Effects of anti‐myosin drugs on anaphase chromosome movement and cytokinesis in crane‐fly primary spermatocytes

Abstract

To investigate whether myosin is involved in crane-fly primary spermatocyte division, we studied the effects of myosin inhibitors on chromosome movement and on cytokinesis. With respect to chromosome movement, the myosin ATPase inhibitor 2,3-butanedione 2-monoxime (BDM) added during autosomal anaphase reversibly perturbed the movements of all autosomes: autosomes stopped, slowed, or moved backwards during treatment. BDM added before anaphase onset altered chromosome movement less than when BDM was added during anaphase: chromosome movements only rarely were stopped. They often were normal initially and then, if altered at all, were slowed. To confirm that the effects of BDM were due to myosin inhibition, we treated cells with ML-7, a drug that inhibits myosin light chain kinase (MLCK), an enzyme necessary to activate myosin. ML-7 affected anaphase movement only when added in early prometaphase: this treatment prevented chromosome attachment to the spindle. We treated cells with H-7 as a control for possible non-myosin effects of ML-7. H-7, which has a lower affinity than ML-7 for MLCK but a higher affinity than ML-7 for other potential targets, had no effect. These data confirm that the BDM effect is on myosin and indicate that the myosin used for chromosome movement is activated near the start of prometaphase. With respect to cytokinesis, BDM did not block furrow initiation but did block subsequent contraction of the contractile ring. When BDM was added after initiation of the furrow, the contractile ring either stalled or relaxed. ML-7 blocked contractile ring contraction when added at all stages after autosomal anaphase onset, including when added during cytokinesis. H-7 had no effect. These results confirm that the effects of BDM are on myosin and indicate that the myosin used for cytokinesis is activated starting from autosomal anaphase and continuing throughout cytokinesis.

Phospholipase C and myosin light chain kinase inhibition define a common step in actin regulation during cytokinesis

“…In control cells, cytokinesis was initiated by invagination of the equatorial plasma membrane to form a furrow between the nascent daughter cells (Figure 2A, B) [see Additional files 1, 2] [10,29]. Ingression continued unabated until the cleavage furrow reached a minimum diameter of one to several micrometers (Figure 2E).…”

“…A concentration range of 75–80 μM was chosen because lower concentrations had little or no effect on cytokinesis ([29]; not shown). In both crane-fly and Drosophila spermatocytes, treatment with 75 μM (crane-fly) or 80 μM ( Drosophila ) ML-7 caused cleavage furrow regression ([29]; Figure 5A) [see Additional file 9].…”

“…A concentration range of 75–80 μM was chosen because lower concentrations had little or no effect on cytokinesis ([29]; not shown). In both crane-fly and Drosophila spermatocytes, treatment with 75 μM (crane-fly) or 80 μM ( Drosophila ) ML-7 caused cleavage furrow regression ([29]; Figure 5A) [see Additional file 9]. Treatment of Drosophila spermatocytes with ML-7 was reversible upon washout (Figure 5B) [see Additional file 9], unlike treatment with U73122, ET-18-OCH 3 or NEM; however, after resuming, ingression arrested prematurely in 21 out of 33 cells.…”

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

“…We also often have followed several spermatocytes consecutively from metaphase through anaphase, or prometaphase through anaphase, with no apparent deleterious changes in the later cells. Crane-fly spermatocytes in clots act the same as spermatocytes smeared under oil using every criterion we have looked at, namely time intervals between nuclear membrane breakdown and anaphase (Forer and Pickett-Heaps, 1998b;LaFountain et al, 2001;Silverman-Gavrila and Forer, 2001), velocities of autosome movements during anaphase (Forer, 1965;Schaap and Forer, 1979;Yin and Forer, 1996;Ilagan and Forer, 1997;LaFountain et al, 2001;Forer, 2003, spermatocytes under oil, compared with Forer andPickett-Heaps, 1998b;Silverman-Gavrila and Forer, 2001;, spermatocytes in clots), time intervals between onset of autosomal anaphase and subsequent sexchromosome anaphase (Schaap andForer, 1979, under oil, compared with Forer andPickett-Heaps, 1998b;Silverman-Gavrila and Forer, 2001, in clots), behaviours of severed spindle fibres and associated chromosomes after ultraviolet microbeam irradiation of spindle fibres (Forer, 1965(Forer, , 1966Wilson and Forer, 1988;Spurck et al, 1997, under oil, compared with Forer et al, 2003, and general appearances of cells and chromosomes. Nor does periodic flow of solution past the cells seem to harm the cells: anaphase velocities are not perturbed by flow of Ringer's solution or DMSO (Forer and Pickett-Heaps, 1998a, b).…”

“…For example, when living crane-fly spermatocytes are smeared under halocarbon oil the chromosomes and other components are seen clearly, but the cells do not adhere to coverslips and cannot be treated readily with aqueous media. When held in a fibrin clot, however, the cells remain in place and do not change shape on being perfused with drugs (Wilson and Forer, 1997;Silverman-Gavrila and Forer, 2001;Forer and Pickett-Heaps, 1998a;Saul et al, 2004;, or with lysis buffer for immunofluorescence (Wilson and Forer, 1997;SilvermanGavrila and Forer, 2001;Forer et al, 2003;Saul et al, 2004;, or with fixative . 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.…”