Determining Molecular Mechanisms of Cell Division in Fission Yeast by Testing Major Assumptions of the Search, Capture, Pull, and Release Model of Contractile-Ring Assembly

Determining Molecular Mechanisms of Cell Division in Fission Yeast by Testing Major Assumptions of the Search, Capture, Pull, and Release Model of Contractile-Ring Assembly

Bibliographic Details
Main Author: Coffman, Valerie Chest
Language:English
Published: The Ohio State University / OhioLINK 2013
Subjects:
Cellular Biology
Genetics
Molecular Biology
cytokinesis
node
contractile ring
formin
myosin
fission yeast
centromere
Cnp1
Cse4
kinetochore
quantitative microscopy
cell division
tetrad fluorescence microscopy
Online Access:http://rave.ohiolink.edu/etdc/view?acc_num=osu1366202358
id ndltd-OhioLink-oai-etd.ohiolink.edu-osu1366202358
record_format oai_dc
collection NDLTD
language English
sources NDLTD
topic Cellular Biology
Genetics
Molecular Biology
cytokinesis
node
contractile ring
formin
myosin
fission yeast
centromere
Cnp1
Cse4
kinetochore
quantitative microscopy
cell division
tetrad fluorescence microscopy
spellingShingle Cellular Biology
Genetics
Molecular Biology
cytokinesis
node
contractile ring
formin
myosin
fission yeast
centromere
Cnp1
Cse4
kinetochore
quantitative microscopy
cell division
tetrad fluorescence microscopy
Coffman, Valerie Chest
Determining Molecular Mechanisms of Cell Division in Fission Yeast by Testing Major Assumptions of the Search, Capture, Pull, and Release Model of Contractile-Ring Assembly
author Coffman, Valerie Chest
author_facet Coffman, Valerie Chest
author_sort Coffman, Valerie Chest
title Determining Molecular Mechanisms of Cell Division in Fission Yeast by Testing Major Assumptions of the Search, Capture, Pull, and Release Model of Contractile-Ring Assembly
title_short Determining Molecular Mechanisms of Cell Division in Fission Yeast by Testing Major Assumptions of the Search, Capture, Pull, and Release Model of Contractile-Ring Assembly
title_full Determining Molecular Mechanisms of Cell Division in Fission Yeast by Testing Major Assumptions of the Search, Capture, Pull, and Release Model of Contractile-Ring Assembly
title_fullStr Determining Molecular Mechanisms of Cell Division in Fission Yeast by Testing Major Assumptions of the Search, Capture, Pull, and Release Model of Contractile-Ring Assembly
title_full_unstemmed Determining Molecular Mechanisms of Cell Division in Fission Yeast by Testing Major Assumptions of the Search, Capture, Pull, and Release Model of Contractile-Ring Assembly
title_sort determining molecular mechanisms of cell division in fission yeast by testing major assumptions of the search, capture, pull, and release model of contractile-ring assembly
publisher The Ohio State University / OhioLINK
publishDate 2013
url http://rave.ohiolink.edu/etdc/view?acc_num=osu1366202358
work_keys_str_mv AT coffmanvaleriechest determiningmolecularmechanismsofcelldivisioninfissionyeastbytestingmajorassumptionsofthesearchcapturepullandreleasemodelofcontractileringassembly
_version_ 1719419177564897280
spelling ndltd-OhioLink-oai-etd.ohiolink.edu-osu13662023582021-08-03T05:21:47Z Determining Molecular Mechanisms of Cell Division in Fission Yeast by Testing Major Assumptions of the Search, Capture, Pull, and Release Model of Contractile-Ring Assembly Coffman, Valerie Chest Cellular Biology Genetics Molecular Biology cytokinesis node contractile ring formin myosin fission yeast centromere Cnp1 Cse4 kinetochore quantitative microscopy cell division tetrad fluorescence microscopy Cytokinesis is the final step of the cell-division cycle, resulting in formation of two daughter cells. The fission yeast <i>Schizosaccharomyces pombe</i> is an ideal model organism to study eukaryotic cytokinesis, because the components, mechanisms, and regulatory pathways are well conserved. Cells from yeast to humans assemble the contractile ring of actin, myosin-II, formin, and other proteins, which constricts to divide the cells. We tested some of the basic assumptions of the search, capture, pull, and release (SCPR) model to explain the mechanism of contractile-ring assembly during cytokinesis in fission yeast. The SCPR model requires that the formin Cdc12 is present in at least 30 nodes to nucleate actin filaments that are captured by myosin-II in neighboring nodes. Live microscopy shows that Cdc12 localizes to a broad band of 30 to 50 dynamic nodes, where actin filaments are nucleated in random directions. Consistent with the SCPR model, myosin motor activity is required to condense the nodes into a contractile ring, based on slower or absent node condensation in <i>myo2-E1</i> and <i>rng3-65</i> mutants. The SCPR model also predicts the number of Cdc12 present per node. To determine the stoichiometry of proteins in nodes, we used in vivo fluorescent standards. A popular standard is the histone H3 variant CENP-A, Cse4 in budding yeast. Anaphase centromere clusters contained about 4-fold more Cse4 in <i>S. cerevisiae</i> and about 40-fold more CENP-A (Cnp1) in <i>S. pombe</i> than assumed. These results suggest CENP-A molecules exceed the number of kinetochore-microtubule attachment sites on each chromosome. We counted proteins in the cytokinesis nodes, based on the revised standard, and measured their dynamics to understand their assembly and architecture. We found a modular and hierarchical node assembly in which stable node components scaffold dynamic proteins. We also found an average of 3 dimers per Cdc12 node, consistent with the SCPR model predictions. This work characterizing the assembly of precursor nodes defines important steps and molecular players for contractile-ring assembly.Finally, we found a novel role for the formin For3 during cytokinesis. The fission yeast has three formins that nucleate actin filaments. Only Cdc12 is essential for contractile-ring assembly, but For3 nucleates about 10% of the actin filaments at the division site. Two temperature-sensitive alleles of <i>cdc12</i>, <i>cdc12-112</i> and <i>cdc12-299</i>, lack the ability to nucleate actin filaments in vitro but localize normally to nodes. However, the nodes condense into clumps rather than rings at the restrictive temperature. Clump formation in the absence of Cdc12 nucleation activity suggests another source of actin. Node movements in <i>cdc12-112 for3Δ</i> double mutant suggest that actin filaments nucleated by For3 could be utilized to pull nodes together into clumps. NH<sub>2</sub>-terminal truncations of Cdc12 require actin filaments nucleated by For3 for localization, and are unable to divide successfully in <i>for3Δ</i>, pointing to a predominant role for medially-nucleated actin filaments in contractile-ring assembly. These results reveal a novel role for For3 at the division site and cooperation between two formins for cytokinesis. Taken together, these data provide strong support for the SCPR model of contractile-ring formation in cytokinesis. 2013-07-24 English text The Ohio State University / OhioLINK http://rave.ohiolink.edu/etdc/view?acc_num=osu1366202358 http://rave.ohiolink.edu/etdc/view?acc_num=osu1366202358 unrestricted This thesis or dissertation is protected by copyright: all rights reserved. It may not be copied or redistributed beyond the terms of applicable copyright laws.

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