Skeletor, a Novel Chromosomal Protein That Redistributes during Mitosis Provides Evidence for the Formation of a Spindle Matrix

Abstract

A spindle matrix has been proposed to help organize and stabilize the microtubule spindle during mitosis, though molecular evidence corroborating its existence has been elusive. In Drosophila, we have cloned and characterized a novel nuclear protein, skeletor, that we propose is part of a macromolecular complex forming such a spindle matrix. Skeletor antibody staining shows that skeletor is associated with the chromosomes at interphase, but redistributes into a true fusiform spindle structure at prophase, which precedes microtubule spindle formation. During metaphase, the spindle, defined by skeletor antibody labeling, and the microtubule spindles are coaligned. We find that the skeletor-defined spindle maintains its fusiform spindle structure from end to end across the metaphase plate during anaphase when the chromosomes segregate. Consequently, the properties of the skeletor-defined spindle make it an ideal substrate for providing structural support stabilizing microtubules and counterbalancing force production. Furthermore, skeletor metaphase spindles persist in the absence of microtubule spindles, strongly implying that the existence of the skeletor-defined spindle does not require polymerized microtubules. Thus, the identification and characterization of skeletor represents the first direct molecular evidence for the existence of a complete spindle matrix that forms within the nucleus before microtubule spindle formation.

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.…”

“…Skeletor is associated with chromosomes at interphase, but preceding microtubule spindle formation and nuclear lamina breakdown, it redistributes into a true fusiform spindle at prophase. During metaphase the Skeletor defined spindle and the microtubule spindles are coaligned and when embryos are treated with nocodazole to disassemble microtubules, the Skeletor spindle persists (Walker et al, 2000). Thus, many of the features of the Skeletor defined spindle are consistent with the spindle matrix hypothesis.…”

“…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) …”

“…The involvement of a spindle matrix that can act as a stationary substrate to stabilize the spindle during force production, and microtubule sliding has long been proposed (Pickett-Heaps et al, 1982;; however, direct evidence for its existence has remained elusive (Scholey et al, 2001;Wells, 2001;Bloom, 2002;Kapoor and Compton, 2002;Johansen and Johansen, 2002). Recently, a putative spindle matrix protein, Skeletor, was identified in Drosophila (Walker et al, 2000). Skeletor is associated with chromosomes at interphase, but preceding microtubule spindle formation and nuclear lamina breakdown, it redistributes into a true fusiform spindle at prophase.…”

Drosophila JIL-1 kinase mediates histone H3 Ser10 phosphorylation, maintains higher order chromatin structure, and is implicated in dosage compensation

“…Embryo staining was performed essentially as previously described f Walker et al, 2000). Embryos (0-5 hour) were dechorionated in a 50% Chlorox solution for about 2 min, washed with 0.7M NaCl/0.2% Triton X-100, and fixed in a 1:1 heptane/fixative mixture for 20 min with vigorous shaking at room temperature.…”