Analysis of a nuclear complex of proteins that form a spindle matrix during mitosis in Drosophila

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Repository URL:
10.31274/rtd-180817-4156; 10.31274/rtd-180813-4156
Rath, Uttama
Iowa State University; Digital Repository @ Iowa State University,
Biochemistry; biophysics; and molecular biology; Molecular; cellular; and developmental biology
thesis / dissertation description
A spindle matrix is a macromolecular structure hypothesized to provide a stationary support or strut for the anchorage of motor proteins during force generation, microtubule sliding, and chromosome segregation in mitosis. Previous work in Drosophila has shown Skeletor, as the first putative molecular candidate of spindle matrix structure since it possesses many features consistent with the proposed spindle matrix structure.;We have identified two more putative spindle matrix candidate proteins, Chromator and Megator, using yeast two-hybrid interaction assay, cross-immunoprecipitation and immunocytochemistry. Chromator is a novel and essential chromodomain containing protein that interacts directly with Skeletor and extensively colocalizes with it throughout the cell cycle. During interphase Chromator colocalizes with Skeletor on the chromosomes, and then redistributes on to the Skeletor defined spindle during metaphase and anaphase. Furthermore, RNAi depletion of Chromator in S2 cells leads to abnormal microtubule spindle morphology and to chromosome segregation defects, thus suggesting that Chromator plays a role in proper spindle dynamics during mitosis. We have generated new Chromator mutant alleles to further aid in the functional analysis of its roles in force production and chromosome segregation during mitosis.;Megator (Bx34 antigen) is a Drosophila Tpr ortholog and has an extended N-terminal coiled-coil domain. During interphase Megator is localized to the nuclear rim and occupies the intranuclear space surrounding the chromosomes. However, during mitosis Megator reorganizes and aligns together with Skeletor and Chromator into a fusiform spindle structure. The Megator metaphase spindle persists in the absence of microtubule spindles, thus strongly implying that the existence of the Megator-defined spindle does not require polymerized microtubules. Furthermore, deletion construct analysis in S2 cells revealed that the NH2-terminal coiled-coil region of Megator self assembles into large spheres indicating its ability to form polymers and serve as the structural basis for the putative spindle matrix complex.