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.
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.
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