DSK1, a kinesin-related protein involved in anaphase spindle elongation, is a component of a mitotic spindle matrix.

DSK1 is a kinesin-related protein that is involved in anaphase spindle elongation in the diatom Cylindrotheca fuisiformis [Wein et al., 1996: J. Cell Biol. 113:595-604]. DSK1 staining appeared to be concentrated in the gap that forms as the two half-spindles separate, suggesting that DSK1 may be part of a non-microtubule spindle matrix. We set out to investigate this possibility using three-dimensional high-resolution fluorescence microscopy, and biochemical methods of tubulin extraction. Three-dimensional fluorescence microscopy reveals that DSK1 remains in the midzone after the bulk of the microtubules from the two half-spindles have left the region. Biochemical studies show that CaCl2 extraction of tubulin from a mitotic spindle preparation does not extract similar proportions of DSK1 protein. Immunofluorescence confirms that this CaCl2 extraction leaves behind spindle-like bars that are recognized by anti-DSK1, but not by anti-tubulin antibodies. We conclude that DSK1 is part of, or attached to, a non-microtubule scaffold in the diatom central spindle. This discovery has implications for both the structural organization of the mitotic spindle and the mechanism of spindle elongation.

DSK1, a novel kinesin-related protein from the diatom Cylindrotheca fusiformis that is involved in anaphase spindle elongation.

We have identified an 80-kD protein that is involved in mitotic spindle elongation in the diatom Cylindrotheca fusiformis. DSK1 (Diatom Spindle Kinesin 1) was isolated using a peptide antibody raised against a conserved region in the motor domain of the kinesin superfamily. By sequence homology, DSK1 belongs to the central motor family of kinesin-related proteins. Immunoblots using an antibody raised against a non-conserved region of DSK1 show that DSK1 is greatly enriched in mitotic spindle preparations. Anti-DSK1 stains in diatom central spindle with a bias toward the midzone, and staining is retained in the spindle midzone during spindle elongation in vitro. Furthermore, preincubation with anti-DSK1 blocks function in an in vitro spindle elongation assay. This inhibition of spindle elongation can be rescued by preincubating concurrently with the fusion protein against which anti-DSK1 was raised. We conclude that DSK1 is involved in spindle elongation and is likely to be responsible for pushing hal-spindles apart in the spindle midzone.

Inhibition of anaphase spindle elongation in vitro by a peptide antibody that recognizes kinesin motor domain.

Isolated central spindles or spindles in detergent-permeabilized cells from the diatom Cylindrotheca fusiformis can undergo ATP-dependent reactivation of spindle elongation in vitro. We have used a peptide antibody raised against a 10-amino acid portion common to the kinesin superfamily motor domain to look for kinesin-like motor activity during anaphase B of mitosis. The peptide antibody localizes to central spindles. Upon ATP reactivation of spindle elongation, antigens recognized by the antibody are associated exclusively with the central spindle midzone where antiparallel microtubules of each half-spindle overlap. The antibody recognizes several polypeptides by immunoblot using isolated spindle extracts. One of these polypeptides behaves like kinesin with respect to nucleotide-specific binding to and release from taxol-stabilized microtubules. Preincubation of the spindle model with the peptide antibody inhibits subsequent ATP reactivation of spindle elongation. Coincubation of the peptide antibody with peptide antigen rescues spindle function. These results support a role for kinesin-related protein(s) in spindle elongation (anaphase B) of mitosis and suggest that one or several polypeptides that we have identified in spindle extracts may fulfill this function.

Biochemical heterogeneity in xeroderma pigmentosum complementation group E.

Cells from two patients with xeroderma pigmentosum complementation group E (XP-E) have been shown to lack an activity which binds specifically to UV-irradiated DNA (Chu and Chang, 1988). We investigated the occurrence of this binding activity in cell strains from nine additional, unrelated XP-E patients and found that all but one of these strains contained normal levels of the binding protein. Furthermore, the binding activity from these XP-E strains was indistinguishable from that of normal controls in thermal stability, behavior on ion-exchange chromatography, and electrophoretic mobility of protein-DNA complexes, indicating that there were no gross structural alterations in the protein. The association of XP-E with a deficiency in DNA-damage binding protein in cells from 3 of 12 XP-E patients (compared to 0 of 20 non-XP-E controls) is statistically significant (p less than 0.05), but there is no obvious correlation between the biochemical defect and the clinical or cellular characteristics of individual patients. Implications of these findings for the role of the binding protein in XP-E are discussed.