Blebbistatin and blebbistatin-inactivated myosin II inhibit myosin II-independent processes in Dictyostelium

Abstract

Blebbistatin, a cell-permeable inhibitor of class-II myosins, was developed to provide a tool for studying the biologic roles of myosin II. Consistent with this use, we find that blebbistatin inhibits three myosin II-dependent processes in Dictyostelium (growth in suspension culture, capping of Con A receptors, and development to fruiting bodies) and does not inhibit growth on plates, which does not require myosin II. As expected, macropinocytosis (myosin I-dependent), contractile vacuole activity (myosin V-dependent), and phagocytosis (myosin VII-dependent), none of which requires myosin II, are not inhibited by blebbistatin in myosin II-null cells, but, unexpectedly, blebbistatin does inhibit macropinocytosis and phagocytosis by cells expressing myosin II. Expression of catalytically inactive myosin II in myosin II-null cells also inhibits macropinocytosis and phagocytosis. Both blebbistatin-inhibited myosin II and catalytically inactive myosin II form cytoplasmic aggregates, which may be why they inhibit myosin II-independent processes, but neither affects the distribution of actin filaments in vegetative cells or actin and myosin distribution in dividing or polarized cells. Blebbistatin also inhibits cell streaming and plaque expansion in myosin II-null cells. Our results are consistent with myosin II being the only Dictyostelium myosin that is inhibited by blebbistatin but also show that blebbistatin-inactivated myosin II inhibits some myosin II-independent processes and that blebbistatin inhibits other activities in the absence of myosin II.

Blebbistatin (1), a cell-permeable inhibitor of myosin II ATPase activity, promises to be a very useful tool for identifying and studying myosin II-dependent processes in cells. As for any pharmacologic agent, however, extended use of blebbistatin will depend on the specificity and generality of its inhibition of class-II myosins. Thus far, blebbistatin's effect has been tested in vitro (1, 2) on only a few representatives of four of the 18 myosin classes that, together, contain >200 myosins. Blebbistatin inhibits myosin II from striated muscle, vertebrate nonmuscle cells, and Dictyostelium by ≈95%, with an IC₅₀ of 0.5-5 μM. Smooth muscle and Acanthamoeba myosin II are only incompletely inhibited at blebbistatin concentrations as high as 160 μM, with IC₅₀ values of ≈80 μM, and blebbistatin does not inhibit either rat myosin Ib, Acanthamoeba myosin IC, mouse myosin V, or bovine myosin X. It is not practical to test the blebbistatin sensitivity of all of the myosins or to determine whether blebbistatin might inhibit enzymes other than myosins, whether some functions of myosin II might not require enzymatic activity (and, therefore, not be inhibited by blebbistatin), or whether enzymatically inactive myosin II might directly or indirectly affect any non-myosin II-dependent cell function in all cells.

The social amoeba Dictyostelium discoideum provides a useful system for assessing some of these variables. Dictyostelium contains a limited number of myosins: seven class-I, one class-II, two class-V, one class-VII, and two myosins (myoG and myoM) that do not belong to any established class (http://dictybase.org). At least some of the functions of Dictyostelium class-I, -II, -V, and -VII myosins, but not of myoG and myoM, are known and myosin II-null cells have been extensively studied. If blebbistatin is specific for myosin II in Dictyostelium, (i) blebbistatin should have no effect on myosin II-null cells, (ii) blebbistatin should inhibit all of the myosin II-dependent functions of cells expressing myosin II, and (iii) blebbistatin-inhibited wild-type cells should be essentially identical to myosin II-null cells, unless the presence of enzymatically inactive myosin II affects the phenotype, which can be evaluated by expressing enzymatically inactive myosin II in myosin II-null cells.

After confirming that blebbistatin inhibits the basal and actin-activated ATPase activities of purified Dictyostelium myosin II, we evaluated the effects of blebbistatin on three cellular activities known to require myosin II [growth in suspension culture, capping of Con A receptors, and development to fruiting bodies (3-7)]; on five processes that do not require myosin II (8) [growth on plates, cell streaming, macropinocytosis, which involves class-I myosins (9, 10), contractile vacuole function, which involves class-V myoJ (11), and phagocytosis in suspension, which is myosin VII-dependent (12, 13)]; and on the organization of the actomyosin cytoskeleton. In all of the experiments, we compared the effects of blebbistatin on myosin II heavy chain-null (HS1) cells, HS1 cells expressing wild-type myosin II heavy chain (WT), and HS1 cells expressing an enzymatically inactive myosin II heavy chain mutant (E476K) (14).

Materials and Methods

Cells. HS1 cells, which lack the endogenous copy of myosin II heavy chain (15), were cultured at 21°C in HL5 medium supplemented with 60 μg/ml penicillin and streptomycin (16). Dictyostelium WT, GFP-WT, and inactive myosin II heavy chain mutant GFP-E476K cDNAs were electroporated into HS1 cells by using a Bio-Rad gene pulser (17). Individual clones were selected and maintained in the presence of 12 μg/ml geneticin (G418) sulfate in HL5 medium containing 60 μg/ml each penicillin and streptomycin.

Drug Treatment. S-(-)-blebbistatin (lot no. 5-SHG-28-2) was purchased from Toronto Research Chemicals (Downsview, ON, Canada); (-)-blebbistatin (lot no. B62655 and (+)-blebbistatin (lot no. 62052) were purchased from EMD Biosciences (San Diego, CA). Blebbistatin was dissolved in DMSO to make a 100-mM stock solution, and cells were treated with 50, 100, or 150 μM blebbistatin, as stated, or the same volume of DMSO. S-(-)-blebbistatin from Toronto Research Chemicals was used in all experiments except where otherwise stated. In all of the biological experiments, flasks containing cells were kept in the dark and/or covered with aluminum foil to prevent photoinactivation and phototoxicity (18). All biological assays were repeated and confirmed at least three times.

As determined by HPLC on a Chiracel OD-H column (Daicel, Fort Lee, NJ) eluted with a 90:10 mixture of n-hexane:isopropanol and quantified by UV absorption (Fig. 10, which is published as supporting information on the PNAS web site), the S-(-)-blebbistatin was actually a mixture of 59% (-)-enantiomer and 41% (+)-enantiomer; (+)-blebbistatin and (-)-blebbistatin were 100% pure.

Growth Rates. Cells grown in suspension culture on a rotary shaker at 145 rpm were counted daily by using a hemocytometer. After 3 days in suspension culture, cells were fixed and stained with 1 μg/ml 4,6-diamidino-2-phenylindole to visualize nuclei, and 100 cells were scored. Cells were also grown on etched coverslips in 6-well culture clusters, and the same fields of cells were counted every 12 h. Nuclei of cells grown on coverslips were stained and counted as above.

Capping of Con A Receptors and Myosin II and Immunomicroscopy. Cells were washed and resuspended in starvation buffer, 20 mM morpholinoethanesulfonic acid, pH 6.8, allowed to attach to a coverslip and then incubated for 15 min with 50 μl of tetramethyl rhodamine isothiocyanate-conjugated Con A (Molecular Probes), 30 μg/ml (8). For myosin II redistribution after capping, cells were incubated with Con A for 3 min to allow capping, incubated with or without blebbistatin for 25 min, fixed and permeabilized in acetone/1% formalin at -20°C for 15 min, and mounted directly to visualize the Con A caps or reacted with anti-Dictyostelium myosin II antibody (8) for colocalization of myosin II by indirect immunofluorescence. For localization of myosin and F-actin, GFP-WT cells were preincubated with blebbistatin for at least 30 min. Cells were fixed with acetone at -20°C for 3 min and incubated with rhodamine phalloidin (Molecular Probes). Micrographs were taken on a Zeiss LSM-510 laser scanning fluorescence microscope equipped with a Plan apo ×63 oil objective.

Cell Streaming and Development. For cell streaming, cells were harvested, resuspended at 5 × 10⁶/ml, and 1.5 × 10⁷ cells were plated on 60-mm Petri dishes and allowed to adhere for 30 min (8). The medium was removed by aspiration and 2 ml of starvation buffer, containing 100 μM blebbistatin or an equal volume of DMSO, was carefully applied and the cells placed in the dark for 6-8 h. Development was assessed 48 h after spotting small aliquots of amoebae on black filters (Millipore HABP4700) saturated with starvation buffer with and without 100 μM blebbistatin. Images were recorded with a Nikon SMZ-U dissection microscope.

Plaque Expansion. A suspension of heat-killed Klebsiella aerogenes containing fewer than 100 Dictyostelium cells was seeded onto black filters saturated with HL5 medium in 60-mm Petri dishes (8). Either blebbistatin, 150 μM, or an equivalent volume of DMSO was added to both the mixture and the HL5 medium-saturated pads. Plaque sizes were imaged after 5 days in the dark in a humid chamber at 21°C.

Phagocytosis, Macropinocytosis, and Contractile Vacuole Activity. For phagocytosis (8), cells were collected, washed in Sorenson's buffer (16 mM KH₂PO₄,2mMNa₂HPO₄, pH 6.1), diluted to 5 × 10⁶ cells per ml and incubated for 1 h at room temperature with 150 μM blebbistatin or an equivalent volume of DMSO on a rotary shaker at 100 rpm. Washed Fluoresbrite yellow orange carboxylate-conjugated polystyrene-latex beads (1 μm in diameter; Polysciences) were added at a ratio of 300 beads per cell. At the indicated times, 1 ml of cells was added to 2 ml of ice-cold Sorenson's buffer to stop phagocytosis, and undigested beads were removed by centrifugation through 10 ml of a 20% polyethyleneglycol cushion. Cell pellets were washed and resuspended in 3 ml of 50 mM Na₂HPO₄, pH 9.2, and lysed by addition of Triton X-100 to 0.4%, and fluorescence was measured with excitation and emission wavelengths of 529 and 546 nm, respectively.

To assess adherence of beads to the cell surface, cells were incubated as described for the phagocytosis assay with Fluoresbrite carboxylate YG-conjugated polystyrene-latex beads (1 μm in diameter; Polysciences), and unwashed cells were immediately visualized in a chambered cover glass system by using a Zeiss LSM-510 confocal microscope.

For macropinocytosis (19), cells were harvested and resuspended at a density of 5 × 10⁶ cells per ml in HL5 medium, and 20 ml of cells were transferred to a 50-ml flask and incubated for 1 h on an orbital shaker at room temperature with 150 μM blebbistatin or an equivalent volume of DMSO; tetramethylrhodamine isothiocyanate-dextran (Sigma-Aldrich) was then added to a final concentration of 2 mg/ml. All of the following steps were carried out on ice. At the indicated times, samples of 1 ml were removed and transferred to a 1.5-ml tube containing 100 μl of 0.4% trypan blue. The tubes were vortexed and centrifuged at 2,800 rpm for 3 min in a Microfuge centrifuge, and the supernatant was carefully aspirated. The pellets were washed and resuspended in 1 ml Sorenson's buffer, and fluorescence was measured with excitation and emission wavelengths of 544 nm and 574 nm, respectively.

To assay contractile vacuole activity, washed cells were suspended in distilled water (10⁴ cells per ml) containing 100 μM blebbistatin or an equal volume of DMSO. At the indicated times, 10 μl were mixed with heat-killed K. aerogenes and seeded onto black filters saturated with HL5 medium, and plaques were counted after 5 days (8).

Protein Purification and ATPase Assays. Dictyostelium myosin II (20) and rabbit skeletal muscle actin (21) were purified as described. Bacterially expressed, polyHis-tagged myosin light chain kinase carrying an activating T166E mutation was purified by affinity chromatography on Ni-nitrilotriacetic acid resin (22). Protein concentrations were determined by the Bradford method.

Steady-state ATPase activities were determined at 30°C by measuring ³²Pi released from [³²P]ATP (23). The reaction mixtures contained 20 mM imidazole (pH 7.5), 25 mm KCl, 4 mM MgCl₂, 2 mM [³²P]ATP, and 50 μg/ml myosin, with or without F-actin and blebbistatin as indicated. Reactions were started by the addition of phosphorylated myosin (14).

Electron Microscopy. Purified Dictyostelium myosin II was dialyzed against assembly buffer (50 mM NaCl/10 mM Hepes, pH 7.5/2 mM MgCl₂/1 mM DTT). When present, blebbistatin was added to both the protein solution and dialysis buffer. Negatively stained images were obtained as described (24).

Results

As reported (2), we found that blebbistatin inhibits the actin-activated MgATPase of Dictyostelium myosin II with an IC₅₀ of 7 μM and 95% inhibition at 100 μM (Fig. 1A). As was reported for skeletal muscle myosin II (25) and nonmuscle myosin IIB (26), blebbistatin also inhibits the basal (non-actin-activated) MgATPase of Dictyostelium myosin II (Fig. 1 A), with a slightly lower IC₅₀. These experiments were carried out with S-(-)-blebbistatin from Toronto Research Chemicals, which we found to be a mixture of 59% (-) and 41% (+) enantiomers (see Materials and Methods). A sample of 100% (+)-enantiomer (EMD Biosciences) was found to have essentially no inhibitory activity (Fig. 1 A). Therefore, the IC₅₀ for (-)-blebbistatin would likely be ≈3-4 μM with 95% inhibition at 60 μM, although the pure (-)-blebbistatin was slightly less active than the mixture of (-)- and (+)-blebbistatin (Fig. 1 A).

Fig. 1.

Blebbistatin inhibits Dictyostelium myosin II. (A) S-(-)-blebbistatin (Toronto Research Chemicals) was used except for data indicated by + and -, which were (+)- and (-)-blebbistatin (EMD Biosciences), respectively. Myosin head concentration, 160 nM; F-actin concentration, 25 μM (filled circles); no F-actin (open circles). (B) Myosin head concentration, 160 nM; blebbistatin concentration, 8 μM. K_actin is the concentration of F-actin at half-maximal ATPase activity.

Inhibition by blebbistatin is due entirely to a reduction in k_cat with no change in K_actin (Fig. 1B). These results are consistent with blebbistatin binding to the myosin-ADP-Pi complex, inhibiting the rate of release of P_i and trapping myosin in an F-actin weak-binding state, as has been proposed from more detailed studies of skeletal muscle myosin II (25) and nonmuscle myosin IIB (26).

Effect of Blebbistatin on Myosin II-Dependent Activities. Like myosin II-null (HS1) and E476K cells, blebbistatin-inhibited WT cells do not divide in suspension culture (Fig. 2A) resulting in large, multinucleated cells (Fig. 2B); 100 μM blebbistatin is required for complete inhibition. Blebbistatin also inhibits both the development of WT cells beyond the mound stage (Fig. 3) and the ability of WT cells to cap Con A receptors (Fig. 4A). In both assays, blebbistatin-inhibited WT cells resemble HS1 and E476K cells (Figs. 3 and 4). When added after completion of Con A-capping, blebbistatin inhibits the redistribution of myosin II that normally is complete in <20 min (Fig. 4B). S-(-)-blebbistatin from Toronto Research Chemicals was used in these experiments but control experiments with pure (-)- and (+)-blebbistatin showed that only the (-)-isomer is active (Fig. 11, which is published as supporting information on the PNAS web site).

Fig. 2.

Blebbistatin inhibits growth and cytokinesis of Dictyostelium in suspension culture. (A) Growth in suspension culture of WT cells and WT cells in the presence of 50 and 100 μM blebbistatin (Bb) compared with HS1 cells and HS1 E476K cells. (B) Representative images of nuclei-stained cells and the number of nuclei per cell after 3 days. (Bar, 100 μm.)

Fig. 3.

Blebbistatin inhibits development of WT Dictyostelium to fruiting bodies. Images were taken 48 h after WT cells, WT cells, WT cells in buffer containing 100 μM blebbistatin (Bb), HS1 cells, and HS1 cells expressing E476K were spotted on black filters saturated with starvation buffer. (Bar, 500 μm.)

Fig. 4.

Blebbistatin inhibits Con A-induced capping and redistribution of myosin II after capping in the absence of blebbistatin. (A) Images taken 15 min after rhodamine-labeled Con A (red) was added to cells in starvation buffer in the absence or presence of 150 μM blebbistatin (Bb). (B) WT cells expressing GFP-myosin II were incubated with rhodamine-labeled Con A for 3 min to allow the Con A (red) and myosin II (green) to co-cap and then for another 25 min with (Bb) or without blebbistatin (Mes). (Bar, 5 μm.)

Effect of Blebbistatin on Myosin II-Independent Activities: Growth on Plates, Cell Streaming, and Plaque Expansion. As expected, blebbistatin does not inhibit growth or cytokinesis of WT or HS1 cells on plates, and E476K cells grow as well as WT and HS1 cells (Fig. 5A). The cells are of similar size and mostly mononucleate (Fig. 5B).

Fig. 5.

Blebbistatin does not inhibit growth of Dictyostelium on plates. (A) WT, HS1, E476K cells, and WT cells in the presence of 100 μM blebbistatin (Bb) grow equally well on plates. (B) Representative images of cells stained with 4,6-diamidino-2-phenylindole and quantification of nuclei per cell.

Although HS1 and E476K cells plated in non-nutrient medium stream somewhat less well than WT cells (Fig. 6A), HS1 and E476K cells form essentially normal mounds (Fig. 4). However, streaming of all three cells lines is severely affected by blebbistatin (Fig. 6A); in the presence of blebbistatin, the cells form many more streams leading to very much smaller mounds (not shown) than in the absence of blebbistatin.

Fig. 6.

Blebbistatin inhibits cell streaming and plaque expansion in the absence of myosin II. (A) Streams formed by WT, HS1, and E476K cells in the absence and presence of 100 μM blebbistatin 7 h after plating in starvation buffer. (B) Plaques of WT, HS1, and E476K cells imaged 5 days after cells were plated with a suspension of dead K. aerogenes in HL-5 medium in the absence or presence of 150 μM blebbistatin. (Bar, 400 μm.)

Similarly, in the plaque expansion assay, HS1 cells form somewhat smaller, and E476K cells very much smaller, plaques than WT cells (Fig. 6B), indicative of a nonessential role for myosin II in plaque expansion. However, even though myosin II is not essential, and is not present in HS1 and E476K cells, blebbistatin substantially inhibits plaque formation by all three cell lines (Fig. 6B). Because blebbistatin inhibits cell streaming and plaque formation in HS1 (myosin II-null) and E476K cells as well as WT cells, blebbistatin must inhibit one or more activities that do not involve myosin II. Control experiments show that the effects of blebbistatin are due entirely to the (-)-isomer, the (+)-isomer having little if any detectable effect (Fig. 11).

Macropinocytosis, Contractile Vacuole Activity, and Phagocytosis. Macropinocytosis, contractile vacuole activity, and phagocytosis involve class I, class V, and class VII myosins, respectively, but none has been shown to require myosin II. Consistent with this finding, we find that HS1 cells are as active, or nearly so, as WT cells in all three functions (Fig. 7 A, B, and C). Importantly, blebbistatin has little, if any, effect on macropinocytosis, contractile vacuole function, or phagocytosis by HS1 cells, indicating that blebbistatin does not inhibit class I, V, or VII myosins. Surprisingly, however, blebbistatin strongly inhibits (≈85%) both macropinocytosis and phagocytosis by WT cells (Fig. 7 A and C); blebbistatin does not inhibit contractile vacuole activity (Fig. 7B). Blebbistatin inhibits ingestion of beads but not their adherence to the cell surface (Fig. 7D), which is inhibited in myosin VII-null cells (12, 13). Expression of inactive myosin II (E476K) in HS1 cells also inhibits macropinocytosis (≈50%) and phagocytosis (≈70%) (Fig. 7 A and C). These results suggest that the two endocytic processes are inhibited indirectly by enzymatically inactive myosin II, both blebbistatin-inhibited WT myosin II and catalytically inactive E476K. Again, the pure (-)-isomer was active and the (+)-isomer inactive (Fig. 11).

Fig. 7.

Effects of blebbistatin on macropinocytosis, contractile vacuole activity, and phagocytosis. (A) Blebbistatin (Bb) inhibits macropinocytosis of rhodamine-labeled dextran by WT but not by HS1 cells. Fluorescence is in arbitrary units normalized to protein concentration. (B) Blebbistatin (Bb) does not inhibit contractile vacuole activity as assessed by survival of cells in distilled water. (C) Blebbistatin (Bb) severely inhibits phagocytosis of latex beads by WT cells. (D) Confocal images show that blebbistatin (Bb) does not inhibit adherence of latex beads to the cell surface. Blebbistatin concentration: 150 μM. (Bar, 3 μm.)

Effect of Blebbistatin on the Localization of Myosin II and F-Actin. Normally, myosin II is mostly concentrated in the cortex of vegetative cells, but myosin II in blebbistatin-treated WT cells and cells expressing inactive E476K aggregate into patches (Fig. 8); endogenous myosin II in WT cells is affected identically as the GFP-myosin (not shown). Blebbistatin does not inhibit myosin II polymerization into filaments in vitro, nor does it cause the filaments to aggregate (Fig. 8). The effects of pure (-)-isomer were identical to those of the enantiomeric mixture and pure (+)-isomer had no effect (Fig. 11). Even though blebbistatin-inactivated myosin II is mostly aggregated in vegetative cells, it does localize properly to the rear of polarized cells and to the cleavage furrow of dividing cells (Fig. 9). Blebbistatin does not interfere with the localization of F-actin in the cortex of vegetative cells or at the leading edge of motile and dividing cells (Fig. 9).

Fig. 8.

Blebbistatin-inhibited myosin II and E476K mutant myosin II form cytoplasmic aggregates. (Upper) Confocal images of GFP-myosin in WT cells, GFP-WT cells incubated for 1 h with 100 μM blebbistatin (Bb), and HS1 cells expressing GFP-E476K myosin. (Bar, 5 μm.) (Lower) Filaments of WT myosin II in the absence and presence of 100 μM blebbistatin (Bb). (Bar, 100 nm.)

Fig. 9.

Blebbistatin does not inhibit F-actin or myosin II localization in polarized and dividing cells. Confocal images of F-actin (red) and endogenous myosin II (green) in vegetative, polarized (motile), and dividing WT cells in the absence and presence of 100 μM blebbistatin (Bb). (Bar, 10 μm.)

Discussion

Although blebbistatin has been, and will continue to be, a useful tool for elucidating at least some of the biological roles of class-II myosins, one should be cautious in reaching conclusions simply from the effect or lack of effect of blebbistatin on a cellular activity. First, the myosin II in question should be shown by in vitro assays to be essentially completely inhibited by blebbistatin. For example, Acanthamoeba myosin II is only ≈60% inhibited by 160 μM blebbistatin, which is about as high a concentration as can be attained, and smooth muscle myosin II is only ≈50% inhibited by 95 μM blebbistatin (1, 2). Because we find that 50 μM blebbistatin, which inhibits Dictyostelium myosin II by 85%, does not completely inhibit any of the processes that are inhibited by 100 μM blebbistatin, myosin II functions in Acanthamoeba and smooth muscle might be expected to be incompletely, if at all, inhibited by blebbistatin. In the original report on blebbistatin (1), the (+)-blebbistatin enantiomer was said to be inactive but it was subsequently reported (24) that the (+) and (-) enantiomers inhibit myosin ATPase activity similarly. We confirm in this paper that, when pure enantiomers are used, only the (-)-isomer is inhibitory; conclusions that the (+)-enantiomer inhibits myosin II were probably due to the use of incompletely resolved isomers. The (+)-enantiomer should be used as a negative control in biological experiments.

Our finding that blebbistatin does not inhibit macropinocytosis, contractile vacuole activity, or phagocytosis in Dictyostelium myosin II-null cells, which require myosins I, V, and VII, respectively) adds to the still incomplete evidence that class-II myosins may be the only myosins inhibited by blebbistatin. However, we find that blebbistatin dramatically and paradoxically inhibits macropinocytosis and phagocytosis, processes that do not require myosin II, in wild-type cells that contain myosin II. Macropinocytosis and phagocytosis are also inhibited by expressing the inactive myosin II mutant E476K in myosin II-null cells. Because both E476K and blebbistatin-inhibited myosin II aggregate in vegetative cells, it may be that the aggregated myosin indirectly inhibits macropinocytosis and phagocytosis, neither of which requires myosin II. This conclusion is strengthened by the fact that (+)-blebbistatin, which does not induce myosin II aggregation, does not inhibit macropinocytosis or phagocytosis in WT cells.

Blebbistatin-aggregated myosin II is still capable of relocalizing appropriately in polarized and dividing cells, as is F-actin, whose localization in vegetative cells is also unaffected by blebbistatin. Thus, myosin II localization in polarized and dividing cells does not seem to depend on either its ATPase activity or strong binding to F-actin, but relocalization of the myosin II that co-caps with Con A, which is inhibited by blebbistatin, probably requires either or both myosin ATPase activity or myosin association with F-actin.

Finally, blebbistatin seems not to be an absolutely specific inhibitor of myosin II because it inhibits cell streaming and plaque expansion by HS1 cells. Because HS1 cells do not express any myosin II, inhibition of these processes in HS1 cells must be due to inhibition of something other than myosin II. These effects of blebbistatin are not completely nonspecific, however, because they occur only with the (-)-enantiomer.

In summary, then, although our data provide additional evidence that class-II myosins are the only myosins that are inhibited by blebbistatin, not all class II myosins may be sufficiently sensitive to blebbistatin for it to be a useful tool for identifying myosin II-dependent activities in all species and cell lines. Moreover, blebbistatin must be able to inhibit something in Dictyostelium other than myosin II (to explain its inhibition of cell streaming and plaque expansion in HS1 cells, which do not express myosin II), and blebbistatin-inhibited myosin II can indirectly inhibit some myosin II-independent processes in Dictyostelium (e.g., macropinocytosis and phagocytosis), probably as a result of blebbistatin-induced aggregates of myosin II. These results indicate the need for some caution in interpreting the effects of blebbistatin in other cells.