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1 Rostock University, Rostock, Germany
2 Dartmouth College, Hanover, NH
In mammalian cells, Rho proteins Rho/Rac/Cdc42 regulate the formation of the actin cytoskeleton in stress fibers, lamellipodia, and filopodia (1). One of the ways in which Rho proteins mediate effects on the actin cytoskeleton is via the Rho-ROK/Rho kinase-myosin phosphatase pathway. 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-mediated activities. In addition, Rho-family proteins have also been shown to regulate the actin cytoskeleton during cell division (3).
To study the role of Rho proteins in myosin-II mediated vesicle transport during the M-phase of the cell cycle, we used Y27632 to inhibit Rho kinase activity in extracts of clam oocytes. We have shown previously that actin filaments assemble spontaneously in such extracts and organize into a three-dimensional network of interconnected filaments (4). This self-organized network of actin filaments resembles the cytokinetic ring of dividing cells in the following ways: (i) it exhibits myosin-II-mediated, anti-parallel sliding of actin filaments (4, 5), and (ii) it assembles during the M-phase of the cell cycle. Fortuitously, actin filaments in the extracts co-align to form bundles (10–20 parallel filaments) that are visible by AVEC-DIC microscopy. We have also shown that vesicles are moved along the actin filaments by a class II myosin motor (4). In this report, we show that a specific inhibitor of Rho kinase, Y27632, blocks vesicle transport in these extracts, thereby providing additional evidence that vesicle movement on actin in these extracts is mediated by myosin-II.
Extracts prepared from mature oocytes arrested at the G2/M phase of the cell cycle were snap frozen and stored at -80 °C. To begin an experiment, the pH of the cytoplasmic extracts was shifted from 6.8 to 7.2 by diluting 2-fold with pH-8 buffer to initiate progression through M-phase. 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 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, vesicle transport was measured at regular time points to determine whether motile activity varied upon entry into M-phase of the cell cycle. Vesicle transport was measured by counting the number of vesicles moving per video field per min (v/f/m; motile activity) at 15-min time intervals. We found that the motile activity was high during the first 15 min of incubation at 18 °C, then declined during the 15–30-min interval but rose again and remained stably high between 45 and 60 min (Fig. 1A). Motile activity declined again after 120 min at 18 °C. The actin network, as revealed by rhodamine-phalloidin staining, did not change during the 2-h period of incubation. To determine when the extracts were in the M-phase of the cell cycle, a Xenopus sperm-nucleus shape-change assay was performed. Xenopus sperm nuclei were added to the extract, stained with DAPI, and observed by fluorescence microscopy at regular intervals during incubation. In the initial period, the Xenopus sperm nuclei remained elongated, but they began to expand and appear uniformly bright at 30 min (Fig. 1B). At 45 min, the nuclei assumed an irregular shape as the chromosomes condensed. Chromosome condensation is diagnostic of M-phase. The nuclei remained irregular in shape with condensed chromosomes from 45–120 min, the period when motile activity was high (Fig. 1A). Based on this assay, extracts incubated for 45 to 60 min were judged to be in M-phase.
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In summary, these data show that Rho kinase is directly involved in vesicle transport. Because there was no effect on the actin cytoskeleton in this case, the downstream target of Rho kinase is mostly likely myosin II. This is the only myosin-mediated pathway known to be regulated by Rho GTPases. The downstream effector of the Rho proteins is most likely myosin light chain phosphatase (1). Therefore, we can conclude that the Rho-ROK/Rho kinase-myosin phosphatase pathway regulates vesicle transport during M-phase in clam oocytes.
This work was supported by NSF Grant IBN-0131470 and MBL Shifman award to CJD.
Literature Cited
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