Phospholipase C and myosin light chain kinase inhibition define a common step in actin regulation during cytokinesis
© Wong et al; licensee BioMed Central Ltd. 2007
Received: 06 February 2007
Accepted: 17 May 2007
Published: 17 May 2007
Phosphatidylinositol 4,5-bisphosphate (PIP2) is required for successful completion of cytokinesis. In addition, both PIP2 and phosphoinositide-specific phospholipase C (PLC) have been localized to the cleavage furrow of dividing mammalian cells. PLC hydrolyzes PIP2 to yield diacylglycerol (DAG) and inositol trisphosphate (IP3), which in turn induces calcium (Ca2+) release from the ER. Several studies suggest PIP2 must be hydrolyzed continuously for continued cleavage furrow ingression. The majority of these studies employ the N-substituted maleimide U73122 as an inhibitor of PLC. However, the specificity of U73122 is unclear, as its active group closely resembles the non-specific alkylating agent N-ethylmaleimide (NEM). In addition, the pathway by which PIP2 regulates cytokinesis remains to be elucidated.
Here we compared the effects of U73122 and the structurally unrelated PLC inhibitor ET-18-OCH3 (edelfosine) on cytokinesis in crane-fly and Drosophila spermatocytes. Our data show that the effects of U73122 are indeed via PLC because U73122 and ET-18-OCH3 produced similar effects on cell morphology and actin cytoskeleton organization that were distinct from those caused by NEM. Furthermore, treatment with the myosin light chain kinase (MLCK) inhibitor ML-7 caused cleavage furrow regression and loss of both F-actin and phosphorylated myosin regulatory light chain from the contractile ring in a manner similar to treatment with U73122 and ET-18-OCH3.
We have used multiple inhibitors to examine the roles of PLC and MLCK, a predicted downstream target of PLC regulation, in cytokinesis. Our results are consistent with a model in which PIP2 hydrolysis acts via Ca2+ to activate myosin via MLCK and thereby control actin dynamics during constriction of the contractile ring.
Cell proliferation and growth require the coordination of cell signaling pathways. PLC plays an important role in cell signaling, mediating transduction of signals from a variety of intracellular and extracellular stimuli [1, 2]. PLC-dependent hydrolysis of PIP2 produces the second messengers DAG and IP3 (reviewed in ). IP3 binds specific receptors on the ER to mobilize calcium (Ca2+) from internal stores. DAG and Ca2+ activate protein kinase C (PKC), which stimulates cell growth . In addition, Ca2+ itself facilitates diverse cellular events including membrane trafficking, contractility and proliferation . PLC-dependent pathways thus play key roles in promoting cell growth.
Using inhibitors affecting PI metabolism, we previously showed that continuous PI cycling is required for cytokinesis . In particular, treatment of cells with U73122 resulted in regression of the cleavage furrow and failure of cytokinesis [9, 10]. The effects of U73122 suggested PIP2 hydrolysis is required for normal cytokinesis. Indeed, cytokinesis failed in cells treated with an IP3 receptor antagonist or in which intracellular Ca2+ was chelated by BAPTA-AM . Perhaps most strikingly, our data indicated that PIP2 hydrolysis may be required to maintain F-actin in the contractile ring .
U73122 may exert non-specific effects, however, because several lines of evidence suggest that U73122 may affect phosphoinositide cycling or Ca2+ levels independent of PLC inhibition. In rabbit platelets, U73122 caused up to 50% reduction in the levels of phosphatidylinositol 4-phosphate (PIP) and PIP2, but not PI, indicating that U73122 may inhibit both PI and PIP kinases. Although IP3 levels were reduced in agonist-stimulated cells, consistent with inhibition of PLC, this may have been an indirect effect of a shortage in substrate availability . In U73122-treated mouse fibroblasts and rat neutrophils, inhibition of Ca2+ influx across the plasma membrane contributed to the suppression of Ca2+ levels [12, 13]. In contrast, U73122 actually increased Ca2+ release from internal stores in rat pancreatic acinar cells and rat liver microsomes [14, 15].
A second PLC inhibitor, the lysophosphatidylcholine analogue ET-18-OCH3 (Figure 1D; ), is of interest in cancer therapy because of its demonstrated anti-tumor properties . ET-18-OCH3 and related compounds inhibit phosphatidylinositol 3-kinases and PKC [18–21] and selectively promote tumor cell apoptosis [17, 22]. Like U73122, ET-18-OCH3 also has other off-target effects, including release of Ca2+ from intracellular stores . However, unlike U73122, ET-18-OCH3 is inserted in the plasma membrane, and its effects may be due in part to alteration of membrane lipid and protein composition [24–26]. In both human histiocytic lymphoma and tumorigenic rat liver cells, ET-18-OCH3 inhibits cytokinesis [27, 28].
To determine if U73122 inhibits cytokinesis via PLC, we compared the effects of U73122 with those of ET-18-OCH3 and NEM. We further examined the role of a potential downstream target of PLC activity, MLCK. We found that inhibition of MLCK perturbed cytokinesis in a manner similar to inhibition of PLC by U73122 and ET-18-OCH3. Our results are consistent with a model in which PLC and MLCK act in the same pathway to maintain the integrity of the contractile ring, implicating MLCK as a target of Ca2+ regulation in this process.
PLC inhibition causes cleavage furrows to regress
Additional file 1: Meiosis in a crane-fly spermatocyte. Time-lapse phase-contrast micrographs of a dividing crane-fly primary spermatocyte. Times are hr:min:s. Movie corresponds to Figure 2A. (MPG 2 MB)
Additional file 3: Inhibition of PLC with U73122 causes cleavage furrow regression in a dividing crane-fly spermatocyte. Dividing crane-fly spermatocyte treated with 4.3 μM U73122 at 1 min 36 sec (14:51:00). Note that this cell was followed for only a short time after treatment. Times are hr:min:s. Movie corresponds to Figure 2C. (MPG 1 MB)
Additional file 5: Inhibition of PLC with ET-18-OCH3 causes cleavage furrow regression in a dividing crane-fly spermatocyte. Dividing crane-fly spermatocyte treated with 35 μM ET-18-OCH3 at 13:32:00. Times are hr:min:s. Movie corresponds to Figure 3A. (MPG 803 KB)
Additional file 6: Inhibition of PLC with ET-18-OCH3 causes cleavage furrow regression in a dividing Drosophila spermatocyte. Dividing Drosophila spermatocyte treated with 35 μM ET-18-OCH3 at 5 min. 35 sec. Times are hr:min:s:ms. Movie corresponds to Figure 3B. (MPG 1 MB)
Alkylation stops furrow ingression but does not cause regression
Additional file 7: The alkylating agent NEM inhibits cytokinesis in a dividing crane-fly spermatocyte. Dividing crane-fly spermatocyte treated with 1 mM NEM at 15:02:00. Times are hr:min:s. Movie corresponds to Figure 4A. (MPG 1 MB)
Myosin Light Chain Kinase (MLCK) inactivation causes cleavage furrows to regress
PLC and MLCK activities are required to maintain actin at the cleavage furrow
PLC and MLCK activities are required to maintain activated myosin
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.
We showed that two different PLC inhibitors, U73122 and ET-18-OCH3, have similar effects on cleavage furrow stability, F-actin localization and myosin regulatory light chain phosphorylation, indicating a role for PLC activity in cleavage furrow ingression. Moreover, inhibition of MLCK with ML-7 affects cytokinesis in a similar manner, suggesting that PIP2 hydrolysis and Ca2+ release stimulate non-muscle myosin II to maintain actin-myosin interactions and stability of the contractile ring.
Live spermatocyte preparation
Preparation of crane-fly spermatocytes in fibrin clots has been described in detail elsewhere ; a modified protocol was used to examine Drosophila spermatocytes . Briefly, testes from fourth instar crane-fly (Nephrotoma suturalis (Loew)) or third instar Drosophila melanogaster were dissected in Insect Ringer's buffer (0.13 M NaCl, 5 mM KCl, 1 mM CaCl2, and 5 mM KH2PO4 and 7 mM Na2HPO4·7H2O, pH 6.8) and transferred to a small drop of 3–10% fibrinogen in Insect Ringer's buffer on a glass coverslip. Testes were then pierced with an insect needle, allowing the cells to flow out. After waiting ~30 seconds for the cells to settle, 2–5 μl of bovine thrombin was added to form a clot. The clot was inverted onto a drop of Insect Ringer's buffer in a perfusion chamber and sealed with wax or silicon grease. Cells were perfused with Insect Ringer's buffer (with or without pharmacological agents) throughout the experiment.
All experiments were performed at room temperature (25°C). Stock solutions of 0.01 M U73122 (1-[6-[[17 β-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione; Calbiochem) or U73343 (1-[6-[[17 β-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-2,5-pyrrolidine-dione; Calbiochem), 0.03 M ET-18-OCH3 (1-O-Octadecyl-2-O-methyl-rac-glycero-3-phosphorylcholine; Biomol Research Laboratories, Inc., Plymouth Meeting, PA), 0.05 M ML-7 (1-[5-Iodonaphthalene-1-sulfonyl]-1H-hexahydro-l,4-diazepine·HCl; Sigma-Aldrich) or 6 M NEM (Sigma-Aldrich) were prepared in dimethylsulfoxide (DMSO) (Sigma-Aldrich) and stored at -20°C. Prior to treatment, aliquots of each drug were thawed and diluted to the appropriate final concentration with Insect Ringer's buffer. To ensure that any observed effects were not due to the solvent, control cells were treated with the same final concentration of DMSO.
Phase-contrast and fluorescence microscopy
Live Drosophila spermatocytes were imaged using an upright Zeiss Axioplan 2 microscope with a Zeiss Apochromat 100× oil immersion objective lens (NA 1.4). Phase-contrast and fluorescent images were recorded with an Axiocam CCD camera using AxioVision software for image acquisition. Live crane-fly spermatocytes were followed using a phase-contrast inverted Nikon microscope with a Nikon 100× oil-immersion objective (NA 1.3). Real-time images were recorded on videotape or on DVD.
Immunostaining and fluorescence microscopy
Immunostaining and imaging of crane-fly and Drosophila spermatocytes were performed essentially as described  except that Drosophila spermatocytes were fixed with 4% paraformaldehyde (Electron Microscopy Sciences) rather than glutaraldehyde. Drosophila spermatocytes were stained with Alexa 488 phalloidin (Molecular Probes) for F-actin, 4',6-diamidino-2-phenylindole (DAPI; Molecular Probes) for DNA and 1:400 rabbit polyclonal antibody against phospho-Ser19-Sqh (from Luke Alphey ) and were mounted in Fluorescence Mounting Medium (Daxocytomation). Crane-fly spermatocytes were stained using 2.2 μM Alexa 488 phalloidin for F-actin (Molecular Probes) and 1:4000 YL1/2 rat monoclonal antibody against tyrosinated tubulin  and were mounted in Mowiol. Immunofluorescence images of Drosophila spermatocytes were collected with a Zeiss Axioplan 2 microscope and Axiovision 4.2 software. Confocal images of crane-fly spermatocytes were collected with Fluoview (Olympus) software, and were processed further using Image J (public domain software available at http://rsb.info.nih.gov/ij/) and Adobe Photoshop. All illustrations were prepared using Adobe Photoshop and were adjusted for image presentation only (brightness, contrast).
Analysis of data
1-O-Octadecyl-2-O-methyl-rac-glycero-3-phosphorylcholine; edelfosine (PLC inhibitor)
- ML-7 1-[5-Iodonaphthalene-1-sulfonyl]-1H-hexahydro-l:
4-diazepine·HCl (MLCK inhibitor)
myosin light chain kinase
- PIP2 phosphatidylinositol 4:
Spaghetti squash (Drosophila myosin regulatory light chain)
- U73122 1-[6-[[17 β-3-methoxyestra-1:
3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (PLC inhibitor)
- U73343 1-[6-[[17 β-3-methoxyestra-1:
3,5(10)-trien-17-yl]amino]hexyl]-2,5-pyrrolidine-dione (inactive isomer of U73122)
The authors gratefully acknowledge Daniel Saul for carrying out initial experiments using ET-18-OCH3 on crane-fly spermatocytes, Luke Alphey for providing anti-phospho-Sqh antibody and John Ashkenas for comments on the manuscript. This research was generously supported by Ontario Graduate Scholarships (to RW and to LF), SickKids Restracomp funding (to RW), a Discovery Grant from the Canadian Natural Sciences and Engineering Research Council (to AF) and a Terry Fox Foundation grant from the National Cancer Institute of Canada (to JAB).
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