The essential open reading frame YNL152w (now called INN1) of Saccharomyces cerevisiae was previously identified in a screen for negative regulators of cell integrity signaling. Subsequent studies and data from genome-wide functional analyses suggested, that the encoded protein plays a role in cell division. This was further addressed in the thesis presented here. Functional Inn1-GFP fusions were shown to co-localize with the contractile actomyosin ring component Myo1 during cytokinesis. Mutants depleted for Inn1 failed to form a primary septum, but did not affect the dynamics of the cytokinetic actin ring (CAR). This has been attributed to the inability to couple plasma membrane ingression (hence Inn1) to CAR constriction, a phenomenon also found by Sanchez-Diaz et al. (2008). Further investigations focused on the question of how Inn1 is recruited to the bud neck and identified the cytokinetic regulators Hof1 and Cyk3, which act in concert for this purpose. Each of them contains a SH3 domain, which interacts with the proline-rich carboxy-terminal part of Inn1. Localization studies and genetic analyses indicate that Inn1 acts downstream of Hof1 and Cyk3. Either the simultaneous repression of HOF1 and CYK3 gene expression or the deletion of their SH3 domains was lethal, with a concomitant failure to localize Inn1-GFP to the division site. Overproduction of either, Hof1 or Cyk3 perturbed the dynamics of Inn1-GFP distribution, which followed that of the overproduced proteins. This atypical CAR-independent localization of Inn1 supports a presumed role of Hof1 and Cyk3 in an alternative cytokinesis pathway to form a primary septum. Since INN1 is also a multicopy suppressor of a myo1 deletion, this further supports the notion that Inn1 may be required for plasma membrane ingression, also in CAR-independent cytokinesis. Preliminary data suggest, that the protein Vrp1 is responsible for the required removal of Inn1 from the bud neck after completion of cytokinesis. The essential amino-terminal C2 domain of Inn1 may mediate plasma membrane ingression by interaction with the membrane lipid phosphatidic acid, observed in biochemical studies. Alternatively, the C2 domain has been suggested to modulate chitin synthesis in the primary septum by modulating Chs2 activity (Nishihama et al., 2009).

No description available.

At the beginning of mitosis, chromosomes are condensed and segregated to facilitate correct alignment later in cytokinesis. Condensin is the pentameric enzyme responsible for this DNA compaction and is composed of two structural maintenance of chromosomes (SMC) subunits and three non-SMC subunits. Condensin mutations generate chromosomal abnormalities due to improper segregation, leading to genome instability and eventual malignant transformation of the cell. Cdc2 phosphorylation of the non-SMC subunits, CAP-G, CAP-D2, and CAP-H, has been demonstrated to be important for condensin supercoiling activity and function. While these subunits are thought to be phosphorylated by Cdc2, the exact sites have not yet been identified and characterized. The basis of this research was to determine the Cdc2 phosphorylation sites in the CAP-G subunit of the condensin enzyme and to characterize the functional significance of the sites in the regulation of condensin activity using site-directed mutagenesis and immunofluoresence microscopy. While DNA condensation represents a critical step early in mitosis, formation of the mitotic spindle represents a vital event leading to the division of a cell into two daughter cells in a process known as cytokinesis. Protein regulating cytokinesis 1 (PRC1) is a mitotic protein essential for cytokinesis that participates in formation of the mitotic spindle in a phosphorylation dependent manner. PRC1 possesses microtubule bundling properties. Loss of PRC1 leads to mis-segregation of chromosomes and abnormal cytokinesis. HSF2 is a transcription factor known to be important in development and differentiation. Previous research has determined that HSF2 plays a significant mechanistic role in the process of hsp70i gene bookmarking during mitosis. Bookmarking is an epigenetic phenomenon whereby certain gene promoters remain uncompacted, in contrast to the majoritiy of genomic DNA during mitosis. This lack of compaction allows quick reassembly to a transcriptionally competent in G1 of the cell cycle and ensures the ability of the cell to induce expression of the cytoprotective hsp70i protein. HSF2 and PRC1 were found to interact in a yeast-two hybrid screen. Given the importance of both of these proteins during mitosis, this study seeks to characterize the HSF2/PRC1 interaction and determine the potential role for PRC1 in hsp70i gene bookmarking.

Animals, fungi, and amoebas require an actomyosin contractile ring at the division site to perform cytokinesis. The contractile ring initiates and guides the invagination of the plasma membrane as it forms new barriers between the nuclei at the cell equator. Defects in the contractile ring can result in misdirected, delayed, or premature cytokinesis, which leads to abnormal chromosome numbers. Aneuploidies resulting from failed cytokinesis sometimes lead to aggressive forms of cancer. This dissertation was motivated by the goal of better understanding the properties of the contractile ring and how it drives cytokinesis. Actomyosin is initially recruited to the cell equator through the coordination of scaffolding factors, actin-binding proteins, and signaling cascades. Subsequently, the sliding of actin filaments by myosin reshapes the resulting meshwork into a compact ring. Once fully assembled, the contractile ring establishes tension, which leads the plasma membrane inward. The primary motor proteins in the contractile ring of animal cells are class-II nonmuscle myosins, which typically function as bipolar filaments. Filament assembly is activated by phosphorylation and plays a central role in myosin function during cytokinesis. However, many underlying processes that regulate contractile ring function are poorly understood. Current models of cytokinesis have been based on mechanistic insights provided by two decades of work in the fission yeast system Schizosaccharomyces pombe. In fission yeast, the class-II myosin Myo2 provides the major source of motor activity in the contractile ring. Myo2 is two-headed and has a rod-like tail, which is consistent with other class-II myosins. Yet, it was unknown whether Myo2 assembles into filaments, or how phosphorylation affects its activity. To investigate these features, recombinant Myo2 was purified from the baculovirus/Sf9 insect cell expression system. Hydrodynamic measurements were used to examine whether Myo2 forms filaments. These sedimentation velocity data gave no indication that Myo2 self-assembles under the typical physiological salt concentrations, which suggests that Myo2 is unlike any class-II myosin known to date. Myo2 was also treated in vitro with its native kinase Pak1. Phosphorylation of Myo2 molecules had no effect on self-assembly, however it reduced actin-binding in motility assays and increased steady-state ATPase rates by two fold. Our results imply that the function and regulation of fission yeast Myo2 during cytokinesis depends on a specific scaffolding scheme at the plasma membrane, which has not been observed in other eukaryotes. Another interest of this dissertation was how the contractile ring is regulated during cytokinesis. We examined one cytokinesis protein, Cyk3, believed to mediate between the ring and extracellular processes. Genetics and live cell imaging analyses indicated that Cyk3 functions through a catalytically-inactive enzyme domain, which implicated Cyk3's involvement in one of the primary cytokinesis signaling pathways. This dissertation sheds new light on core aspects of how fission yeast undergo cytokinesis, especially with respect to the mechanism of Myo2 activity in the contractile ring. Characterizing the physical and enzymatic properties of an essential myosin in a simple organism should provide insights into cytokinesis in higher organisms.

Thesis advisor: David Burgess / This doctoral thesis addresses two central topics divided into separate chapters. In Chapter 1: The cortical response to RhoA is regulated during mitosis, experimental findings using sea urchin embryos are presented that demonstrate that the small GTPase RhoA participates in positive signaling for cell division and that this activity is negatively regulated prior to anaphase. In a second series of experiments, myosin phosphatase is shown to be a central negative regulator of myosin activity during the cell cycle through metaphase of mitosis and experimental findings support the conclusion that myosin phosphatase opposes RhoA signaling until anaphase onset. These experiments also reveal that myosin activation alone is insufficient to stimulate cortical contractions during S phase and during metaphase arrest following activation of the spindle checkpoint. In Chapter 2: Annotation of cytoskeletal and motility proteins in the sea urchin genome assembly, as part of a collaborative project, homologs of cytoskeletal genes and gene families were derived and annotated from the sea urchin genome assembly. In addition, phylogenetic analysis of multiple gene families is presented based on these findings. / Thesis (PhD) — Boston College, 2008. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

A thesis submitted to the Faculty of Science, University of Witwatersrand, Johannesburg, South Africa, in fulfilment of the requirements for the Degree of Doctor of Philosophy, This thesis presented as a series of manuscripts 1993 / In this study the morphologic, cytokinetic, biochemical, and molecu1w: consequences of low-dose continuous Puromycin-exposure were examined in HL-60 cells, and in a variety of malignant and non-malignant human and murine cell types. Puromycin (PM) is a composite of the amino nucleoside dimethyladenosine and tyrosine-o-methylether. Functionally it is an analogue of the terminal aminoacyl-adenosine portion of aminoacyl-tRNA, more specifically of tyrosyl..tRNA. At high concentrations 5-S0#tg/ml (10-100#tM) PM has been found to block protein synthesis completely by causing the premature release from the ribosomes of truncated peptide chains which are bound to PM through their carboxyl termini. The nascent PMGpeptide complexes (PMPs) are rapidly degraded through a ubiquitin-dependent pathway and are of interest because of (i) their potential to compete for degradation with the natural substrates of the ubiquitin-dependent pathway, including cyclin B, and (ii) because their structure predicts an inhibitory effect on tyrosine kinase activity. In the current' study then, special consideration was given to the effect of PM on the cell cycle, on apoptosis (programmed. cell death), and on tyrosine kinase activity, As a means of comparison, certain of these effects were also examined with respect to another translation inhibitor Cycloheximide (CHX), to two other substituted purines Puromycin Amino nucleoside (PAN) and 6..Dimethylaminopurine (6-DMAP), as well as to the cyclophospbamide derivative Mafosfamide (ASTA Z 7557). / GR2017

The maturation of early endosomes into multivesicular bodies (MVBs) and subsequent trafficking to lysosomes is an important event for the control and silencing of endocytosed membrane receptors. The endosomal-sorting complex required for transport (ESCRT) proteins appear to play a key role in this event. Phosphatidylinositol lipids including PtdIns(3,5)P2 have been implicated in the MVB-lysosomal pathway and an ESCRT-III component CHMP3 binds to this lipid in vitro. The purpose of this thesis was to investigate the link between ESCRT proteins, PtdIns(3,5)P2 and endo-lysosomal trafficking. Firstly, a protein expressed by Salmonella, which is a phosphatase that acts on PtdIns(3,5)P2, was investigated as a potential tool for manipulating cellular PtdIns(3,5)P2 levels. Our results suggest that it is potentially a useful tool for this purpose and that expression of SopB perturbs endosome to lysosome trafficking. These findings provide further evidence for a role of PtdIns(3,5)P2 in endo-lysosomal trafficking.

This work discusses both mechanisms underlying mitochondrial quality control and cytokinesis in the budding yeast Saccharomyces cerevisiae. As these topics are quite different, their presentation has been divided into two parts, "Part I: Mitochondrial Remodeling Through the Proteasome is Critical for Mitochondrial Quality Control in Budding Yeast" and "Part II: Aim44p Regulates Phosphorylation of Hof1p to Promote Contractile Ring Closure During Cytokinesis in Budding Yeast." In Part I, we show that the proteasome is critical for cellular fitness in response to chronic, low levels of mitochondrial reactive oxygen species (ROS) in budding yeast. Deleting DOA1, which is required for ubiquitin-mediated degradation, UFD5, which promotes proteasome gene expression, or NAS2, which promotes proteasome regulatory particle assembly, increases the sensitivity of yeast to chronic, low levels of mitochondrial ROS. In contrast, deleting ATG32, a gene required for mitophagy, other autophagy genes, non-essential chaperones including prohibitins, or mitochondrial proteins including the Lon protease (Pim1p) or YME1, does not affect cellular fitness under these conditions. Doa1p binds with Cdc48p and Vms1p, which associates with mitochondria and promotes extraction of ubiquitinated proteins from the organelle for proteasomal degradation in a pathway called mitochondria-associated degradation (MAD). Elevated mitochondrial ROS increases protein ubiquitination, ubiquitination of the mitochondrial protein aconitase and expression of key MAD proteins. Interestingly, down-regulating ER-associated degradation (ERAD), which shares some common proteins with MAD, can promote cell growth under conditions of elevated mitochondrial ROS. Finally, deletion of DOA1 results in increased sensitivity of yeast and yeast mitochondria to oxidative stress. Mitochondria in doa1 null cells are more oxidized than mitochondria in wild-type or atg32 null cells under conditions of elevated mitochondrial ROS. Moreover, deletion of DOA1 results in a decrease in chronological lifespan. These findings support a critical role for the proteasome and MAD in mitochondrial quality control, which in turn affects cellular fitness, in response to chronic, low levels of mitochondrial ROS. In Part II, we show that the protein product of YPL158C, Aim44p, undergoes septin-dependent recruitment to the site of cell division. Aim44p co-localizes with Myo1p, the type II myosin of the contractile ring, throughout most of the cell cycle. The Aim44p ring does not contract when the actomyosin ring closes. Instead, it forms a double ring that associates with septin rings on mother and daughter cells after cell separation. Deletion of AIM44 results in defects in contractile ring closure. Aim44p co-immunoprecipitates with Hof1p, a conserved F-BAR protein that binds both septins and type II myosins and promotes contractile ring closure. Deletion of AIM44 results in a delay in Hof1p phosphorylation, and altered Hof1p localization. Finally, overexpression of Dbf2p, a kinase that phosphorylates Hof1p and is required for re-localization of Hof1p from septin rings to the contractile ring and for Hof1p-triggered contractile ring closure, rescues the cytokinesis defect observed in aim44 null cells. Our studies reveal a novel role for Aim44p in regulating contractile ring closure through effects on Hof1p.

The final stage of the cell cycle is cell division by cytokinesis, when the cell physically separates into two daughter cells. Improper timing or location of the division site results in incorrect segregation of chromosomes and thus genetically unstable aneuploid cells, which is associated with tumorigenesis. Cytokinesis in animal, fungal and amoeboid cells occurs through the assembly and constriction of an actomyosin contractile ring, a mechanism that dates back about one billion years in the common ancestor of these organisms. However, it is not well understood how the ring generates tension or how the rate of ring constriction is set. Long ago a sliding filament mechanism similar to skeletal muscle was proposed, but definitive evidence for muscle-like sarcomeric order in the ring is lacking. Here we build mathematical models of cytokinesis in the fission yeast Schizosaccharomyces pombe, where the most complete inventory of more than 150 cytokinesis genes have been documented. The models explicitly represent proteins in the contractile ring such as formin, myosin, actin, α-actinin, etc. and implements their quantities, biomechanical properties and organizations from the best available experimental information. At the same time, the models adopt coarse-grain approaches that are able to describe the collective behaviors of thousands of ring components, which include tension production, constriction, and disassembly of the ring. In the first part of this thesis, we modeled the extraordinarily rapid constriction of the partially unanchored ring in fission yeast cell ghosts. Experiments on isolated fission yeast rings showed sections of ring unanchoring from the membrane and shortening ~30-fold faster than normal (1). We demonstrated that anchoring of actin to the plasma membrane generates tension in the fission yeast cytokinetic ring by showing (1) unanchored segments in these experiments were tensionless, and (2) only a barbed-end anchoring of actin can generate tension in the normally anchored ring, and can explain the extraordinary behavior of unanchored segments. Molecularly explicit simulations accurately reproduced experimental constriction rates, and showed a novel non-contractile reeling-in mechanism by which the unanchored segment shortens, despite being tensionless. In the second part of this thesis, we built a highly coarse-grained model to study how ring tension is generated and how structural stability is maintained. Recently, a super-resolution microscopy study of the fission yeast ring revealed that myosins and formins that nucleate actin filaments colocalize in plasma membrane-anchored complexes called nodes in the constricting ring (2). The nodes move bidirectionally around the ring. Here we construct and analyze a coarse-grained mathematical model of the fission yeast ring to explore essential consequences of the recently discovered ring ultrastructure. The model reproduces experimentally measured values of ring tension, explains why nodes move bidirectionally and shows that tension is generated by myosin pulling on barbed-end-anchored actin filaments in a stochastic sliding-filament mechanism. This mechanism is not based on an ordered sarcomeric organization. We show that the ring is vulnerable to intrinsic contractile instabilities, and protection from these instabilities and organizational homeostasis require both component turnover and anchoring of components to the plasma membrane. In the third part of this thesis, we measured ring tension in fission yeast protoplasts. We found ~650 pN tension in wild type cells, ~65% the normal tension in myp2 deletion mutants and ~40% normal tension in myo2-E1 mutant cells with negligible ATPase activity and reduced actin binding. To understand the relation between organization and tension, we developed a molecularly explicit simulation of the fission yeast ring with the above organization. Our simulations revealed a clear division of labor between the 2 myosin-II isoforms, which maintains organization and maximal tension. (1) Myo2 anchors the ring to the plasma membrane, and transmits ring tension to the membrane. (2) Myo2, extending ~100 nm away from the membrane, bundles half (~25) of the actin filaments in the cross-section due to filament packing constraints, as only ~25 filaments are within reach. (3) To increase tension requires that the ring be thickened, as tensions in the ~25 membrane-proximal filaments are close to fracture. (4) Unanchored Myp2 indeed enables thickening, by bundling an additional ~25 filaments and doubling tension. Anchoring of these filaments to the membrane is indirect, via filaments shared with the anchored Myo2. (5) In simulated myo2-E1 rings ~20% of the actin filaments peeled away from the ring and formed Myp2-dressed bridges, as observed experimentally in myo2-E1 cells. (6) The organization in simulated Δmyp2 rings was highly disrupted, with ~ 50% of the actin filaments unbundled. Therefore, beyond their widely recognized job to pull actin and generate tension, myosin-II isoforms are vital crosslinking organizational elements of the ring. Two isoforms in the ring cooperate to organize the ring for maximal actomyosin interaction and tension.

"November 2002" / Bibliography: p. 177-194. / 194 p. : ill., plates (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Molecular Biosciences, 2003