ABSTRACT

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Studies were conducted to determine whether the microtubules present within native spindles isolated from eggs of the surf clam, Spisula solidissima, could bind dynein obtained from axonemes of Tetrahymena thermophila . SDS gel electrophoresis revealed that the high molecular weight polypeptides that make up dynein cosedimented with the isolated spindles . Moreover, the ATPase activity of dynein bound to the spindle microtubules was stimulated approximately sevenfold . The birefringence retardation of spindles incubated without dynein decreased from 1 .4 nm to an undetectable level within 45 min, whereas that of spindles incubated for the same period of time with dynein was 1 .0 nm,^"70% of its initial value, thereby indicating that dynein stabilized spindle birefringence. Ultrastructural analysis revealed that each spindle microtubule was decorated with four to seven dynein arms attached by their "B" end, that which cross-bridges the B-subfiber within native axonemes . In addition, the polarity of the spindle microtubules could be determined by the orientation of the bound dynein arms . The results of these studies suggest that the half-spindle is composed of microtubules possessing the same polarity .Microtubules possess structural polarity, a property which may be involved in their ability to elicit directional, intracellular movements. Within the axoneme, microtubule doublets are of the same polarity, with their assembly or "plus" end located distal to the cell body (1, 5, 7) . The dynein arms on the Asubfiber of one microtubule doublet cyclically cross-bridge the B-subfiber of the adjacent doublet (15), causing it to slide toward the distal end of the axoneme (44) . Accordingly, the direction of microtubule sliding may be a manifestation of intrinsic microtubule polarity .It has been proposed that, during mitosis, sliding between microtubules of opposite polarity occurs and results in the separation of the mitotic poles (31,34,38) and in the procession of the chromosomes to the poles (34,38) . Recent studies involving serial sections and subsequent microtubule reconstruction of the central spindle of Diatoma vulgare during metaphase and anaphase demonstrate a change in microtubule profile, indicating that two groups of interdigitating, antiparallel microtubules may slide with respect to each other and result in spindle elongation (32,33,35) .Microtubule sliding within the mitotic apparatus would require the presence of a force-generating protein, and evidence exists for the participation of a dyneinlike ATPase in generating mitotic movements . For example, spindles isolated from sea urchin eggs contain both Mg"-stimulated ATPase activity and

Basal bodies isolated from Chlamydomonas reinhardi will serve as initiation centers for the assembly of chick brain microtubule protein subunits (tubulin) into microtubules. The rate of microtubule assembly is tubulin-concentration dependent; this assembly occurs onto both distal and proximal ends of the basal body mnicrotubules, with distal assembly greatly favored. In vitro assembly of brain tubulin also occurs onto the mid-lateral aspects of the basal bodies, presumably onto the fiber connecting the two basal bodies.

Abstract.Previous studies have shown that pigment granule dispersion and aggregation in melanophores of the African cichlid, Tilapia mossambica, are regulated by protein phosphorylation and dephosphorylation, respectively (Rozdzial, M. M., and L. T. Haimo. 1986. Cell. 47:1061-1070. The present studies suggest that calcineurin, a Ca2÷/calmodulin-stimulated phosphatase, is the endogenous phosphatase that mediates pigment aggregation in melanophores. Aggregation, but not dispersion, is inhibited by okadaic acid at concentrations consistent with an inhibition of calcineurin activity. Inhibition of aggregation in melanophores that have been BAPTA loaded or treated with calmodulin antagonists implicate Ca 2+ and calmodulin, respectively, in this process. Moreover, addition of calcineufin rescues aggregation in lysed melanophores which are otherwise incapable of aggregating pigment. Immunoblotting with an anticalcineurin IgG reveals that calcineurin is a component of the dermis, which contains the melanophores, and indirect immunofluorescence localizes calcineurin specifically to the melanophores. Finally, this antibody, which inhibits calcineurin's phosphatase activity (Tash, J. S., M.