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

Sandberg, Leslie A.; Stafford, Phillip; Langford, George M.

doi:10.2307/1542898

Cited by 6 publications

(9 citation statements)

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“…In fact myosin V and II can colocalize to the same vesicle (Costa et al, 1999;Miller and Sheetz, 2000). More recently myosin II has been found associated with a substantial fraction of axoplasmic organelles and is postulated to play a transport role for those organelles in axons (DeGiorgis et al, 2002), a finding that supports earlier results from antibodies (Sandberg et al, 2000). Likewise, our finding that myosin Va is not required to reseal membrane disruptions in S91 melanoma cells does not rule out that another uncharacterized myosin type V could be needed for resealing in these cells.…”

Section: Role Of Nonmuscle Myosin Iib In Membrane Resealing and Exocysupporting

confidence: 75%

“…3) Myosin II is purified with vesicles (Costa et al, 1999;Miller and Sheetz, 2000). 4) It has been shown that myosin II can carry vesicles along actin filament in vitro (Sandberg et al, 2000). 5) Injection of polyclonal antibody against nonmuscle myosin IIB inhibited neurotransmitter release (Mochida et al, 1994;Mochida, 1995).…”

Section: Role Of Nonmuscle Myosin Iib In Membrane Resealing and Exocymentioning

confidence: 99%

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

Togo¹,

Steinhardt

2004

MBoC

101

112

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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,...

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Section: Role Of Nonmuscle Myosin Iib In Membrane Resealing and Exocysupporting

confidence: 75%

Section: Role Of Nonmuscle Myosin Iib In Membrane Resealing and Exocymentioning

confidence: 99%

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

Togo¹,

Steinhardt

2004

MBoC

101

112

View full text Add to dashboard Cite

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“…Anti-myosin II antibodies and inhibitors of MLC phosphorylation prevented movement of myosin II-containing vesicles on actin filaments in vitro (44,45). Inhibition of myosin II activity or MLC phosphorylation suppressed insulin secretion in pancreatic islet cells (46), granule exocytosis in chromaffin cells (34), and mobilization of synaptic vesicles in presynaptic neurons (47,48).…”

Section: Expression Of a Non-phosphorylatable Mlc2 Mutant Decreases Amentioning

confidence: 99%

Myosin II Regulatory Light Chain Is Required for Trafficking of Bile Salt Export Protein to the Apical Membrane in Madin-Darby Canine Kidney Cells

Chan

Calderón

Swift

et al. 2005

Journal of Biological Chemistry

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“…The purified clam oocyte Myo2 sequenced by Edman chemistry was used to make a rabbit polyclonal antibody (ap205) [Sandberg et al, 2000]. The affinity purified ap205 antibody blocked actin-based vesicle transport in clam oocyte extracts as described below (Table IV).…”

Section: Peptide Sequences Of Clam Nonmuscle Myo2 Show High Homology mentioning

confidence: 99%

“…Consistent with other findings, blebbistatin did not inhibit assembly of actin bundles in these extracts (data not shown). In addition, we treated the extracts with the clam Myo2-specific ap205 antibody [Sandberg et al, 2000] and found that it blocked movement of vesicles in these extracts (Table IV). We also investigated the effect of treatment of extracts with RhoGDP dissociation inhibitor (RhoGDI), which binds GDP-Rho and prevents exchange of GDP for GTP.…”

Section: Inhibition Of Myo2 Function Blocks Vesicle Transport On Afs mentioning

confidence: 99%

Membrane associated nonmuscle myosin II functions as a motor for actin‐based vesicle transport in clam oocyte extracts

DePina

Wöllert

Langford

2007

Cell Motil. Cytoskeleton

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Nonmuscle myosin II (Myo2) has been shown to associate with membranes of the trans-Golgi network and to be involved in Golgi to ER retrograde protein transport. Here, we provide evidence that Myo2 not only associates with membranes but functions to transport vesicles on actin filaments (AFs). We used extracts from unactivated clam oocytes for these studies. AFs assembled spontaneously in these extracts and myosin-dependent vesicle transport was observed upon activation. In addition, actin bundles formed and moved relative to each other at an average speed of 0.30 microm/s. Motion analysis revealed that vesicles moved on the spontaneously assembled AFs at speeds greater than 1 microm/s. The motor on these vesicles was identified as a member of the nonmuscle Myo2 family based on sequence determination by Edman chemistry. Vesicles in these extracts were purified by sucrose gradient centrifugation and movement was reconstituted in vitro using skeletal muscle actin coated coverslips. When peripheral membrane proteins of vesicles including Myo2 were removed by salt stripping or when extracts were treated with an antibody specific to clam oocyte nonmuscle Myo2, vesicle movement was inhibited. Blebbistatin, a Myo2 specific inhibitor, also blocked vesicle movement. Myo2 light chain kinase activity was found to be essential for vesicle movement and sliding of actin bundles. Together, our data provide direct evidence that nonmuscle Myo2 is involved in actin-dependent vesicle transport in clam oocytes.

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

Cited by 6 publications

References 0 publications

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

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

Myosin II Regulatory Light Chain Is Required for Trafficking of Bile Salt Export Protein to the Apical Membrane in Madin-Darby Canine Kidney Cells

Membrane associated nonmuscle myosin II functions as a motor for actin‐based vesicle transport in clam oocyte extracts

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