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

Abstract

Isolated central spindles or spindles in detergent-permeabilized celis from the diatom Cylindrotheca fusifornis can undergo ATP-dependent reactivation of spindle elongation in vitro. We have used a peptide antibody raised agains 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 antiparaHlel microtubuies 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.Diatom spindles are important model systems for describing the rearrangements of microtubules (MTs) that occur during anaphase because the MTs associated with spindle elongation are closely packed in an antiparallel overlapping MT array (the central spindle) and are spatially separated from kinetochore and astral MTs (1, 2). Using functional spindles prepared from diatoms we have demonstrated that forces sufficient to drive half-spindles apart during spindle elongation (anaphase B) must be generated in the zone of antiparallel microtubule overlap (3-5). Similar forces generated by mechanochemical interactions in the zone of microtubule overlap may play a role during mitosis in most eukaryotic cells (6-9). The pennate diatom Cylindrothecafusiformis can be readily grown in bulk cultures suitable for biochemistry and the cells can be arrested in mitosis by a combination of light/dark cell cycle synchronization and drug arrest. At harvest, 40-60% of the cells contain 4-to 5-,Am-long metaphase/early anaphase spindles and interphase MT arrays are absent (10). After isolation or cell permeabilization the two overlapping half-spindles that comprise the central spindle retain the ability to slide apart when incubated with 1 mM

Going mobile: microtubule motors and chromosome segregation.

“…In this regard, the identification of KLP families with distinct motor properties may lead to the identification of KLP "family-specific" inhibitors that can be used to target the elimination of an entire KLP family during cell division. The ability of injected "pan" antibodies to inhibit mitosis, when directed against the most conserved regions of the kinesin motor domain, and hence against multiple kinesins simultaneously, shows the potential for this type of approach (104,117,118).…”

Model for anaphase B: Role of three mitotic motors in a switch from poleward flux to spindle elongation

“…Anaphase B is driven in part by a bipolar kinesin-dependent sliding filament mechanism (2)(3)(4)(5)(6)(7)(8)(9), with the extent of spindle elongation determined by MT polymerization in the overlap zone (2). Poleward flux, the movement of tubulin subunits from the MT plus ends facing the spindle equator to their minus ends at the poles (10)(11)(12)(13)(14), is proposed to constrain the length of metaphase spindles, with subsequent inhibition of depolymerization at the poles converting metaphase flux to anaphase spindle elongation (12,15,16).…”

A kinesin-like protein, KatAp, in the cells of arabidopsis and other plants.

“…Polypeptides with plus-end-directed motor activity also have been reported in isolated phragmoplasts (Asada and Shibaoka, 1994). Plus-end-directed KLPs have been implicated in the operation of the central spindle in diatoms (Hogan et al, 1992(Hogan et al, , 1993 as well as in the function or assembly of Chlamydomonas flagella (see Bernstein and Rosenbaum, 1994;Walther et al, 1994). Recently, several genes (Kat genes) encoding KLPs were identified in Arabidopsis by using PCR (Mitsui et al, 1993(Mitsui et al, , 1994.…”

“…One or more such motors may also play a role in centrosome organization (Endow et al, 1994a). KLPs associated with the kinetochores may be responsible for elements of chromosome motion, including congression and anaphase A and B (Bloom and Endow, 1994;Wordeman and Mitchison, 1995;Hogan et al, 1992Hogan et al, ,1993. Cytoplasmic dynein, a minusend-directed motor, may cooperate with one or more KLPs to accomplish spindle elongation and may also govern or participate in nuclear migration (Eshel et al, 1993;Li et al, 1993;Plamann et al, 1994;Xiang et al, 1994;Saunders et al, 1995;Yeh et ai., 1995;see also Schroer, 1994) and the initial organization or stabilization of the mitotic apparatus (Vaisberg et al, 1993).…”

“…Indeed, Bloom and Endow (1994) have proposed that, in the absence of the focusing action of a discrete centrosome, one or more minus-end-directed motors, such as KatAp, play an important role in organizing the plant spindle poles. It is worth reiterating, however, that KatAp is not localized at the poles in intact cells.The same motor could also function during cytokinesis, maintaining the distance between daughter nuclei by counterbalancing plus-end-directed microtubule motors that would tend to push the nuclei apart through microtubule-microtubule sliding of the kind reported to occur in the central spindle of diatoms (Hogan et al, 1992(Hogan et al, ,1993. Kakimoto and Shibaoka (1989) reported that the distance between daughter nuclei is maintained in isolated phragmoplasts in which microtubules are eliminated during cell lysis.…”