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Item Open Access Anaphase Chromosomes in Crane-Fly Spermatocytes Treated With Taxol (Paclitaxel) Accelerate When Their Kinetochore Microtubules Are Cut: Evidence for Spindle Matrix Involvement With Spindle Forces(Frontiers, 2018-07-24) Forer, Arthur; Sheykhani, Rozhan; Berns, MichaelVarious experiments have indicated that anaphase chromosomes continue to move after their kinetochore microtubules are severed. The chromosomes move poleward at an accelerated rate after the microtubules are cut but they slow down 1–3 min later and move poleward at near the original speed. There are two published interpretations of chromosome movements with severed kinetochore microtubules. One interpretation is that dynein relocates to the severed microtubule ends and propels them poleward by pushing against non kinetochore microtubules. The other interpretation is that components of a putative “spindle matrix” normally push kinetochore microtubules poleward and continue to do so after the microtubules are severed from the pole. In this study we distinguish between these interpretations by treating cells with taxol. Taxol eliminates microtubule dynamics, alters spindle microtubule arrangements, and inhibits dynein motor activity in vivo. If the dynein interpretation is correct, taxol should interfere with chromosome movements after kinetochore microtubules are severed because it alters the arrangements of spindle microtubules and because it blocks dynein activity. If the “spindle matrix” interpretation is correct, on the other hand, taxol should not interfere with the accelerated movements. Our results support the spindle matrix interpretation: anaphase chromosomes in taxol-treated crane-fly spermatocytes accelerated after their kinetochore microtubules were severed.Item Open Access Elastic tethers between separating anaphase chromosomes in crane-fly spermatocytes coordinate chromosome movements to the two poles.(Wiley, 2017-02) Forer, Arthur; Berns, Michael; Sheykhani, RozhanSeparating anaphase chromosomes in crane-fly spermatocytes are connected by elastic tethers, as originally described by [LaFountain et al., 2002]: telomere-containing arm fragments severed from the arms move backwards to the partner telomeres. We have tested whether the tethers coordinate the movements of separating partner chromosomes. In other cell types anaphase chromosomes move faster, temporarily, when their kinetochore microtubules are severed. However, in crane-fly spermatocytes the chromosomes move at their usual speed when their kinetochore microtubules are severed. To test whether the absence of increased velocity is because tethers link the separating chromosomes and coordinate their movements, we cut tethers with a laser microbeam and then cut the kinetochore microtubules. After this procedure, the associated chromosome sped up, as in other cells. These results indicate that the movements of partner anaphase chromosomes in crane-fly spermatocytes are coordinated by elastic tethers connecting the two chromosomes and confirm that chromosomes speed up in anaphase when their kinetochore microtubules are severed.Item Open Access The role of actin and myosin in PtK2 spindle length changes induced by laser microbeam irradiations across the spindle(Wiley, 2013-05) Forer, Arthur; Berns, Michael; Shah, Jagesh; Liaw, Lih-Huei; Gomez, Veronica; Baker, Norman; Sheykhani, RozhanThis study investigates spindle biomechanical properties to better understand how spindles function. In this report, laser microbeam cutting across mitotic spindles resulted in movement of spindle poles toward the spindle equator. The pole on the cut side moved first, the other pole moved later, resulting in a shorter but symmetric spindle. Intervening spindle microtubules bent and buckled during the equatorial movement of the poles. Because of this and because there were no detectable microtubules within the ablation zone, other cytoskeletal elements would seem to be involved in the equatorial movement of the poles. One possibility is actin and myosin since pharmacological poisoning of the actin-myosin system altered the equatorial movements of both irradiated and un-irradiated poles. Immunofluorescence microscopy confirmed that actin, myosin and mono-phosphorylated myosin are associated with spindle fibres and showed that some actin and mono-phosphorylated myosin remained in the irradiated regions. Overall, our experiments suggest that actin, myosin and microtubules interact to control spindle length. We suggest that actin and myosin, possibly in conjunction with the spindle matrix, cause the irradiated pole to move toward the equator and that cross-talk between the two half spindles causes the un-irradiated pole to move toward the equator until a balanced length is obtained.Item Open Access The role of myosin phosphorylation in anaphase chromosome movement(Elsevier, 2013-04) Sheykhani, Rozhan; Shirodkar, Purnata V.; Forer, ArthurThis 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 biphosphorylated 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 2Pmyosin. 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.