Golgi-derived vesicles from developing epithelial cells bind actin filaments and possess myosin-I as a cytoplasmically oriented peripheral membrane protein.
In the intestinal brush border, the mechanoenzyme myosin-I links the microvillus core actin filaments with the plasma membrane. Previous immunolocalization shows that myosin-I is associated with vesicles in mature enterocytes (Drenckhahn, D., and R. Dermietzel. 1988. J. Cell Biol. 107:1037-1048) suggesting a potential role mediating vesicle motility. We now report that myosin-I is associated with Golgi-derived vesicles isolated from cells that are rapidly assembling brush borders in intestinal crypts. Crypt cells were isolated in hyperosmotic buffer, homogenized, and fractionated using differential- and equilibrium-density centrifugation. Fractions containing 50-100-nm vesicles, a similar size to those observed in situ, were identified by EM and were shown to contain myosin-I as demonstrated by immunoblotting and immunolabel negative staining. Galactosyltransferase, a marker enzyme for trans-Golgi membranes was present in these fractions, as was alkaline phosphatase, which is an apical membrane targeted enzyme. Galactosyltransferase was also present in vesicles immuno-purified with antibodies to myosin-I. Villin, a marker for potential contamination from fragmented microvilli, was absent. Myosin-I was found to reside on the vesicle "outer" or cytoplasmic surface for it was accessible to exogenous proteases and intact vesicles could be immunolabeled with myosin-I antibodies in solution. The bound myosin-I could be extracted from the vesicles using NaCl, KI and Na2CO3, suggesting that it is a vesicle peripheral membrane protein. These vesicles were shown to bundle actin filaments in an ATP-dependent manner. These results are consistent with a role for myosin-I as an apically targeted motor for vesicle translocation in epithelial cells.
Fath
K R
KR
Department of Biological Sciences, University of Pittsburgh, Pennsylvania 15260.
Burgess
D R
DR
D000199:Actins / Q000378:metabolism*; D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D001692:Biological Transport; D002451:Cell Compartmentation; D016764:Cell Polarity; D002645:Chickens; D004847:Epithelial Cells; D004848:Epithelium / Q000378:metabolism; D005700:Galactosyltransferases / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism*; D007413:Intestinal Mucosa / Q000648:ultrastructure*; D007425:Intracellular Membranes / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D008871:Microvilli / Q000382:microbiology / Q000648:ultrastructure; D009218:Myosins / Q000378:metabolism*
D000199:Actins; D008565:Membrane Proteins; D005700:Galactosyltransferases; D000469:Alkaline Phosphatase; D009218:Myosins