U73122 is widely accepted as a specific inhibitor of PLC. Nevertheless, its mechanism of action has yet to be elucidated. In some systems, NEM mimics treatment with U73122 , raising the possibility that previously reported effects of U73122 were due to alkylation of off-target proteins rather than inhibition of PLC. To obtain such effects with NEM, Horowitz et al. treated cells with concentrations of NEM forty-times larger than those used for U73122. The authors suggested that higher concentrations of NEM were required because NEM lacks the lipophilic steroid domain of U73122 that targets the NEM moiety to membranes . Consistent with their observations, we found that the concentration required for NEM to block cytokinesis in crane-fly spermatocytes was twenty-times greater than the minimum effective concentration of U73122. However, unlike U73122, NEM did not cause regression of the cleavage furrow or redistribution of F-actin to the poles of the cell. In addition, the effect of U73122 on cytokinesis was reversible in crane-fly spermatocytes at the minimum effective concentration , whereas the effect of NEM was not. In Drosophila spermatocytes, on the other hand, NEM stopped cleavage furrow ingression at a concentration well below the effective concentration of U73122 , suggesting that NEM has more pleiotropic effects. Indeed, NEM treatment resulted in blebbing of the plasma membrane, an effect not seen after treatment with U73122. These observations show that the effects of U73122 are distinct from those of NEM, and suggest that they are due to inhibition of PLC by U73122, although our data do not rule out a mechanism in which U73122 inhibits PLC by alkylation. Effects of other inhibitors strengthen the argument that a PLC-dependent pathway is involved in cytokinesis.
A second PLC inhibitor, ET-18-OCH3, had effects on cytokinesis similar to U73122. ET-18-OCH3 is believed to act by inserting into cellular membranes. Like U73122, ET-18-OCH3 caused cleavage furrow regression and redistribution of F-actin to the poles of the cell. These inhibitors have two known overlapping cellular effects: inhibition of PLC and release of Ca2+ from intracellular stores. In previous experiments using Drosophila spermatocytes, we showed that treatments predicted to greatly influence intracellular Ca2+ levels had little effect on cytokinesis. Treatment of cells with Ca2+ ionophores A23187 or ionomycin did not block cleavage, although they did induce cell contractility and occasional ectopic cell fusion events . In contrast, chelation of intracellular Ca2+ with the cell permeable chelator BAPTA-AM did block cytokinesis, but only when cells were cultured in buffer lacking Ca2+ . Thus, at least Drosophila spermatocytes can divide provided they have either an intracellular or an extracellular source of Ca2+, rendering it unlikely that any effect of ET-18-OCH3 or U73122 on intracellular Ca2+ stores would have blocked cytokinesis in these cells. The simplest explanation for the common effect of U73122 and ET-18-O-CH3 is that PLC activity is required for cytokinesis.
The requirement for PLC activity in cytokinesis appears to be conserved. Several mammalian PLC isoforms, PLCδ1, PLCβ1 and PLCδ3, were found to localize to the cleavage furrows of HeLa, NIH3T3 and MDCK cells during cytokinesis [33, 34]. Moreover, PLCγ is tyrosine phosphorylated, and presumably activated, in dividing sea urchin embryos . U73122, which blocks cleavage in sea urchin embryos , and ET-18-OCH3, which was originally found to interfere with cytokinesis in transformed cells and tumor cell lines [27, 28], were recently found to inhibit cytokinesis in NIH3T3 cells . Although there are currently no genetic studies showing a role for individual PLCs in cytokinesis in any system, this is likely due to redundancy, as most organisms contain multiple PLCs.
In dividing crane-fly and Drosophila spermatocytes, two PLC inhibitors, U73122 and ET-18-OCH3, cause cleavage furrow regression (; this study). Inhibition of PLC would be expected to result in an increase in PIP2, concomitant with a decrease in the second messengers DAG and IP3. PIP2 is required for cytokinesis in Drosophila spermatocytes and mammalian cells [10, 36, 37]. However, it is unclear if an increase in PIP2 would be deleterious to the cell. On the other hand, inhibition of the IP3 receptor with 2-APB or chelation of Ca2+ with BAPTA-AM blocked continued cleavage in Drosophila spermatocytes and zebrafish embryos [10, 38, 39]. Furthermore, we previously showed that treatment of spermatocytes with a Ca2+ ionophore prevented the cells from responding to U73122, strongly suggesting Ca2+ is a key downstream effector of PIP2 hydrolysis during cytokinesis . Effects of the MLCK inhibitor ML-7 confirm this interpretation.
MLCK, a potential target of Ca2+ during cytokinesis, phosphorylates myosin regulatory light chain on Ser-19, and to a lesser extent on Thr-18. This diphosphorylation activates non-muscle myosin II, allowing it to form bipolar thick filaments postulated to constrict F-actin in the contractile ring [40–42]. Although recent experiments have focused on roles for other myosin activating kinases, Rho kinase and citron kinase, in cytokinesis , MLCK may also have a crucial role in this process. Indeed, treatment of both crane-fly and Drosophila spermatocytes with the MLCK inhibitor ML-7 reversibly blocked cytokinesis (; this study). Similarly, ML-7 interferes with cytokinesis in sea urchin embryos  and with maintenance of F-actin in the contractile ring in mammalian cells . Strikingly, ML-7 caused cleavage furrow regression and redistribution of actin filaments in a manner similar to U73122 and ET-18-OCH3 (this report). Furthermore, treatment with all three inhibitors caused loss of phosphorylated myosin regulatory light chain (phospho-Sqh) from the equator of the cell. Although ML-7 is reported to have other targets in vitro (e.g., PKA and PKC; ), the most straightforward interpretation of our results is that all three inhibitors interfere with cytokinesis by blocking MLCK activity, myosin regulatory light chain phosphorylation and myosin II contractility.