The long-range goal of the project is to understand the mechanism of cytokinesis in enough detail to make useful mathematical models of the process that can predict the results of future experiments. Remarkably, we are close to this goal for the fission yeast S. pombe owing to the experimental advantages of this organism. Our 2008 model for contractile ring assembly from cytokinesis organizing centers called nodes faithfully accounts for prior experimental observations and our subsequent work. Over the past 4 years we tested new models for the formation of cytokinesis nodes from two types of interphase nodes and for the constriction and turnover of the contractile ring. Simulations of these three models show that we have a good understanding of the physical events relating to cytokinesis around the entire cell cycle. The constriction model generates the tension observed in live cells and explains why the constant turnover of both actin filaments and myosin is required for constriction. This work puts us in position to ask well-informed questions about the mechanisms that control each of the transitions in the process. Our first goal for the next award period is to determine the structure of nodes, the cytokinesis-organizing centers of fission yeast.
We aim to determine how ten different proteins are organized in interphase and cytokinesis nodes, including node protein Blt1p, exchange factor Gef2p, cell cycle kinases Cdr1p and Cdr2p, anillin Mid1p, two myosin-II isoforms, F-BAR Cdc15p, formin Cdc12p and IQ-GAP Rng2p. We will combine information from (i) biochemical and biophysical characterization of each protein, (ii) SAXS and x-ray crystallography of selected protein domains and (iii) super-resolution fluorescence microscopy of live cells. The second goal is to characterize the life cycles of the two types of interphase nodes and their combination to form cytokinesis nodes. Observations of cells with mutations in regulatory proteins will reveal how the cell cycle controls the transitionsin the node cycle such as the disappearance of type 1 nodes during mitosis. The third goal is to use super-resolution microscopy of live cells, modeling and effects of mutations to characterize the dynamics of the protein components of the contractile ring as it constricts and disassembles. These projects are powered by four technical innovations. (1) Our method to count fluorescent molecules in confocal images is the basis of our quantitative approach to microscopy of live cells. (2) We have taken quantitative microscopy to a new level with a novel method to measure affinities in live cells. (3) A superior photoswitchable protein and high-speed image acquisition allowed us to make real time super-resolution microscopy routine for live fission yeast. (4) We expanded our mathematical models of cytokinesis to include the formation of cytokinesis nodes from two types of interphase nodes and constriction of the contractile ring. Given the evolutionary conservation of many of the participating molecules, I believe that studies of fission yeast will establish the basic molecular pathways controlling cytokinesis in other eukaryotes.
We aim to understand the mechanism of cytokinesis in the model organism, fission yeast, using a combination of innovative microscopic methods to make quantitative measurements in live cells and simulations of mathematical models to test and improve our hypotheses.
| Arasada, Rajesh; Sayyad, Wasim A; Berro, Julien et al. (2018) High-speed superresolution imaging of the proteins in fission yeast clathrin-mediated endocytic actin patches. Mol Biol Cell 29:295-303 |
| Friend, Janice E; Sayyad, Wasim A; Arasada, Rajesh et al. (2018) Fission yeast Myo2: Molecular organization and diffusion in the cytoplasm. Cytoskeleton (Hoboken) 75:164-173 |
| Dey, Sumit K; Pollard, Thomas D (2018) Involvement of the septation initiation network in events during cytokinesis in fission yeast. J Cell Sci 131: |
| Akamatsu, Matthew; Lin, Yu; Bewersdorf, Joerg et al. (2017) Analysis of interphase node proteins in fission yeast by quantitative and superresolution fluorescence microscopy. Mol Biol Cell 28:3203-3214 |
| Pollard, Thomas D (2017) Nine unanswered questions about cytokinesis. J Cell Biol 216:3007-3016 |
| Pollard, Thomas D (2017) What We Know and Do Not Know About Actin. Handb Exp Pharmacol 235:331-347 |
| Laplante, Caroline; Pollard, Thomas D (2017) Response to Zambon et al. Curr Biol 27:R101-R102 |
| Pollard, Thomas D (2016) Actin and Actin-Binding Proteins. Cold Spring Harb Perspect Biol 8: |
| Laplante, Caroline; Huang, Fang; Bewersdorf, Joerg et al. (2016) High-Speed Super-Resolution Imaging of Live Fission Yeast Cells. Methods Mol Biol 1369:45-57 |
| Courtemanche, Naomi; Pollard, Thomas D; Chen, Qian (2016) Avoiding artefacts when counting polymerized actin in live cells with LifeAct fused to fluorescent proteins. Nat Cell Biol 18:676-83 |
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