In vivo effects of ortho-vanadate on spindle structure and dynamics of locust spermatocytes I

Abstract

After external application of vanadate, a potent inhibitor of several ATPases including dynein, the following effects on living spermatocytes I are detectable: spherical metaphase cells change to a lemon shape due to a concentration dependent elongation of the spindle, apparently achieved by pulling the plasma membrane-inserted poles apart, presumably through the assistance of cytoskeletal filaments. The observed dismembering of the spindle seems to be due to the separation of the half-spindle fibres, composed of usually interdigitating kinetochore microtubules (kMTs), free MTs (fMTs) and polar MTs (pMTs). As revealed by microcinematographic recordings, the lengthening of the half-spindles is accompanied by counter-clockwise twisting movements of the polar regions which, after prolonged vanadate treatment, lead to the formation of filiform appendices. Bundles of 5 nm microfilaments, which could be identified by indirect immunofluorescence microscopy (IIF) as actin, are concentrated within these appendices. In spite of a certain derangement of spindle architecture, half of the metaphases in 1 mM vanadate are capable of entering anaphase, but the rates of chromosome-to-pole movement have changed depending on the incubation time and the cell shape developed, respectively. Thus, chromosomes move with the highest speed in lemon-shaped cells but lag in cells with filiform appendices. However, it remains an open question whether the acceleration of chromosome migration is the result of spindle dismemberment or whether the slowing of anaphase motion is the consequence of a far-reaching displacement of the filamentous component from the spindle framework.