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Department of Biological Sciences, Dartmouth College, Hanover, New Hampshire 03755
1 Rostock University, Germany
Small GTP-binding proteins (G-proteins) of the Rho family have been shown to regulate cytoskeleton reorganization, whereas G-proteins of the Rab family have been shown to regulate intracellular trafficking and vesicle transport (1). In mammalian cells, Rho proteins Rho/Rac/Cdc42 regulate the formation of stress fibers, lamellipodia, and filopodia. One of the ways in which Rho proteins mediate effects on the cytoskeleton is via the Rho-ROK/Rho kinase-myosin phosphatase pathway (Fig. 1A). In this pathway, myosin light-chain phosphatase (MyoP) is phosphorylated by ROK/Rho kinase and is thereby inhibited (2). The net result is the activation of myosin-II and the sustained contraction of smooth muscle cells, even after the Ca2+ concentration is decreased.
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Fortuitously, actin filaments within the pseudo-contractile ring co-aligned to form bundles (10–20 parallel filaments) that were thick enough to visualize by AVEC-DIC microscopy. Therefore, sliding of actin filaments could be monitored and studied in networks reconstituted in vitro. The term contraction is used to refer to the myosin-II-mediated sliding of actin filaments in the network; the network itself does not shorten. In this report, we show that RhoGDI blocked filament sliding and vesicle transport in these extracts.
Extracts were prepared from mature oocytes obtained from gravid female clams. The cytoplasmic extracts were diluted 2-fold to a final protein concentration of about 15 mg/ml and clarified to remove large membrane aggregates. Nocodazole (30 µM) was added to the extracts to block microtubule assembly, an ATP regenerating system was added to maintain ATP levels, and the preparation was incubated for 45 min at 18 °C to assemble actin filaments and reconstitute motor activity. 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.
In control extracts, bundles of actin filaments in the pseudo-contractile ring (Fig. 1C, control) were observed to slide relative to each other. The advancing tips of sliding bundles were tracked at speeds greater than 0.2 µm/s for distances greater than 25 µm. An average of 2 sliding actin bundles/field/min were observed (Fig. 1B, control). In addition, movement of vesicles on actin filaments was observed. In a given video field that measured 25 µm2, 46 ± 2 vesicles/min (n = 2) moved at an average speed of 1.0 µm/s (Fig. 1B). The difference in speed of vesicle movement and filament sliding provided evidence that vesicle movement was not due to the passive attachment of vesicles to sliding filaments. In addition, vesicle movement was observed on stationary actin filaments tightly bound to the glass coverslip.
To determine the effect of RhoGDI on filament sliding and vesicle transport mediated by myosin-II, we incubated extracts with 200 nM bacterially expressed RhoGDI (gift from S. Kuznetsov). Vesicle transport was measured by counting the number of vesicles moving/video field/min (v/f/m; motile activity) at 15-min time intervals after GDI addition. RhoGDI inhibited motile activity by 72% (13 ± 1 v/f/m, n = 3) and filament sliding by 68% (Fig. 1B). Pseudo-contractile ring formation was similar to the controls (Fig. 1C). On the other hand, 200 nM RabGDI affected neither motile activity nor filament sliding; the motile activity and number of sliding filament bundles were similar to the controls (Fig. 1B). In addition, bundles of actin filaments in pseudo-contractile rings were similar to the control extracts (Fig. 1C). These studies showed that blocking Rho proteins with RhoGDI inhibited myosin-II-mediated vesicle transport and filament sliding. We conclude, therefore, that Rho proteins are required for contraction of the contractile ring during cytokinesis.
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. These studies provide evidence that the two activities mediated by myosin-II in these extracts are activated by Rho GTPases. The downstream effector of the Rho proteins is most likely myosin light-chain phosphatase (5). Therefore, because inhibition of Rho proteins blocked cytokinesis, the Rho-ROK/Rho kinase-myosin phosphatase pathway appears to regulate cell division in clam oocytes.
Supported by NSF Grant IBN-0131470 and MBL Shifman award to RFT.
Literature Cited
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  T. Wollert, A. S. DePina, C. J. DeSelm, and G. M. Langford Rho-kinase Is Required for Myosin-II-Mediated Vesicle Transport During M-Phase in Extracts of Clam Oocytes Biol. Bull., October 1, 2003; 205(2): 195 - 197. [Full Text] [PDF]
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