Both actin and myosin inhibitors affect spindle architecture in PtK1 cells: does an actomyosin system contribute to mitotic spindle forces by regulating attachment and movements of chromosomes in mammalian cells?

Abstract

Immunocytochemical techniques are used to analyze the effects of both an actin and myosin inhibitor on spindle architecture in PtK(1) cells to understand why both these inhibitors slow or block chromosome motion and detach chromosomes. Cytochalasin J, an actin inhibitor and a myosin inhibitor, 2, 3 butanedione 2-monoxime, have similar effects on changes in spindle organization. Using primary antibodies and stains, changes are studied in microtubule (MT), actin, myosin, and chromatin localization. Treatment of mitotic cells with both inhibitors results in detachment or misalignment of chromosomes from the spindle and a prominent buckling of MTs within the spindle, particularly evident in kinetochore fibers. Evidence is presented to suggest that an actomyosin system may help to regulate the initial and continued attachment of chromosomes to the mammalian spindle and could also influence spindle checkpoint(s).

Measurements of forces produced by the mitotic spindle using optical tweezers

“…Briefly, the cells were incubated at 37°C with 5% CO2. A fibrinogen-thrombin clot was used as previously described to adhere the cells to the coverslip before irradiation (Forer and Pickett-Heaps, 2005; Snyder et al. , 2010, Sheykhani et al , 2013).…”

MCAK regulates chromosome alignment but is not necessary for preventing aneuploidy in mouse oocyte meiosis I

“…Alternatively, chromosome misalignment might reflect a more general effect of MCAK perturbation upon spindle MTs. The mechanisms of chromosome positioning in oocyte meiosis I remain relatively poorly studied, although it is interesting in this context that recent studies have revealed that microfilaments surround the spindle in mouse meiosis I (Azoury et al, 2008;Li et al, 2008;Schuh and Ellenberg, 2008) and that actin/myosin perturbation causes chromosome misalignment in some systems (Snyder et al, 2009).…”

“…Alternatively, chromosome misalignment might reflect a more general effect of MCAK perturbation upon spindle MTs. The mechanisms of chromosome positioning in oocyte meiosis I remain relatively poorly studied, although it is interesting in this context that recent studies have revealed that microfilaments surround the spindle in mouse meiosis I (Azoury et al, 2008;Li et al, 2008;Schuh and Ellenberg, 2008) and that actin/myosin perturbation causes chromosome misalignment in some systems (Snyder et al, 2009).Most importantly, our experiments employing MCAK depletion, RAMFLhyp-RFP expression, both together, or MCAK depletion paired with monastrol-induced spindle collapse, all failed to uncover a requirement for MCAK in preventing segregation errors, indicating that, unlike in mitosis, MCAKmediated error correction at the kinetochore is not necessary to prevent aneuploidy in oocyte meiosis I. Although we cannot formally exclude the possibility that undetectable levels of residual pre-existing MCAK might persist in the morpholino experiments, the additional use of RAMFLhyp-RFP supports the notion that MCAK is dispensable.…”

The regulation of the actin cytoskeleton and cell cycle progression

“…Both actin depolymerising drugs such as cytochalasin D, cytochalasin J and latrunculin B (Fabian and Forer, 2007;Forer and Pickett-Heaps, 1998;Snyder et al), and actinpolymerising drugs such as jasplakinolide (Xie and Forer, 2008), has been found to alter anaphase chromosome movement, further supporting our idea that the alterations of actin structures by addition of Y27632 and C3 transferase may alter force distribution with the cell that is required for proper timing of chromosome separation.…”