Effects of myosin-II antibody on actin-dependent vesicle transport in extracts of clam oocytes.

Abstract

The movement of vesicles and organelles in cells has been shown to occur on both microtubules and actin filaments (1, 2). The transport of vesicles on microtubules permits movement over long distances in cells, while the short-range transport of vesicles on actin filaments produces the fine and precise movements required to position vesicles at membrane sites for capture, docking, or fusion (3, 4). The transport of vesicles on actin filaments requires vesicle-associated myosins, some of which have been identified. Myosin V, for example, transports endoplasmic reticulum (ER) vesicles (5-7) and synaptic vesicles (8) in neurons and is involved in the capture of melanosomes at the tips of mouse melanocytes (9). In addition, myosin V is involved in the transport of lysosomes in some cells (10), secretory vesicles in yeast (11), and melanosomes in Xenopus melanophores (12). Myosin II, also known as conventional myosin, is vesicleassociated in extracts of clam (Spisula solidissima) oocytes (4, 13). In addition, myosin II was reported to be associated with Golgi vesicles (14), but the antibody used in these studies cross-reacts with coatomer proteins on Golgi-derived vesicles (15-17). Therefore, the role of myosin II in vesicle transport remains controversial. In this study we performed antibody-inhibition experiments with extracts of clam oocytes to provide additional evidence that myosin II is a vesicle motor. Clam oocyte extracts are well characterized and have been used extensively to study cell cycle events including cyclin synthesis and degradation, as well as ubiquitin-mediated proteolysis of cyclins (18). Extracts were prepared from quiescent oocytes by the method of Rud rman et al., (18) with slight modifications. Fluorescence microscopy was used to view rhodamine-phalloidin stained actin filaments in the extracts, and AVEC-DIC microscopy was used to assay vesicle transport on actin filaments. Motile activity in extracts was determined by counting the number of vesicles moving per minute per field (v/m/f). Antibody-inhibition experiments were performed with an antibody made to oocyte yo in II. The myosin II in oocyte extracts was precipitated with ammonium sulfate, dissolved in buffer (20 mM Tris pH 7.2, 1 mM EDTA-K, 0.2 mM CaCl2, 1 mM DTT), and dialyzed overnight at 4?C. The pure fraction of the myosin II was prepared by running the solubilized fractions on SDS-gels and eluting the myosin II heavy chain band. The eluted protein was used f r antibody production in rabbit. The serum of the immunized rabbit was collected, and the polyclonal antibody (ap-205) was purified on protein A beads. The antibody (ap-205) recognized a single band at 205 kD on west n blots of oocyte extracts (Fig. 1A) and in myosin IIenr ched fractions (Fig. lB). The protein recognized by ap-205 wa also recognized by a myosin II antibody, M2.42 (Fig. 1A), produced to a peptide in the head domain of Acanthamoeba