THE EFFECT OF CYTOCHALASIN J ON KINETOCHORE STRUCTURE IN PTK₁CELLS IS MITOTIC CYCLE DEPENDENT

Abstract

Mitotic PtK1 cells were arrested in mitosis with nocodazole to determine the effect of cytochalasin J (CJ) on kinetochore structure in arrested and nocodazole-released cells. In previous studies it was shown that CJ had a more pronounced effect on alteration of kinetochore structure and spindle microtubule (MT) architecture when applied during prophase or prometaphase. In this study, mitotic cells were treated at preanaphase for 10 min with 1 microg/ml nocodazole, or in 1 microg/ml nocodazole and 10 microg/ml CJ to allow for the advancement of the 'mitotic clock'. Thus it can be determined if either changes in the timing of mitosis, the maturation of the kinetochore, and/or the lack of MT connection to the kinetochore affects the ability of CJ to detach or alter the attachment of chromosomes to the developing spindle. Preanaphase cells treated with 1 microg/ml nocodazole for 10 min and released into 10 microg/ml CJ showed significant changes in MT organization and kinetochore structure. MTs nucleated at the centrosome are fragmented and kinetochore structure was significantly altered showing only two laminae with few MTs inserted into this structure. Preanaphase cells treated with 1 microg/ml nocodazole and 10 microg/ml CJ for 10 min and released into 10 microg/ml CJ showed similar, but more pronounced, effects on kinetochore structure and spindle MT organization. We interpret these results to suggest that CJ treatment has a greater effect on MT attachment and kinetochore structure in nocodazole pre-treated cells, where the kinetochore structure is mature and the mitotic cycle has been advanced.

Possible roles of actin and myosin during anaphase chromosome movements in locust spermatocytes

“…Y-27632 and BDM were diluted ~1000x from stock solutions made up in Ringers solution; Latrunculin B, cytochalasin D and ML-7 were diluted ~1000x from stock solutions made up in dimethylsulphoxide (DMSO). DMSO at its final concentration of 0.1% has no effect on locust spermatocytes (e.g., Daub and Hauser, 1988), similar to the lack of effect of DMSO on crane-fly spermatocytes (e.g., Pickett-Heaps, 1998, at 0.2% DMSO, andLaFountain et al, 1992, at 1% DMSO) or on PtK cells (Wrench and Snyder, 2001, at 0.4% DMSO). The perfusion of Ringers solution itself had no effect on locust spermatocyte division either, similar to results in crane-fly spermatocytes (Forer and Pickett-Heaps, 1998).…”

Fibrin clots keep non-adhering living cells in place on glass for perfusion or fixation

“…Other cells such as tissue culture cells are maintained in aqueous medium, adhere to coverslips, and readily can be treated with altered medium, but even these cells can present problems. For example, they round up when treated with anti-actin drugs, complicating or preventing observations on internal events (Yvon et al, 2001); holding tissue culture cells in place in a clot prevents them from changing shape and allows study of internal events (Snyder and Cohen, 1995;Wrench and Snyder, 2001).…”

“…The clot technique requires a physiological saline solution to keep alive those particular cells one wants to study, so it probably can be applied to all cell types for which a physiological saline solution is known. Embedment in a fibrin clot has been used successfully to hold in place crane-fly spermatocytes (Forer and Pickett-Heaps, 1998a), Haemanthus endosperm cells (Czaban and Forer, 1992), flea-beetle spermatocytes (Forer and Wilson, 2000;Wilson et al, 2003), locust spermatocytes , Drosophila spermatocytes (Wong et al, 2005), PtK cells (Snyder and Cohen, 1995;Wrench and Snyder, 2001), Chlorella, Paramecium and sea urchin zygotes (Inada et al, 1977), mouse oocytes (Hunt et al, 1995;Hodges et al, 2002;Hunt et al, 2003), sea urchin zygotes and various erythrocytes (Lee et al, 1998), and Mesostoma spermatocytes (Forer and Pickett-Heaps, unpublished).…”