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Department of Biological Sciences, Dartmouth College, Hanover, New Hampshire 03755
Cell division requires the construction of a contractile ring of actin filaments attached to the plasma membrane at the site of cleavage. Bipolar myosin-II filaments in the contractile ring generate sliding of anti-parallel bundles of actin filaments, thereby constricting the cell (1). A recent study of cell division in Dictyostelium (2) showed that myosin-II filaments are recruited to the contractile ring cell by "cortical flow." The underlying mechanism of cortical flow is not known. We inhibited the motor activity of myosin-II in cell-free extracts of clam (Spisula solidissima) oocytes with a function-blocking myosin-II-specific antibody to investigate the mechanism of movement of myosin-II to the contractile ring.
Cytoplasmic extracts were prepared from mature oocytes obtained from gravid female clams (3,4). The clarified extracts were diluted 2-fold (protein concentration about 15 mg/ml) and adjusted to pH 7.2. Nocodazole (50 µm) was added to the extracts to block microtubule assembly, and an ATP regenerating system was added to maintain ATP levels. The final extract was incubated for 45 min at 18 °C to initiate transition into the meiotic phase of the cell cycle. Rhodamine-phalloidin (0.5 µM) was added to stain the actin filaments, and the myosin II motor activity was monitored by AVEC-DIC and fluorescence microscopy (5).
Actin bundles detectable by AVEC-DIC microscopy assembled spontaneously in the meiotic phase extracts and formed interactive three-dimensional networks or pseudo-contractile rings by a mechanism of self-organization (6). Two types of myosin-dependent movement associated with the actin networks were observed. First, overlapping bundles of actin filaments in the network were observed to slide along each other. The advancing tips of sliding bundles were tracked at speeds greater than 0.2 µm/s for more than 25 µm before disappearing out of the field of view (Fig. 1A). A similar sliding motion produced by bipolar myosin-II filaments is generally accepted as the mechanism by which the contractile ring constricts the cell during cytokinesis. Therefore, these self-organized actin networks or pseudo-contractile rings exhibited one of the principal properties ascribed to the contractile ring.
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To demonstrate that both filament sliding and vesicle transport were dependent on myosin-II, we performed antibody inhibition experiments with a rabbit-polyclonal antibody raised to myosin-II from clam oocytes. The Protein-A-purified, myosin-II-specific antibody recognized a single band on immunoblots of the oocyte extracts. Inhibition of vesicle transport was determined by comparing motile activity after antibody addition with motile activity in controls. Motile activity was measured by counting the number of moving vesicles per video field per minute (v/f/m). The control extracts showed high levels of motile activity (122 v/f/m) for periods of 60 min or more. However, addition of the myosin-II-specific polyclonal antibody caused a concentration-dependent inhibition of motile activity (Fig. 1B). Vesicle transport was inhibited by 95% (5 v/f/m) at an antibody concentration of 1 mg/ml (Fig. 1C). The antibody inhibition experiments provided direct evidence that vesicle transport was mediated by myosin-II.
At concentrations of 0.75 and 1.0 mg/ml, the myosin-II-specific antibody inhibited the formation of the 3-D network (Fig. 1D). With fluorescence microscopy, pseudo-contractile rings of actin bundles could be seen in the control extracts, but they were absent at these two antibody concentrations (Fig. 1D). The antibody did not inhibit the assembly of actin filaments but blocked the association of filaments into bundles; therefore, the myosin-II antibody blocked the self-organizing and bundle-sliding activities observed in the control extracts. The concentration of myosin-II in these extracts was estimated to be in the range of 0.1–0.2 mg/ml, so an antibody concentration of 1 mg/ml was about 5-fold higher than the concentration of myosin. The antibody is polyclonal, and only a subset of IgG molecules are expected to bind at sites that block motor function; thus we judge the antibody concentration required for inhibition of filament sliding and vesicle transport to be within the expected range.
The generation of sliding forces between actin filaments is a well-established activity of bipolar filaments of myosin-II. Therefore, the observation that actin filaments self-organized and moved in an anti-parallel fashion in these extracts fits current models of the contractile ring. Sliding of actin filaments is assumed to occur in intact contractile rings, but it has not been observed. These studies provide a direct view of the sliding activity that occurs in self-organized actin networks that mimic contractile rings. Self-organized networks in cell-free extracts such as these may be the only means available to observe myosin-II-mediated sliding of actin bundles like those in the contractile ring.
The other novel observation in these experiments was the myosin-II-dependent movement of vesicles. Myosin-II has not previously been shown to be a vesicle motor. However, the movement of vesicles to the contractile ring has been documented, and myosin-II is known to move toward the equator by cortical flow (2). Myosin-II-mediated vesicle transport on cortical actin filaments may provide a mechanism by which myosin-II filaments arrive at the contractile ring. Such a model is not consistent with several published reports. Yumura and Uyeda (7), for example, demonstrated that myosin-II molecules that lack ATPase activity are recruited to the equator. In addition, headless myosin-II localizes to the equator (8,9). These observations suggest that myosin filaments are transported as passive passengers to the actin cortex rather than through their own motor activity. Our studies, on the other hand, provide some of the first evidence that myosin-II binds specifically to vesicles and drives vesicle movement. The motor activity of myosin-II may thus be another mechanism by which bipolar myosin-II filaments are recruited to the cortex and to the contractile ring.
Footnotes
1 Rostock University, Germany. 
Literature Cited
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