Characterization of two novel spindle matrix proteins in Drosophila, Chromator and Megator

Wang, Dong
Major Professor
Kristen M. Johansen
Jorgen Johansen
Committee Member
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Zoology and Genetics
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Zoology and Genetics

A spindle matrix has been proposed to help organize and stabilize microtubules during mitosis. This concept has gained new interest due to the identification of Skeletor as a potential component of the spindle matrix. Skeletor is associated with chromosomes during interphase but reorganizes into a spindle-like structure during early prophase before the nuclear envelope breaks down and microtubules enter the nucleus. The Skeletor spindle extends from pole to pole during metaphase coaligning with the microtubule spindle and persists even after nocodazole treatment dissembles the microtubules. These features make Skeletor an ideal candidate as a spindle matrix protein.;In this study, I report the characterization of two other spindle matrix proteins in Drosophila, Chromator and Megator. Chromator was identified in two independent yeast two-hybrid screens using a Skeletor fragment. The physical interaction between Skeletor and Chromator was confirmed by in vitro pull-down and coimmunoprecipitation experiments. Chromator and Skeletor colocalize on the spindle during metaphase with Chromator in addition having centrosomal localization. Chromator is heavily concentrated on the centrosomal region and spindle midzone during telophase, which is different from the pole to pole distribution pattern of Skeletor. A P element insertion allele for Chromator was identified with homozygous animals showing highly fragmented polytene chromosomes implying possible Chromator function in maintaining chromosome integrity.;The second protein, Megator, was originally identified as the Bx34 antigen and was proposed to be a possible homolog of mammalian protein TPR. Our results show that Megator shares the same distribution pattern with Skeletor during most of the cell cycle and physically interact with Chromator. A P element insertion allele of Megator was identified with recessive lethal phenotype indicating that Megator is an essential gene.;Our data show that Skeletor, Megator and Chromator form a multiprotein complex being potentially part of the spindle matrix. We propose that this complex is essential for spindle matrix function and Megator is the structural component.