Nonmuscle Myosin IIA and IIB Have Distinct Functions in the Exocytosis-dependent Process of Cell Membrane Repair

Abstract

Vesicle generation, recruitment, and exocytosis are essential for repairing disruptions of cell membranes. The functions of nonmuscle myosin IIA and IIB in this exocytotic process of membrane repair were studied by the antisense technique. Knockdown of myosin IIB suppressed wound-induced exocytosis and the membrane resealing process. Knockdown of myosin IIA did not suppress exocytosis at an initial wound and had no inhibitory effect on the resealing at initial wounds but did inhibit the facilitated rate of resealing normally found at repeated wounds made at the same site. COS-7 cells, which lack myosin IIA, did not show the facilitated response of membrane resealing to a repeated wound. S91 melanoma cells, a mutant cell line lacking myosin Va, showed normal membrane resealing and normal facilitated responses. We concluded that myosin IIB was required for exocytosis and therefore cell membrane repair itself and that myosin IIA was required in facilitation of cell membrane repair at repeated wounds. Myosin IIB was primarily at the subplasmalemma cortex and myosin IIA was concentrated at the trans-Golgi network consistent with their distinct roles in vesicle trafficking in cell membrane repair. INTRODUCTIONMyosins are a large family of structurally diverse molecular motors. To date, at least 15 structurally distinct classes of myosin heavy chains have been identified (Sellers, 2000;Berg et al., 2001). Conventional nonmuscle myosin II is comprised of two genetically distinct isoforms referred to as myosin IIA and IIB (Simons et al., 1991). Different isoforms of myosin II localize differently within individual cells, and these different distributions in the cell suggest that the two proteins have some important functional differences (Maupin et al., 1994;Rochlin et al., 1995; Kelley et al., 1997). In nonmuscle cells, myosin II has diverse functions including cytoplasmic contractility (Condeelis and Taylor, 1977), cytokinesis (DeLozanne and Spudich, 1987;Knecht and Loomis, 1987), capping of cell-surface components (Pasternak et al., 1989), polarization of cell locomotion (Wessels et al., 1988), and neurite outgrowth (Wylie et al., 1998;Wylie and Chantler, 2001). Myosin II is also suggested to be involved in membrane trafficking within the cell. It has been proposed that myosin IIB is involved in exocytosis, because microinjection of polyclonal antibody against myosin IIB suppressed neurotransmitter release (Mochida et al., 1994;Mochida, 1995). Although it has not been clear how many populations of vesicles bud off the trans-Golgi network (TGN), the p200/myosin II protein, analogous to nonmuscle myosin IIA heavy chain, has been reported to be on a specific subset of TGN-derived vesicles (Narula et al., 1992;Narula and Stow, 1995;Ikonen et al., 1997;Musch et al., 1997;Heimann et al., 1999). However, there are conflicting reports about whether p200/myosin II is an essential participant in the vesicle budding reaction (Musch et al., 1997;Simon et al., 1998). It has also been proposed that unconventional myosins type I, V,...

Powering membrane traffic in endocytosis and recycling

“…The specificities of these associations have to be established firmly, but they support other studies that have observed that myosin II is functionally required for the final phase of exocytosis. For example, the fusion of lysosomes with the plasma membrane during wound repair depends on myosin IIb 122 . Also, in chromaffin cells, a mutant regulatory myosin light chain decreases docking and exocytosis 123 , and inhibitors of myosin light chain kinase or the myosin ATPase reduce the exocytosis of tear secretory vesicles in lacrimal acinar epithelial cells 124 .…”

Myosin IIA is required for cytolytic granule exocytosis in human NK cells

“…Although myosin IIA was the isoform identifi ed as a component of the WIP -WASp -containing multiprotein complex at the synapse by vMALDI tandem mass spectroscopy ( 10 ), a smaller amount of myosin IIB was also seen by Western blot (unpublished data). In this light, nonmuscle myosin IIA and IIB have been shown to have distinct roles in vesicle exocytosis during cell membrane repair in fi broblasts ( 20 ). Myosin IIA had a proposed role in vesicle traffi cking, and IIB in membrane resealing.…”

Conventional myosins – unconventional functions

“…In Swiss 3T3 cells, M2B-depletion suppressed wound-induced exocytosis and the membrane resealing process (Togo and Steinhardt 2004), an effect not seen following M2A knockdown. Consistent with these observations, S91 melanoma cells (which lack myosin 5a) showed a normal membrane resealing process (Togo and Steinhardt 2004). These results suggest that M2B plays a distinct role in vesicle trafficking.…”

“…For example, migrating growth cones of rat superior cervical ganglion neurons exhibit a differential distribution of M2A and M2B (Rochlin et al 1995), M2B being the more peripheral. M2B is also found to be peripheral to M2A in lamellipodia of motile cultured Xenopus A6 cells (Kelley et al 1996) and in Swiss 3T3 fibroblasts (Togo and Steinhardt 2004). By contrast, when monolayer cultures of bovine aortic endothelial cells migrate into a scratch wound, M2A is found at the leading edge of motile cells whereas M2B is found at the rear, moving the back end forwards (Kolega 1998(Kolega , 2003.…”

“…Black arrows indicate the direction of retrograde actin flow, known to be powered by one or more of the three myosin 2 motors of breaks to the plasma membrane, an essential process that promotes resealing because it decreases membrane tension (Togo et al 2000). In Swiss 3T3 cells, M2B-depletion suppressed wound-induced exocytosis and the membrane resealing process (Togo and Steinhardt 2004), an effect not seen following M2A knockdown. Consistent with these observations, S91 melanoma cells (which lack myosin 5a) showed a normal membrane resealing process (Togo and Steinhardt 2004).…”