This proposal focuses on the use of molecular genetics, live and fixed cell image analysis, and in vitro biochemistry to investigate the regulation of microtubule dynamics in the early C. elegans embryo. Two aspects of C. elegans MT dynamics regulation have received substantial attention, resulting in important contributions of general significance for cell biology research. First, when astral MTs reach the cortex of early embryonic cells in C. elegans, they undergo catastrophe, a general property of astral MT dynamics in animal cells. The subsequent depolymerization of astral MTs appears to be tightly linked to the generation of cortically localized, dynein-dependent pulling forces on astral MTs that properly position mitotic spindles during the early asymmetric cell divisions that pattern embryonic cell fates. Second, ubiquitylation by an E3 ligase targets the MT severing complex katanin for destruction after the completion of oocyte meiotic cell divisions, allowing for the stable assembly of much longer MTs during mitotic cell division in the early embryo. Two of the three Specific Aims in this proposal focus on the first topic: the regulation of cortical MT catastrophe during mitosis. In the past funding period, we discovered a conserved and cortically localized protein, called EFA-6 that is both necessary and sufficient for cortical MT catastrophe in early embryonic cells. We know of no other cortically localized protein that promotes cortical astral MT catastrophe in any system, or of any protein that has been shown in vivo to so uniformly promote cortical MT catastrophe.
In Specific Aim 1, we use (i) molecular genetics approaches to investigate whether EFA-6 orthologs in other animal phyla also promote cortical MT catastrophe;(ii) in vitro MT polymerization assays to determine if EFA-6 directly interacts with MTs to promote catastrophe;and (iii) genetic and biochemical approaches to identify proteins that interact with EFA-6 to influence MT dynamics. Remarkably, EFA-6 is not essential: homozygous efa-6(-) mutants are viable and healthy.
In Specific Aim 2, we investigate nine more conserved but non-essential genes that like efa-6 were found in modifier screens as suppressors of temperature-sensitive mutants with defects in mitotic cell division. We also propose to identify additional regulators of MT dynamics by conducting a genome-wide screen of all conserved but non-essential C. elegans genes. Our focus on identifying roles for non-essential genes during early embryogenesis is a unique and innovative exploration of an important new frontier in C. elegans genetics research. Finally, our third Specific Aim focuses on the second topic: katanin destruction after the completion of meiotic cell divisions. Specifically, we will investigate the importance of sub-cellular localization dynamics as a mechanism for regulating E3 ligase assembly and thereby properly timing katanin destruction. To investigate the temporal and spatial regulation of E3 ligase assembly, we will use an innovative method, called the Proximity Ligation Assay, to detect when and where E3 ligase components assemble into active complexes. To our knowledge, this new technique has not been applied to the investigation of protein- protein interactions in C. elegans. In sum, the experiments we propose in our three Specific Aims will provide important new insights into the fundamentally important topic of MT dynamics and its regulation during eukaryotic cell division.

Public Health Relevance

The early C. elegans embryo is an appealing model system for the study of the microtubule cytoskeleton, both because of its powerful genetics, and because one can use live cell imaging to investigate microtubule dynamics and function in wild-type and mutant embryos. Furthermore, all of the genes we propose to study are widely conserved in other organisms including humans. In many cases, vertebrate orthologs of these genes also are required for mitotic cell division, and thus are relevant to our understanding and ability to detect and treat cancers and other important human diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM049869-20
Application #
8706158
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Deatherage, James F
Project Start
1994-05-01
Project End
2016-07-31
Budget Start
2014-08-01
Budget End
2015-07-31
Support Year
20
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of Oregon
Department
Type
Organized Research Units
DUNS #
City
Eugene
State
OR
Country
United States
Zip Code
97403
Sugioka, Kenji; Fielmich, Lars-Eric; Mizumoto, Kota et al. (2018) Tumor suppressor APC is an attenuator of spindle-pulling forces during C. elegans asymmetric cell division. Proc Natl Acad Sci U S A 115:E954-E963
Sugioka, Kenji; Bowerman, Bruce (2018) Combinatorial Contact Cues Specify Cell Division Orientation by Directing Cortical Myosin Flows. Dev Cell 46:257-270.e5
Mok, Calvin A; Au, Vinci; Thompson, Owen A et al. (2017) MIP-MAP: High-Throughput Mapping of Caenorhabditis elegans Temperature-Sensitive Mutants via Molecular Inversion Probes. Genetics 207:447-463
Sugioka, Kenji; Hamill, Danielle R; Lowry, Joshua B et al. (2017) Centriolar SAS-7 acts upstream of SPD-2 to regulate centriole assembly and pericentriolar material formation. Elife 6:
Severson, Aaron F; von Dassow, George; Bowerman, Bruce (2016) Oocyte Meiotic Spindle Assembly and Function. Curr Top Dev Biol 116:65-98
Lowry, Josh; Yochem, John; Chuang, Chien-Hui et al. (2015) High-Throughput Cloning of Temperature-Sensitive Caenorhabditis elegans Mutants with Adult Syncytial Germline Membrane Architecture Defects. G3 (Bethesda) 5:2241-55
Connolly, Amy A; Sugioka, Kenji; Chuang, Chien-Hui et al. (2015) KLP-7 acts through the Ndc80 complex to limit pole number in C. elegans oocyte meiotic spindle assembly. J Cell Biol 210:917-32
Du, Zhuo; He, Fei; Yu, Zidong et al. (2015) E3 ubiquitin ligases promote progression of differentiation during C. elegans embryogenesis. Dev Biol 398:267-79
Phillips, Patrick C; Bowerman, Bruce (2015) Cell biology: scaling and the emergence of evolutionary cell biology. Curr Biol 25:R223-R225
Keikhaee, Mohammad R; Nash, Eric B; O'Rourke, Sean M et al. (2014) A semi-dominant mutation in the general splicing factor SF3a66 causes anterior-posterior axis reversal in one-cell stage C. elegans embryos. PLoS One 9:e106484

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