Actin and myosin inhibitors block elongation of kinetochore fibre stubs in metaphase crane-fly spermatocytes

Summary. We used an ultraviolet microbeam to cut individual kinetochore spindle fibres in metaphase crane-fly spermatocytes. We then followed the growth of the “kinetochore stubs”, the remnants of kinetochore fibres that remain attached to kinetochores. Kinetochore stubs elongate with constant velocity by adding tubulin subunits at the kinetochore, and thus elongation is related to tubulin flux in the kinetochore microtubules. Stub elongation was blocked by cytochalasin D and latrunculin A, actin inhibitors, and by butanedione monoxime, a myosin inhibitor. We conclude that actin and myosin are involved in generating elongation and thus in producing tubulin flux in kinetochore microtubules. We suggest that actin and myosin act in concert with a spindle matrix to propel kinetochore fibres poleward, thereby causing stub elongation and generating anaphase chromosome movement in nonirradiated cells. Correspondence: A. Forer, Biology Department, York University, 4700 Keele Street, Toronto, ON M3J 1P3, Canada.     

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

Summary. 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. Supplementary material to this paper is available in electronic form at http://dx.doi.org/10.1007/s00709-005-0094-6 Correspondence and reprints: Biology Department, York University, 4700 Keele Street, Toronto, ON M3J 1P3, Canada.     

The role of myosin phosphorylation in anaphase chromosome movement

This work deals with the role of myosin phosphorylation in anaphase chromosome movement. Y27632 and ML7 block two different pathways for phosphorylation of the myosin regulatory light chain (MRLC). Both stopped or slowed chromosome movement when added to anaphase crane-fly spermatocytes. To confirm that the effects of the pharmacological agents were on the presumed targets, we studied cells stained with antibodies against mono- or bi-phosphorylated myosin. For all chromosomes whose movements were affected by a drug, the corresponding spindle fibres of the affected chromosomes had reduced levels of 1P- and 2P-myosin. Thus the drugs acted on the presumed target and myosin phosphorylation is involved in anaphase force production.Calyculin A, an inhibitor of MRLC dephosphorylation, reversed and accelerated the altered movements caused by Y27632 and ML-7, suggesting that another phosphorylation pathway is involved in phosphorylation of spindle myosin. Staurosporine, a more general phosphorylation inhibitor, also reduced the levels of MRLC phosphorylation and caused anaphase chromosomes to stop or slow. The effects of staurosporine on chromosome movements were not reversed by Calyculin A, confirming that another phosphorylation pathway is involved in phosphorylation of spindle myosin.     

Ultraviolet microbeam irradiations of spindle fibres in crane-fly spermatocytes and newt epithelial cells: Resolution of previously conflicting observations

Summary In order to resolve apparent differences in reported experiments, we directly compared the effects of ultraviolet (UV) microbeam irradiations on the behaviour of spindle fibres in newt epithelial cells and crane-fly spermatocytes, using the same apparatus for both cell types. This work represents the first time that irradiated crane-fly spermatocytes have been followed using a high-NA objective and video-enhancement of images. In both cell types, irradiation of a kinetochore fibre in metaphase produced an area of reduced birefringence (ARB), known to be devoid of spindle microtubules (MTs). Subsequently the kinetochore-ward edge of the ARB moved poleward with average velocities of 0.5 m/min (n=20) in spermatocytes and 1.1 m/min (n=6) in epithelial cells. The poleward edge of the ARB rapidly disappeared when viewed using a ×100, high-NA objective but generally remained visible when viewed with a ×32, low-NA objective; this difference suggests that MTs poleward from the ARB disperse vertically out of the narrow depth of field of the ×100 objective but that many remain encompassed by that of the ×32 objective. The primary difference in response between the two cell types was in the behaviour of the spindle poles after an ARB formed. In spermatocytes the spindle maintained its original length whereas in epithelial cells the pole on the irradiated side very soon moved towards the chromosomes, after which the other pole did the same and a much shortened functional metaphase spindle was formed.     

Checkpoint control in crane-fly spermatocytes: unattached chromosomes induced by cytochalasin D or latrunculin treatment do not prevent or delay the start of anaphase

Summary Variable numbers of bivalents and sex chromosomes do not attach to the spindle when prophase or early prometaphase cranefly spermatocytes (2n=8) are treated with cytochalasin D or latrunculin. The unattached bivalents lie in the cytoplasm or at the spindle pole, and they do not delay onset of autosomal anaphase; sometimes they disjoin at the same time as the attached bivalents, so they respond to the global signals that initiate anaphase. Unattached sex chromosomes do not delay autosomal anaphase, either. Of various interpretations of these data, we think the best explanation is that the checkpoint system responds to physical rather than chemical cues; we think that the spindle is a tensegral structure, that chromosomes need to interact with the spindle in order to be recognised by the anaphase-onset checkpoint control, and that the physical interaction of chromosomes with spindle acts as a signalling network. Cytochalasin D and latrunculin treatments delay onset of sex chromosome anaphase (which normally occurs about 15 min after autosomal anaphase) and cause altered patterns of sex-chromosome segregation.