Characterization of three spindle matrix proteins, Megator, EAST and Asator, in Drosophila
Two previously identified proteins Skeletor and Chromator provide the molecular evidence for the existence of a spindle matrix complex in Drosophila. The spindle matrix has been proposed to be a macromolecular complex, which plays a role in organization and stabilization of the mitotic spindle as well as in providing structural support for counterbalancing force production.;In this dissertation, I present the identification and characterization of three more putative spindle matrix candidate proteins, Megator, EAST and Asator. Megator, an ortholog of the mammalian TPR protein, contains a large coiled-coil domain at its NH2-terminus and an acidic, non-structural COOH-terminus. Immunohistochemistry studies show that Megator localizes to the nuclear rim and interchromatin region during interphase, while it reorganizes and coaligns with Skeletor and Chromator to form a fusiform spindle structure during metaphase. The Megator defined spindle structure persists when microtubules are depolymerized by cold or nocodozale treatment, indicating the Megator spindle is independent of the microtubule spindle. Expression of the NH 2-terminal truncated protein in S2 cells shows the coiled-coil domain can form a large spherical structure in cytoplasm, suggesting it is capable of self-assembly, thus suggesting that Megator may serve a structural role in spindle matrix.;EAST is a novel protein that does not contain any predicted functional motifs. It was previously shown to form an expandable nuclear endoskeleton at interphase. Our immunocytochemical studies demonstrate that EAST colocalizes with Megator at the intranuclear space surrounding the chromosomes at interphase and redistributes during mitosis to colocalize with spindle matrix proteins, Skeletor and Megator. Immunoprecipitation experiments indicate that EAST and Megator are in the same protein complex. We propose that Megator and EAST interact to form a nuclear endoskeleton and as well are important components of the putative spindle matrix complex during mitosis.;The third protein, Asator, a conserved tau-tubulin kinase family member, was identified in yeast two-hybrid screen using a Megator NH2-terminal fragment and the physical interaction between Megator and Asator was confirmed by in vitro pull-down assays. Immunocytochemistry demonstrates that V5/GFP tagged Asator protein colocalizes with tubulin throughout the cell cycle. Asator is distributed in the cytoplasm at interphase and relocated to the spindle structure during mitosis. Analysis of an Asator mutant line indicates Asator is an essential gene. These findings suggest that Asator plays a role in proper microtubule dynamics during the cell cycle.