✍ Scribed by LaFountain, James R. ;Siegel, Alan J. ;Rickards, Geoffrey K.
No coin nor oath required. For personal study only.
Cytochalasin D (CD) was applied to crane-fly spermatocytes at late diakinesis with the aim of perturbing actin structure and actin function, thereby testing the hypothesis that intranuclear chromosome movement during late diakinesis is actin-based. Isolated testes were incubated in a range of CD concentrations (2-100 µM) for 1 or 2 h. None of those treatments resulted in cessation of prophase movements in living cells. An immediate effect of 10-100 µM CD at late diakinesis was the formation of highly refractile, actin-containing cables within the nonchromosomal nucleoplasm. No such cables were observed in vehicle-treated control cells. CD treatments caused autosomal bivalents in unusually large numbers of spermatocytes to become aggregated into densely-packed clusters; for example, with 40 µM CD about 80% of late diakinesis spermatocytes had clustered autosomes, vs. about 25% clustering in untreated cells. We conclude from these data that the mechanism of chromosome positioning at the nuclear envelope is CD-sensitive. Rhodamine-conjugates of phalloidin and DNase I were used to assess the status of actin in untreated cells as well as the effect of CD on actin distribution. Differences in nucleoplasmic staining with phalloidin and DNase I conjugates suggest that nucleoplasm at late diakinesis contains actin in a nonfilamentous form.
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