In my thesis I aimed to contribute to the understanding of the mechanism of cytokinesis in C. elegans embryos. I wanted to analyze the relative contributions of different spindle parts – microtubule asters and the midzone - to cytokinesis furrow positioning. I developed a UV laser-based severing assay that allows the spatial separation of the region midway between the asters and the spindle midzone. The spindle is severed asymmetrically between one aster and the midzone. I found that the spindle provides two consecutive signals that can each position a cytokinesis furrow: microtubule asters provide a first signal, and the spindle midzone provides a second signal. The use of mutants that do not form a midzone suggested that the aster-positioned furrow is able to divide the cell alone without a spindle midzone. Analysis of cytokinesis in hypercontracile mutants suggests that the aster-positioned cytokinesis furrow and the midzone positioned furrow inhibit each other by competing for cortical contractile elements. I then wanted to identify the molecular pathway responsible for cytokinesis furrow positioning in response to the microtubule asters. To this end, I performed an RNAi screen, which identified a role for LET-99 in cytokinesis: LET-99 appeared to be required for aster-positioned cytokinesis but not midzone-positioned cytokinesis. LET-99 localizes as a cortical band that overlaps with the cytokinesis furrow. Mechanical displacement of the spindle demonstrated that the spindle positions cortical LET-99 at the site of furrow formation. The furrow localization of LET-99 depended on G proteins, and consistent with this finding, G proteins are also required for aster-positioned cytokinesis. (Anlage: Quick time movies, 466, 67 MB)

The essential gene YNL152w was previously found in a screen designed to isolate putative negative regulators of the S. cerevisiae Pkc1p pathway. Activity assays were performed with a lexA-RLM1-lacZ integrated reporter in different ynl152w truncated mutants. In contrast to the original screen, there were no differences or the activities were even lower in some mutants. To analyze the consequences of different expression levels, YNL152w was expressed under the control of the GAL1/10 promoter. Growth curves were performed under high, intermediate and low expression levels. Strikingly, both conditional strains were able to grow under repressing conditions. However, an aberrant morphology was found suggesting that the cells are indeed affected by low amounts of Ynl152w protein. A series of successive Ynl152wp C-terminal truncations was analyzed to determine cell viability and to investigate the function of the protein. Remarkably, about 2/3 of the protein were dispensable to confer viability. Microscopic analyses of constructs revealed an aberrant morphology characteristic of a cytokinesis defective mutant, with the appearance of swollen cells and formation of big aggregates. Interestingly, the phenotype was more pronounced in the larger truncations. Coherent with these results time-lapse experiments with a large truncated mutant showed a stabilization of the SH3 protein Hof1p at the bud neck. This protein is involved in septum formation and has been reported as a binding partner of YNL152w. The phenotypes observed in the truncated mutants could be attributed to the presence of 4 proline rich motifs. Such motifs have been reported to interact with SH3 domains. An internal deletion of an aspartate rich domain present in the Ynl152wp sequence also displayed a phenotype very similar to that of the largest truncations. Therefore, this domain may play an important role in Ynl152wp function.

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Cytokinesis is defined as the physical division of one cell into two and occurs at the end of the cell cycle. Gestation and development are defined by dividing cells; as an organism develops, cells must duplicate their genetic material, divide, and form two daughter cells. This process is fundamental to all life on our planet. Here, I present work that builds upon our understanding of cytokinesis, focusing on the differential requirements for cytokinesis in different cell types in the early C. elegans embryo, specifically, the P2 cell of the 4-cell embryo. The textbook view of cytokinesis is that all animal cells divide using the same molecular machinery. Yet, growing evidence supports both cell type-specific regulation of cytokinesis and cell type-specific consequences for cytokinesis failure. The 4-cell C. elegans embryo is a powerful model for studying cell type-specific differences in cytokinesis as the cells are already programmed to form distinct cell linages, and previously, we identified cell type-specific regulation of cytokinesis at the 4-cell stage. We weakened the contractile ring using a temperature sensitive (ts) diaphanous formin/CYK-1 mutant. Under this condition, the two anterior cells (ABa and ABp) always failed in cytokinesis, whereas the two posterior cells (EMS and P2) divided successfully at a high frequency, even without detectable F-actin in the cell division plane. Here we focus on the cell type-specific protection of cytokinesis in the P2 germ precursor cell, required to produce all gametes in the adult worm. Using a candidate-based RNAi mini-screen to identify genes required for protection of P2 cytokinesis in the formin(ts) embryos, we identified members of the CCCH Zn2+-finger protein family that are enriched in P2 and required for proper germ cell fate specification. Depletion of MEX-1, PIE-1, or POS-1 led to loss of cytokinetic protection and P2 cytokinesis failure in formin(ts) mutants, but not in control embryos. While depletion of MEX-1 affected multiple cell types, PIE-1 and POS-1 acted exclusively in the P2 cell. Further analysis revealed these germ fate regulators protect cytokinesis by preventing excessive accumulation of septin/UNC-59 and its binding partner, anillin/ANI-1, on the cell cortex in the P2 cell division plane, both negative regulators of actomyosin constriction during cytokinesis in many contexts. We further found that co-depletion of septin and PIE-1 was sufficient to both reduce anillin levels at the P2 division plane and restore cytokinetic protection of P2 in formin(ts) mutant embryos. Thus, germ fate specification protects the P2 germ precursor cell from cytokinesis failure upon damage to the actin cytoskeleton at least in part by controlling the levels of septin and anillin at the division plane.

Thesis advisor: Maitreyi E. Das / Polarization allows cells to form and maintain morphologies necessary for their diverse functions during processes such as growth, division, differentiation, and migration. Signaling proteins such as the family of small Rho GTPases promote polarization by spatiotemporally regulating cytoskeleton dynamics and coordinating membrane trafficking. Here, we investigate and define roles of the Rho GTPase Cdc42 in promoting polarization in S. pombe. As fission yeast, S. pombe cells grow from their cell ends during interphase and divide by medial fission to produce two new daughter cells. As cell-walled organisms, growth and division require intricate remodeling and expansion of the cell wall via incorporation of new membrane and proteins at these polarized sites. Thus, growth and division require specific sequences of membrane trafficking events to deliver and remove cargo at appropriate times and locations. During cytokinesis, fission yeast cells divide by synthesizing new cell wall called the septum to medially bisect the cell. The septum is synthesized behind the actomyosin ring to aid its constriction. Once ring constriction completes and the septum matures, the septum is partially digested to physically separate the daughter cells. Previous work has shown that Cdc42 promotes the delivery of specific but not all septum-synthesizing enzymes as well as septum-digesting enzymes, but it was not known how Cdc42 activation is regulated at the division site to temporally coordinate these processes. Here, we show that the Cdc42 GAPs Rga4 and Rga6 promote proper septum synthesis and timely cell separation by locally decreasing Cdc42 activation during late cytokinesis. This work also reveals a role for Cdc42 in regulating clathrin-mediated endocytosis, both at the division site as well as at growing cell ends. To further explore this role, we systematically examined the behaviors of endocytic actin patches in mutants of Cdc42 regulators and compared these dynamics to wild-type controls. This characterization led to the observation that endocytic patches are best formed to induce successful patch internalization at sites of polarization where Cdc42 is active. In this work, we show that Cdc42 activation promotes proper endocytic patch behavior in a dose-dependent manner and that Cdc42 regulates endocytosis via its downstream effector, the Pak1 kinase. We also demonstrate that Cdc42 and Pak1 activity promote endocytosis through at least two pathways which regulate branched actin formation. First, we show that Cdc42 and Pak1 promote proper endocytic actin patch formation. Secondly, we show that Pak1-mediated phosphorylation of the endocytic Type I myosin promotes timely internalization of endocytic actin patches. / Thesis (PhD) — Boston College, 2024. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Une cellule se divise en deux par le processus de cytokinèse. Elle requiert la coordination de plusieurs composants pour éviter la formation des cellules potentiellement cancéreuses. Premièrement, un anneau contractile (AC) dépendant de l’actine et de Rho-GTP diminue le diamètre de la cellule jusqu’à la formation d’une structure plus stable indépendante de l’actine, l’anneau du midbody (AM) qui guide l’éventuelle séparation des cellules sœurs. Diaphanous (Dia) est une formine dépendante de Rho responsable de l’agencement des filaments d’actine non ramifiés qui se localise à l’AC et est essentielle à la cytokinèse. Nous avons étudié le rôle de Dia pendant la cytokinèse par microscopie de haute résolution en temps réel pour suivre le comportement dynamique des protéines fluorescentes (PF) dans des cellules de Drosophile S2. Une construction fonctionnelle de Dia-PF est recrutée à l’AC et l’AM indépendamment de l’actine mais est absente dans l’AM mature. Dia quitte l’AM au même temps où l’AM dévient indépendant d’actine. La déplétion de Dia par ARN interférant ralentit la constriction de l’AC, augmente les oscillations et, dans 70% des cas, les cellules échouent la cytokinèse pendant la constriction, suggérant que Dia a un rôle dans l’organisation de l’AC. LifeAct-PF, une sonde pour F-actine, dévoile une diminution des filaments d’actine spécifique à l’AC des cellules dépourvues de Dia pendant que Anilline-PF et Myosine-PF sont recrutées en puncta. Ces résultats soutiennent un modèle où Dia nuclée des filaments d’actine qui permettent l’organisation dynamique de l’AC et la perte de Dia régule la transition à l’AM stable indépendant d’actine. / Cytokinesis is the intricate process by which eukaryotic cells divide in two. It involves the coordination of many components in order to avoid the formation potentially cancerous cells. Initially, a Rho GTPase- and actomyosin-dependent contractile ring (CR) drives constriction at the cell equator until a stable actin-independent midbody ring (MR) forms and ultimately guides the separation of the two sister cells. Diaphanous (Dia), is a Rho-dependent formin that nucleates unbranched actin filaments, localises to the cleavage furrow and is required for cytokinesis. We have examined the role of Dia during cytokinesis by time lapse video microscopy of Drosophila S2 cells expressing markers tagged with fluorescent proteins (FPs). A functional Dia-FP was recruited to the CR independently of actin and stayed in the nascent MR, but was absent from the mature MR. The timing of its disappearance coincided with the transition of the MR to an actin-independent structure. RNAi-mediated depletion of Dia slowed furrow ingression, enhanced furrow oscillations and, in 70% of the failures, prevented furrow completion, consistent with a role for Dia in CR organization. The F-actin probe, LifeAct-FP, revealed a decrease in F-actin in Dia-depleted cells specifically at the CR while Anillin-FP and Myosin-FP were aberrantly recruited in punctate structures. Our findings are consistent with a model in which Dia nucleates actin filaments at the CR to maintain the dynamic organization of the actin-dependent CR and that the regulated loss of Dia from the nascent MR guides the formation of the stable, actin-independent MR.