dynamics of the formin for3p in actin cable assembly. Curr Biol 16, 1161-1170. Mata, J., and Nurse, P. (1997). tea1 and the microtubular cytoskeleton are important for generating global spatial order within the fission yeast cell. Cell 89, 939-949. Maundrell, K. (1990). nmt1 of fission yeast. A highly transcribed gene completely repressed by thiamine. J Biol Chem 265, 10857-10864. McCollum, D., Balasubramanian, M.K., Pelcher, L.E., Hemmingsen, S.M., and Gould, K.L. (1995). Schizosaccharomyces pombe cdc4+ gene encodes a novel EF-hand protein essential for cytokinesis. The Journal of cell biology 130, 651-660. Moreno, S., Klar, A., and Nurse, P. (1991). Molecular genetic analysis of fission yeast Schizosaccharomyces pombe. Methods Enzymol 194, 795-823. Morre, D.J., Merritt, W.D., and Lembi, C.A. (1971). Connections between mitochondria and endoplasmic reticulum in rat liver and onion stem. Protoplasma 73, 43-49. Moseley, J.B., and Goode, B.L. (2006). The yeast actin cytoskeleton: from cellular function to biochemical mechanism. Microbiol Mol Biol Rev 70, 605-645. Moseley, J.B., Mayeux, A., Paoletti, A., and Nurse, P. (2009). A spatial gradient coordinates cell size and mitotic entry in fission yeast. Nature 459, 857-860. Motegi, F., Arai, R., and Mabuchi, I. (2001). Identification of two type V myosins in fission yeast, one of which functions in polarized cell growth and moves rapidly in the cell. Mol Biol Cell 12, 1367-1380. Mulholland, J., Preuss, D., Moon, A., Wong, A., Drubin, D., and Botstein, D. (1994). Ultrastructure of the yeast actin cytoskeleton and its association with the plasma membrane. J Cell Biol 125, 381-391. Mulvihill, D.P., Petersen, J., Ohkura, H., Glover, D.M., and Hagan, I.M. (1999). Plo1 kinase recruitment to the spindle pole body and its role in cell division in Schizosaccharomyces pombe. Molecular biology of the cell 10, 2771-2785. Nakano, K., Imai, J., Arai, R., Toh, E.A., Matsui, Y., and Mabuchi, I. (2002). The small GTPase Rho3 and the diaphanous/formin For3 function in polarized cell growth in fission yeast. J Cell Sci 115, 4629-4639. O'Hara, L., Han, G.S., Peak-Chew, S., Grimsey, N., Carman, G.M., and Siniossoglou, 142 S. (2006). Control of phospholipid synthesis by phosphorylation of the yeast lipin Pah1p/Smp2p Mg2+-dependent phosphatidate phosphatase. The Journal of biological chemistry 281, 34537-34548. O'Toole, E.T., Winey, M., and McIntosh, J.R. (1999). High-voltage electron tomography of spindle pole bodies and early mitotic spindles in the yeast Saccharomyces cerevisiae. Mol Biol Cell 10, 2017-2031. Ogata, T., and Yamasaki, Y. (1997). Ultra-high-resolution scanning electron microscopy of mitochondria and sarcoplasmic reticulum arrangement in human red, white, and intermediate muscle fibers. Anat Rec 248, 214-223. Ohashi, M., Mizushima, N., Kabeya, Y., and Yoshimori, T. (2003). Localization of mammalian NAD(P)H steroid dehydrogenase-like protein on lipid droplets. J Biol Chem 278, 36819-36829. Ohsaki, Y., Cheng, J., Suzuki, M., Shinohara, Y., Fujita, A., and Fujimoto, T. (2009). Biogenesis of cytoplasmic lipid droplets: from the lipid ester globule in the membrane to the visible structure. Biochim Biophys Acta 1791, 399-407. Padte, N.N., Martin, S.G., Howard, M., and Chang, F. (2006). The cell-end factor pom1p inhibits mid1p in specification of the cell division plane in fission yeast. Curr Biol 16, 2480-2487. Paoletti, A., and Chang, F. (2000). Analysis of mid1p, a protein required for placement of the cell division site, reveals a link between the nucleus and the cell surface in fission yeast. Mol Biol Cell 11, 2757-2773. Pelham, R.J., Jr., and Chang, F. (2001). Role of actin polymerization and actin cables in actin-patch movement in Schizosaccharomyces pombe. Nat Cell Biol 3, 235-244. Pendin, D., McNew, J.A., and Daga, A. (2011). Balancing ER dynamics: shaping, bending, severing, and mending membranes. Curr Opin Cell Biol 23, 435-442. Peretti, D., Dahan, N., Shimoni, E., Hirschberg, K., and Lev, S. (2008). Coordinated lipid transfer between the endoplasmic reticulum and the Golgi complex requires the VAP proteins and is essential for Golgi-mediated transport. Mol Biol Cell 19, 3871-3884. Phillips, R., Ursell, T., Wiggins, P., and Sens, P. (2009). Emerging roles for lipids in shaping membrane-protein function. Nature 459, 379-385. Pichler, H., Gaigg, B., Hrastnik, C., Achleitner, G., Kohlwein, S.D., Zellnig, G., Perktold, A., and Daum, G. (2001). A subfraction of the yeast endoplasmic reticulum 143 associates with the plasma membrane and has a high capacity to synthesize lipids. Eur J Biochem 268, 2351-2361. Pidoux, A.L., and Armstrong, J. (1993). The BiP protein and the endoplasmic reticulum of Schizosaccharomyces pombe: fate of the nuclear envelope during cell division. J Cell Sci 105 ( Pt 4), 1115-1120. Prinz, W.A., Grzyb, L., Veenhuis, M., Kahana, J.A., Silver, P.A., and Rapoport, T.A. (2000). Mutants affecting the structure of the cortical endoplasmic reticulum in Saccharomyces cerevisiae. The Journal of cell biology 150, 461-474. Prior, M., Shi, Q., Hu, X., He, W., Levey, A., and Yan, R. (2010). RTN/Nogo in forming Alzheimer's neuritic plaques. Neurosci Biobehav Rev 34, 1201-1206. Pruyne, D., Legesse-Miller, A., Gao, L., Dong, Y., and Bretscher, A. (2004). Mechanisms of polarized growth and organelle segregation in yeast. Annu Rev Cell Dev Biol 20, 559-591. Pruyne, D.W., Schott, D.H., and Bretscher, A. (1998). Tropomyosin-containing actin cables direct the Myo2p-dependent polarized delivery of secretory vesicles in budding yeast. J Cell Biol 143, 1931-1945. Puhka, M., Vihinen, H., Joensuu, M., and Jokitalo, E. (2007). Endoplasmic reticulum remains continuous and undergoes sheet-to-tubule transformation during cell division in mammalian cells. J Cell Biol 179, 895-909. Reynwar, B.J., Illya, G., Harmandaris, V.A., Muller, M.M., Kremer, K., and Deserno, M. (2007). Aggregation and vesiculation of membrane proteins by curvature-mediated interactions. Nature 447, 461-464. Riedl, J., Crevenna, A.H., Kessenbrock, K., Yu, J.H., Neukirchen, D., Bista, M., Bradke, F., Jenne, D., Holak, T.A., Werb, Z., et al. (2008). Lifeact: a versatile marker to visualize F-actin. Nat Methods 5, 605-607. Rizzuto, R., Pinton, P., Carrington, W., Fay, F.S., Fogarty, K.E., Lifshitz, L.M., Tuft, R.A., and Pozzan, T. (1998). Close contacts with the endoplasmic reticulum as determinants of mitochondrial Ca2+ responses. Science 280, 1763-1766. Rizzuto, R., and Pozzan, T. (2006). Microdomains of intracellular Ca2+: molecular determinants and functional consequences. Physiol Rev 86, 369-408. Robenek, H., Hofnagel, O., Buers, I., Robenek, M.J., Troyer, D., and Severs, N.J. (2006). Adipophilin-enriched domains in the ER membrane are sites of lipid droplet biogenesis. J Cell Sci 119, 4215-4224. 144 Rodal, A.A., Kozubowski, L., Goode, B.L., Drubin, D.G., and Hartwig, J.H. (2005). Actin and septin ultrastructures at the budding yeast cell cortex. Mol Biol Cell 16, 372-384. Rusinol, A.E., Cui, Z., Chen, M.H., and Vance, J.E. (1994). A unique mitochondria-associated membrane fraction from rat liver has a high capacity for lipid synthesis and contains pre-Golgi secretory proteins including nascent lipoproteins. J Biol Chem 269, 27494-27502. Saitoh, S., Takahashi, K., Nabeshima, K., Yamashita, Y., Nakaseko, Y., Hirata, A., and Yanagida, M. (1996). Aberrant mitosis in fission yeast mutants defective in fatty acid synthetase and acetyl CoA carboxylase. The Journal of cell biology 134, 949-961. Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, 2nd Edn (New York, Cold Spring Harbor Laboratory Press). Santiago-Tirado, F.H., Legesse-Miller, A., Schott, D., and Bretscher, A. (2011). PI4P and Rab inputs collaborate in myosin-V-dependent transport of secretory compartments in yeast. Dev Cell 20, 47-59. Santos-Rosa, H., Leung, J., Grimsey, N., Peak-Chew, S., and Siniossoglou, S. (2005). The yeast lipin Smp2 couples phospholipid biosynthesis to nuclear membrane growth. The EMBO journal 24, 1931-1941. Scarcelli, J.J., Hodge, C.A., and Cole, C.N. (2007). The yeast integral membrane protein Apq12 potentially links membrane dynamics to assembly of nuclear pore complexes. The Journal of cell biology 178, 799-812. Schneider, M.F. (1994). Control of calcium release in functioning skeletal muscle fibers. Annu Rev Physiol 56, 463-484. Schneiter, R., and Cole, C.N. (2010). Integrating complex functions: coordination of nuclear pore complex assembly and membrane expansion of the nuclear envelope requires a family of integral membrane proteins. Nucleus 1, 387-392. Schramm, C., Elliott, S., Shevchenko, A., and Schiebel, E. (2000). The Bbp1p-Mps2p complex connects the SPB to the nuclear envelope and is essential for SPB duplication. The EMBO journal 19, 421-433. Shibata, Y., Voeltz, G.K., and Rapoport, T.A. (2006). Rough sheets and smooth tubules. Cell 126, 435-439. Shibata, Y., Voss, C., Rist, J.M., Hu, J., Rapoport, T.A., Prinz, W.A., and Voeltz, G.K. 145 (2008). The reticulon and DP1/Yop1p proteins form immobile oligomers in the tubular endoplasmic reticulum. The Journal of biological chemistry 283, 18892-18904. Siniossoglou, S., Santos-Rosa, H., Rappsilber, J., Mann, M., and Hurt, E. (1998). A novel complex of membrane proteins required for formation of a spherical nucleus. The EMBO journal 17, 6449-6464. Sohrmann, M., Fankhauser, C., Brodbeck, C., and Simanis, V. (1996). The dmf1/mid1 gene is essential for correct positioning of the division septum in fission yeast. Genes & development 10, 2707-2719. Sparkes, I.A., Frigerio, L., Tolley, N., and Hawes, C. (2009). The plant endoplasmic reticulum: a cell-wide web. Biochem J 423, 145-155. Sprong, H., van der Sluijs, P., and van Meer, G. (2001). How proteins move lipids and lipids move proteins. Nature reviews 2, 504-513. Stefan, C.J., Manford, A.G., Baird, D., Yamada-Hanff, J., Mao, Y., and Emr, S.D. (2011). Osh proteins regulate phosphoinositide metabolism at ER-plasma membrane contact sites. Cell 144, 389-401. Stein, O., and Stein, Y. (1967a). Lipid synthesis, intracellular transport, and secretion. II. Electron microscopic radioautographic study of the mouse lactating mammary gland. J Cell Biol 34, 251-263. Stein, O., and Stein, Y. (1967b). Lipid synthesis, intracellular transport, storage, and secretion. I. Electron microscopic radioautographic study of liver after injection of tritiated palmitate or glycerol in fasted and ethanol-treated rats. J Cell Biol 33, 319-339. Stradalova, V., Blazikova, M., Grossmann, G., Opekarova, M., Tanner, W., and Malinsky, J. (2012). Distribution of cortical endoplasmic reticulum determines positioning of endocytic events in yeast plasma membrane. PLoS One 7, e35132. Tallada, V.A., Tanaka, K., Yanagida, M., and Hagan, I.M. (2009). The S. pombe mitotic regulator Cut12 promotes spindle pole body activation and integration into the nuclear envelope. The Journal of cell biology 185, 875-888. Tamm, T., Grallert, A., Grossman, E.P., Alvarez-Tabares, I., Stevens, F.E., and Hagan, I.M. (2011). Brr6 drives the Schizosaccharomyces pombe spindle pole body nuclear envelope insertion/extrusion cycle. J Cell Biol 195, 467-484. Tange, Y., Hirata, A., and Niwa, O. (2002). An evolutionarily conserved fission yeast 146 protein, Ned1, implicated in normal nuclear morphology and chromosome stability, interacts with Dis3, Pim1/RCC1 and an essential nucleoporin. Journal of cell science 115, 4375-4385. Tatebe, H., Shimada, K., Uzawa, S., Morigasaki, S., and Shiozaki, K. (2005). Wsh3/Tea4 is a novel cell-end factor essential for bipolar distribution of Tea1 and protects cell polarity under environmental stress in S. pombe. Curr Biol 15, 1006-1015. Tauchi-Sato, K., Ozeki, S., Houjou, T., Taguchi, R., and Fujimoto, T. (2002). The surface of lipid droplets is a phospholipid monolayer with a unique Fatty Acid composition. J Biol Chem 277, 44507-44512. Terasaki, M., Jaffe, L.A., Hunnicutt, G.R., and Hammer, J.A., 3rd (1996). Structural change of the endoplasmic reticulum during fertilization: evidence for loss of membrane continuity using the green fluorescent protein. Developmental biology 179, 320-328. Thiele, C., and Spandl, J. (2008). Cell biology of lipid droplets. Curr Opin Cell Biol 20, 378-385. Ueda, H., Yokota, E., Kutsuna, N., Shimada, T., Tamura, K., Shimmen, T., Hasezawa, S., Dolja, V.V., and Hara-Nishimura, I. (2010). Myosin-dependent endoplasmic reticulum motility and F-actin organization in plant cells. Proc Natl Acad Sci U S A 107, 6894-6899. Vance, J.E. (1990). Phospholipid synthesis in a membrane fraction associated with mitochondria. J Biol Chem 265, 7248-7256. Vavylonis, D., Wu, J.Q., Hao, S., O'Shaughnessy, B., and Pollard, T.D. (2008). Assembly mechanism of the contractile ring for cytokinesis by fission yeast. Science (New York, NY 319, 97-100. Vjestica, A., Tang, X.Z., and Oliferenko, S. (2008). The actomyosin ring recruits early secretory compartments to the division site in fission yeast. Molecular biology of the cell 19, 1125-1138. Voeltz, G.K., Prinz, W.A., Shibata, Y., Rist, J.M., and Rapoport, T.A. (2006). A class of membrane proteins shaping the tubular endoplasmic reticulum. Cell 124, 573-586. Voeltz, G.K., Rolls, M.M., and Rapoport, T.A. (2002). Structural organization of the endoplasmic reticulum. EMBO reports 3, 944-950. Wachtler, V., Huang, Y., Karagiannis, J., and Balasubramanian, M.K. (2006). Cell 147 cycle-dependent roles for the FCH-domain protein Cdc15p in formation of the actomyosin ring in Schizosaccharomyces pombe. Mol Biol Cell 17, 3254-3266. Wagner, W., Brenowitz, S.D., and Hammer, J.A., 3rd (2011). Myosin-Va transports the endoplasmic reticulum into the dendritic spines of Purkinje neurons. Nat Cell Biol 13, 40-48. West, M., Zurek, N., Hoenger, A., and Voeltz, G.K. (2011). A 3D analysis of yeast ER structure reveals how ER domains are organized by membrane curvature. J Cell Biol 193, 333-346. West, R.R., Vaisberg, E.V., Ding, R., Nurse, P., and McIntosh, J.R. (1998). cut11(+): A gene required for cell cycle-dependent spindle pole body anchoring in the nuclear envelope and bipolar spindle formation in Schizosaccharomyces pombe. Molecular biology of the cell 9, 2839-2855. Win, T.Z., Gachet, Y., Mulvihill, D.P., May, K.M., and Hyams, J.S. (2001). Two type V myosins with non-overlapping functions in the fission yeast Schizosaccharomyces pombe: Myo52 is concerned with growth polarity and cytokinesis, Myo51 is a component of the cytokinetic actin ring. J Cell Sci 114, 69-79. Witkin, K.L., Chong, Y., Shao, S., Webster, M.T., Lahiri, S., Walters, A.D., Lee, B., Koh, J.L., Prinz, W.A., Andrews, B.J., et al. (2012). The Budding Yeast Nuclear Envelope Adjacent to the Nucleolus Serves as a Membrane Sink during Mitotic Delay. Current biology : CB 22, 1128-1133. Wong, K.C., D'Souza V, M., Naqvi, N.I., Motegi, F., Mabuchi, I., and Balasubramanian, M.K. (2002). Importance of a myosin II-containing progenitor for actomyosin ring assembly in fission yeast. Curr Biol 12, 724-729. Wong, K.C., Naqvi, N.I., Iino, Y., Yamamoto, M., and Balasubramanian, M.K. (2000). Fission yeast Rng3p: an UCS-domain protein that mediates myosin II assembly during cytokinesis. J Cell Sci 113 ( Pt 13), 2421-2432. Wu, J.Q., Sirotkin, V., Kovar, D.R., Lord, M., Beltzner, C.C., Kuhn, J.R., and Pollard, T.D. (2006). Assembly of the cytokinetic contractile ring from a broad band of nodes in fission yeast. The Journal of cell biology 174, 391-402. Wu, M.M., Luik, R.M., and Lewis, R.S. (2007). Some assembly required: constructing the elementary units of store-operated Ca2+ entry. Cell Calcium 42, 163-172. Yam, C., He, Y., Zhang, D., Chiam, K.H., and Oliferenko, S. (2011). Divergent strategies for controlling the nuclear membrane satisfy geometric constraints during 148 nuclear division. Current biology : CB 21, 1314-1319. Yang, Y.S., Harel, N.Y., and Strittmatter, S.M. (2009). Reticulon-4A (Nogo-A) redistributes protein disulfide isomerase to protect mice from SOD1-dependent amyotrophic lateral sclerosis. J Neurosci 29, 13850-13859. Yu, J.W., Mendrola, J.M., Audhya, A., Singh, S., Keleti, D., DeWald, D.B., Murray, D., Emr, S.D., and Lemmon, M.A. (2004). Genome-wide analysis of membrane targeting by S. cerevisiae pleckstrin homology domains. Mol Cell 13, 677-688. Zheng, L., Schwartz, C., Magidson, V., Khodjakov, A., and Oliferenko, S. (2007). The spindle pole bodies facilitate nuclear envelope division during closed mitosis in fission yeast. 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