Redundant mechanisms for anaphase chromosome movements: crane-fly spermatocyte spindles normally use actin filaments but also can function without them

Abstract

Actin inhibitors block or slow anaphase chromosome movements in crane-fly spermatocytes, but stopping of movement is only temporary; we assumed that cells adapt to loss of actin by switching to mechanism(s) involving only microtubules. To test this, we produced actin-filament-free spindles: we added latrunculin B during prometaphase, 9-80 min before anaphase, after which chromosomes generally moved normally during anaphase. We confirmed the absence of actin filaments by staining with fluorescent phalloidin and by showing that cytochalasin D had no effect on chromosome movement. Thus, actin filaments are involved in normal anaphase movements, but in vivo, spindles nonetheless can function normally without them. We tested whether chromosome movements in actin-filament-free spindles arise via microtubules by challenging such spindles with anti-myosin drugs. Y-27632 and BDM (2,3-butanedione monoxime), inhibitors that affect myosin at different regulatory levels, blocked chromosome movement in normal spindles and in actin-filament-free spindles. We tested whether BDM has side effects on microtubule motors. BDM had no effect on ciliary and sperm motility or on ATPase activity of isolated ciliary axonemes, and thus it does not directly block dynein. Nor does it block kinesin, assayed by a microtubule sliding assay. BDM could conceivably indirectly affect these microtubule motors, though it is unlikely that it would have the same side effect on the motors as Y-27632. Since BDM and Y-27632 both affect chromosome movement in the same way, it would seem that both affect spindle myosin; this suggests that spindle myosin interacts with kinetochore microtubules, either directly or via an intermediate component.

asunderIs a Critical Regulator of Dynein–Dynactin Localization duringDrosophilaSpermatogenesis

“…Dynamic localization of dynein at meiotic spindle poles and kinetochores has been observed in grasshopper and crane fly spermatocytes (King et al, 2000;Fabian and Forer, 2005). Localization of dynein-dynactin during Drosophila male meiosis, however, has not been reported to our knowledge.…”

Titin in insect spermatocyte spindle fibers associates with microtubules, actin, myosin and the matrix proteins skeletor, megator and chromator

“…For one, a putative spindle matrix might provide flexibility and elasticity as an underlying component of spindles, and the matrix might even be involved in force production Fabian and Forer, 2005). We used the term 'putative' because there has been no definitive molecular or morphological description of a matrix, except that the Drosophila proteins skeletor, megator and chromator would seem to be components of (or at least markers of) such a matrix.…”

“…Another protein suggested as a structural component of the spindle matrix is lamin B, an intermediate filament present in the interphase nuclear lamina (Tsai et al, 2006). Because mitotic spindles also contain the muscle proteins actin and myosin SilvermanGavrila and Forer, 2003;Robinson and Snyder, 2005), which seem to be involved in force production in anaphase chromosome movement Fabian and Forer, 2005;, we thought that titin might be involved with spindle elasticity, as it is in muscle. Because spindle elasticity might be associated with a spindle matrix, we tested whether titin was present in spindles and whether it was associated with a spindle matrix.…”

The perpetual movements of anaphase

“…The washout of the drugs usually reverted normal chromosome movement. In a subsequent study, somewhat contradicting findings were reported in the same system where addition of latrunculin B before anaphase did not cause an effect on chromosome movement during anaphase [179]. Under these experimental conditions inhibition of myosin with two different drugs, [180].…”