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* Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3200; and Kinesin and myosin have been proposed to
transport intracellular organelles and vesicles to the cell
periphery in several cell systems. However, there has
been little direct observation of the role of these motor
proteins in the delivery of vesicles during regulated exocytosis in intact cells. Using a confocal microscope, we
triggered local bursts of Ca2+-regulated exocytosis by
wounding the cell membrane and visualized the resulting individual exocytotic events in real time. Different temporal phases of the exocytosis burst were distinguished
by their sensitivities to reagents targeting different motor proteins. The function blocking antikinesin antibody SUK4 as well as the stalk-tail fragment of kinesin
heavy chain specifically inhibited a slow phase, while
butanedione monoxime, a myosin ATPase inhibitor, inhibited both the slow and fast phases. The blockage of
Ca2+/calmodulin-dependent protein kinase II with autoinhibitory peptide also inhibited the slow and fast
phases, consistent with disruption of a myosin-actin-
dependent step of vesicle recruitment. Membrane resealing after wounding was also inhibited by these reagents. Our direct observations provide evidence that
in intact living cells, kinesin and myosin motors may
mediate two sequential transport steps that recruit vesicles to the release sites of Ca2+-regulated exocytosis, although the identity of the responsible myosin isoform is
not yet known. They also indicate the existence of three
semistable vesicular pools along this regulated membrane trafficking pathway. In addition, our results provide in vivo evidence for the cargo-binding function of
the kinesin heavy chain tail domain.
Section of
Molecular and Cellular Biology, University of California, Davis, California 95616
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