Membrane associated nonmuscle myosin II functions as a motor for actin-based vesicle transport in clam oocyte extracts.
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.
DePina
Ana S
AS
Marine Biological Laboratory, Woods Hole, Massachusetts, USA.
Wöllert
Torsten
T
Langford
George M
GM
D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D003599:Cytoskeleton / Q000378:metabolism; D006576:Heterocyclic Compounds, 4 or More Rings / Q000009:adverse effects; D008969:Molecular Sequence Data; D024462:Myosin Type II / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D009865:Oocytes / Q000737:chemistry / Q000378:metabolism; D022142:Secretory Vesicles / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D049890:Spisula / Q000378:metabolism*
D000199:Actins; D006576:Heterocyclic Compounds, 4 or More Rings; C472645:blebbistatin; D024462:Myosin Type II