Abstract
Actin and myosin inhibitors often blocked anaphase movements in insect spermatocytes in previous experiments. Here we treat cells with an enhancer of myosin, Calyculin A, which inhibits myosin-light-chain phosphatase from dephosphorylating myosin; myosin thus is hyperactivated. Calyculin A causes anaphase crane-fly spermatocyte chromosomes to accelerate poleward; after they reach the poles they often move back toward the equator. When added during metaphase, chromosomes at anaphase move faster than normal. Calyculin A causes prometaphase chromosomes to move rapidly up and back along the spindle axis, and to rotate. Immunofluorescence staining with an antibody against phosphorylated myosin regulatory light chain (p-squash) indicated increased phosphorylation of cleavage furrow myosin compared to control cells, indicating that calyculin A indeed increased myosin phosphorylation. To test whether the Calyculin A effects are due to myosin phosphatase or to type 2 phosphatases, we treated cells with okadaic acid, which inhibits protein phosphatase 2A at concentrations similar to Calyculin A but requires much higher concentrations to inhibit myosin phosphatase. Okadaic acid had no effect on chromosome movement. Backward movements did not require myosin or actin since they were not affected by 2,3-butanedione monoxime or LatruculinB. Calyculin A affects the distribution and organization of spindle microtubules, spindle actin, cortical actin and putative spindle matrix proteins skeletor and titin, as visualized using immunofluorescence. We discuss how accelerated and backwards movements might arise.
Highlights
Mechanisms of chromosome movements during anaphase have been investigated extensively and several models attempt to explain the forces involved [1,2,3,4]
Calyculin A added in anaphase We tested various concentrations of CalA, from 5 nM to 0.5 μM, in order to determine the lowest concentration that affects chromosome movement
In this study we showed that CalA, an enhancer of myosin, added during anaphase or metaphase, speeds up anaphase chromosome movement
Summary
Mechanisms of chromosome movements during anaphase have been investigated extensively and several models attempt to explain the forces involved [1,2,3,4]. In this article we present data dealing with spindle myosin. Myosin in mitotic cells generally is thought to be involved with cytokinesis, primarily with contractile ring formation and ingression [39,40], and with positioning and orientation of the mitotic spindle [41]. Myosin is present in the spindle [1,15]. Some of the early studies that showed that actin and myosin were present in the spindle discussed a possible role for myosin in force production during anaphase chromosome movement [17,18,4245], but no physiological data were presented.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
