GAB-GFP BASED SYNTHETIC STUDIES ON CYTOKINESIS AND MITOTIC ENTRY REGULATION IN FISSION YEAST YAQIONG TAO (B.Sc SICHUAN UNIVERSITY) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT SCIENCES OF BIOLOGICAL NATIONAL UNIVERSITY SINGAPORE 2014 ! i! OF 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 that have been used in the thesis This thesis has also not been submitted for any degree in any university previously _ Yaqiong Tao 16th Oct 2014 ! ii! Acknowledgement Looking back at my entire period of graduate study, I cannot but think of the wonderful people who have helped me through this journey It is their efforts that made it possible for me to reach the point of writing this manuscript I thank Dr Mohan Balasubramanian, my supervisor His encouragement and advices are invaluable to the work presented here His caring for every student under his mentorship despite changing circumstances is most honorable His strong devotion to science along with his charming personality provides a lasting inspiration and motivation for me I am grateful to my thesis committee members, Drs Cynthia He, Gregory Jedd, and Ronen Zaidel-Bar for their guidance and valuable suggestions I also own gratitude to Drs Jian-Qiu Wu, Sophie Martin, James Mosley, Dan Zhang, Ulrich Rothbauer and Agnes Grallert for sharing yeast strains and plasmids with me I am thankful to Dr Yinyi Huang, Dr Xie Tang, Dr Meredith Calvert, Dr Junqi Huang, Dr Dhivya Subramanian, Dr Anup Padmanabhan, Dr Srinivas Rmanujam, Dr Mithilesh Mishra, Mr Mayagulu Sevugan, Miss Krit Sethi, Miss Nan Shao, Mr Sachin Seshadri and all past members of the Mohan Balasubramanian group for their daily aid, discussion and friendship Special thanks to Dr Junqi Huang and Dr Yinyi Huang for ! iii! advice on my experiments and data processing, and Dr Dhivya Subramanian and Ms Kriti Sethi for their critical reading of my manuscripts The work presented here was funded by the Temasek Life Sciences Laboratory and my study was supported by the scholarship from the National University of Singapore It is my family to whom I am forever grateful for their unconditional love and support I owe the deepest gratitude to my father Qingming Tao, my mother Xingshu Hou and my partner Su Zhu ! iv! Table of Contents Title Page .i DECLARATION! !ii! Acknowledgement! !iii! Table of Contents! !v! List of Tables! !x! List of Figures ! .!xi! List of Abbreviations! ! iii! x List of Publications ! !xiv! Chapter I Introduction! !1! 1.1! Gab in cell biology studies! !4! 1.2 S pombe Cell Cycle and G2/M transition! !7! 1.2.1 S pombe as a model organism! !8! 1.2.2 S pombe cell cycle and mitotic entry! !10! 1.2.2.1 The division cell cycle! !10! 1.2.2.2 Mitotic entry! .!11! 1.2.2.3 Cell size control and G2/M transition! .!13! 1.3 S pombe Cytokinesis! !18! 1.3.1 Assembly of actomyosin ring in S pombe! !19! 1.3.1.1 The molecular basis for ring assembly! !19! 1.3.1.2 Current models regarding actomyosin ring assembly! !21! 1.3.1.3 Completion of cytokinesis! !22! 1.3.2 Division plane positioning! .!24! 1.3.2.1 Mid1p and other overlapping factors! .!24! 1.3.2.2 Nuclear site and division plane positioning! !29! 1.4 Gaps and Objectives! .!30! Chapter II Materials and Methods! !33! 2.1 Yeast strains, media and reagents! .!33! 2.1.1 Yeast strains! .!33! 2.1.2 Growth media! !37! 2.1.3 Drugs and reagents! !38! 2.2 Molecular cloning and yeast genetics! !39! 2.2.1 Molecular cloning! .!39! 2.2.1.1 PCR! .!39! 2.2.1.2 Digestion and ligation! !41! 2.2.1.3 Bacteria transformation and plasmid extraction! !41! 2.2.1.4 Plasmids constructed! .!41! 2.2.2 Yeast genetics! .!46! 2.2.2.1 Yeast strain construction! !46! ! v! 2.2.2.2 Genetic crosses! !49! 2.3 Yeast cell biology! !49! 2.3.1 Fixation and staining! !49! 2.3.2 Microscopy, image processing and data analysis! .!51! 2.3.2.1 Epifluorescence microscopy! .!51! 2.3.2.2 Spinning disk confocal microscopy! !51! 2.3.2.3 Nucleus displacement experiment! !52! 2.3.2.4 SIN inactivation experiment! .!53! 2.3.2.5 General image processing and data analysis! !53! 2.3.2.6 Fluorescence distribution plotting! !54! 2.3.2.7 Fluorescence Recovery after Photobleaching (FRAP)! !55! Chapter III Results! !58! 3.1 The Gab-GFP based protein targeting method! !58! 3.1.1 Construction of the Gab targeting system! !58! 3.1.2 Gab-GFP complex tends to favor the location of the Gab fusion! !61! 3.1.3 Analysis of the Gab fusion targeting effects! !65! 3.2 Rewiring Mid1p independent medial division in S pombe! !68! 3.2.1 Gab-GFP based protein targeting strategy is advantageous over direct protein fusion method! !69! 3.2.2 Medial targeting of Rng2p rescued mid1 mutants! .!75! 3.2.3 Medial actomyosin ring assembly does not require ring components to assemble in an invariant order! !79! 3.2.3.1 A screen for medially targeted cytokinetic proteins that rewired medial cell division! !79! 3.2.3.2 Cdc12p and Myo2p rewired nodes assembling into medial actomyosin rings! !84! 3.2.4 Rewired nodes corrected septum positioning defects in plo1 mutant ! !90! 3.2.5 Mid1p plays multiple roles in fission yeast cytokinesis! !92! 3.2.5.1 Actomyosin ring assembly was delayed in rewired cells! !92! 3.2.5.2 SIN was required for actomyosin ring assembly in rewired cells! !95! 3.2.5.3 Actomyosin ring components were precociously recruited in rewired cells! !99! 3.2.5.4 Dynamics of Myo2p was altered in rewired cells! !105! 3.2.5.5 Mid1p was required for the dynamic coordination between nuclear and division site positions! .!110! 3.3! Cell size sensing by Cdr2p and Pom1p! !115! 3.3.1 Disrupting Cdr2p localization leads to G2 prolongation! !118! 3.3.2 Medial targeting of Pom1p leads to G2 prolongation! !121! Chapter IV Discussion! .!125! 4.1 Gab-GFP binding based targeting strategy! !125! 4.2 Synthetic rewiring studies provided new insights into S pombe cytokinesis! !128! 4.2.1 Gab-GFP binding based strategy is advantageous! !128! ! vi! 4.2.2 Rng2p and actomyosin ring positioning! !130! 4.2.3 Order of assembly of node components is not important! .!131! 4.2.4 Rewired cytokinesis nodes and canonical cytokinesis nodes! !134! 4.2.5 Nuclear and division site positions! .!137! 4.2.6 Conclusions on rewiring studies! !139! 4.3 Cell size sensing and G2/M transition! !141! Chapter V Future directions! !144! 5.1 Building a protein targeting library based on Gab-GFP binding ! .!144! 5.2 Identification of functional epistasis groups in fission yeast cytokinesis! !145! 5.3 Identification of specific Mid1p interacting sites and their binding partners using genetic code expansion strategy! !147! 5.4 Examining Cdr2p as a direct cell-size sensor! .!149! References! !151! ! vii! Summary Understanding the spatial and temporal regulation of proteins is one of the key challenges in studying complex cellular processes in many cell types In this study, I established a protein targeting strategy based on the GabGFP binding affinity, which preferentially allowed spatial manipulation of proteins that are fused to GFP Synthetic protein targeting approach was applied to investigate two essential cellular events during the fission yeast cell cycle, i.e., cytokinesis and mitotic entry Like many higher eukaryotes, the fission yeast cells utilize a contractile actomyosin ring for cell division The Gab-GFP based protein targeting approach synthetically rewired medial cell division in cells lacking Mid1p, a protein thought to be key for medial actomyosin ring assembly in fission yeast By individually targeting the IQGAP-related Rng2p, formin-Cdc12p and type II myosin-Myo2p to the medial cortex, the requirement for Mid1p in organizing cytokinetic nodes for actomyosin ring assembly was bypassed This result also suggests that the order of assembly of ring proteins at the division site is not essential for medial contractile ring assembly I further characterized ring assembly in the rewired cells in which Rng2p, Cdc12p and Myo2p were precociously targeted to medial nodes, and came to several important conclusions A delay in ring assembly was observed in the rewired cells and possible explanations were explored In addition, unlike wild-type cells, these cells not actively ! viii! track the position of the nucleus for ring assembly and cell division These studies suggest that Mid1p performs multiple functions pertaining to cytokinesis – 1) marking the medial cortex for cell division, 2) promoting timely assembly of actomyosin ring in metaphase and 3) dynamically aligning the position of the actomyosin ring to that of the nucleus In a parallel study, the Gab-GFP based protein targeting approach was used to manipulate the spatial localization of the serine/threonine kinase Cdr2p and the DYRK family Pom1p Mistargeting Cdr2p to nonmedial subcellular locations or targeting Pom1p to the medial region both led to a prolonged G2 phase These events were reversed by either depleting the protein phosphatase Clp1p or mutating pom1 to a kinase-dead form pom12 These data provide direct evidence supporting that Cdr2p functions as a read-out for cell size expansion, consistent with models proposed in previous work (Martin and Berthelot-Grosjean, 2009; Moseley et al., 2009) My work also reveals the complicity of multiple inputs in regulating G2-phase / M-phase transition in fission yeast cells ! ix! 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