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© The Rockefeller University Press,
0021-9525/2000//1013 $5.00
The Journal of Cell Biology, Volume 150, Number 5,
, 2000 1013-1026
Original Article |
A Myosin I Is Involved in Membrane Recycling from Early Endosomes
soldati{at}mpimf-heidelberg.mpg.de
Geometry-based mechanisms have been proposed to account for the sorting of membranes and fluid phase in the endocytic pathway, yet little is known about the involvement of the actin–myosin cytoskeleton. Here, we demonstrate that Dictyostelium discoideum myosin IB functions in the recycling of plasma membrane components from endosomes back to the cell surface. Cells lacking MyoB (myoA–/B–, and myoB– cells) and wild-type cells treated with the myosin inhibitor butanedione monoxime accumulated a plasma membrane marker and biotinylated surface proteins on intracellular endocytic vacuoles. An assay based on reversible biotinylation of plasma membrane proteins demonstrated that recycling of membrane components is severely impaired in myoA/B null cells. In addition, MyoB was specifically found on magnetically purified early pinosomes. Using a rapid-freezing cryoelectron microscopy method, we observed an increased number of small vesicles tethered to relatively early endocytic vacuoles in myoA–/B– cells, but not to later endosomes and lysosomes. This accumulation of vesicles suggests that the defects in membrane recycling result from a disordered morphology of the sorting compartment.
Key Words: Dictyostelium • endocytosis • membrane recycling • myosins • sorting
© 2000 The Rockefeller University Press
Abbreviations used in this paper: BDM, butanedione monoxime; cytA, cytochalasin A; MESNA, mercaptoethane sulfonic acid; PM4C4, plasma membrane marker 4C4; PDI, protein disulfide isomerase; SBS, Soerensen buffer containing sorbitol.
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