Chromator, a novel and essential chromodomain protein interacts directly with the putative spindle matrix protein skeletor

Abstract

We have used a yeast two-hybrid interaction assay to identify Chromator, a novel chromodomain containing protein that interacts directly with the putative spindle matrix protein Skeletor. Immunocytochemistry demonstrated that Chromator and Skeletor show extensive co-localization throughout the cell cycle. During interphase Chromator is localized on chromosomes to interband chromatin regions in a pattern that overlaps that of Skeletor. However, during mitosis both Chromator and Skeletor detach from the chromosomes and align together in a spindle-like structure. Deletion construct analysis in S2 cells showed that the COOH-terminal half of Chromator without the chromodomain was sufficient for both nuclear as well as spindle localization. Analysis of P-element mutations in the Chromator locus shows that Chromator is an essential protein. Furthermore, RNAi depletion of Chromator in S2 cells leads to abnormal microtubule spindle morphology and to chromosome segregation defects. These findings suggest that Chromator is a nuclear protein that plays a role in proper spindle dynamics during mitosis.

Megator, an Essential Coiled-Coil Protein that Localizes to the Putative Spindle Matrix during Mitosis inDrosophila

“…The above findings supports the hypothesis that Skeletor and Chromator are members of a macromolecular spindle matrix complex constituted by several nuclear components (Walker et al, 2000;Rath et al, 2004). However, for a spindle matrix to form independently or to form a structural scaffold aligned with the microtubule spindle, one or more of its molecular components would be predicted to have the ability to form polymers.…”

“…Because both Chromator and Skeletor localize to chromosomes as well as to the spindle-like structure, it was not clear whether the physical interactions observed in co-ip and pull-down experiments between these molecules reflected interactions in chromosomal complexes or interactions on the spindlelike structure or both (Rath et al, 2004). However, because Megator is not localized to the chromosomes during interphase or on centrosomes during metaphase through telophase, the molecular interaction of the complex observed likely occurs on the spindle-like structure.…”

“…All procedures for mAb production were performed by the Iowa State University Hybridoma Facility. The anti-Skeletor mAb 1A1 (Walker et al, 2000), antiChromator mAbs 6H11 and 12H9 (Rath et al, 2004), anti-Bx34 antigen mAb Bx34 and polyclonal antiserum (Zimowska et al, 1997), and antilamin mAb ADL195 (Klapper et al, 1997) …”

“…Thus, many of the features of the Skeletor defined spindle are consistent with the spindle matrix hypothesis. Using a yeast two-hybrid screen with Skeletor sequence as bait Rath et al (2004) identified another potential component of a spindle matrix, Chromator, that interacts directly with Skeletor. Chromator contains a chromodomain and colocalizes with Skeletor on the chromosomes at interphase as well as to the Skeletor-defined spindle during metaphase.…”

“…Chromator contains a chromodomain and colocalizes with Skeletor on the chromosomes at interphase as well as to the Skeletor-defined spindle during metaphase. Furthermore, functional assays using P-element insertion mutants and RNAi in S2 cells suggest that Chromator is an essential protein that affects spindle function and chromosome segregation (Rath et al, 2004).…”