Kinesin- and Myosin-driven Steps of Vesicle Recruitment for Ca2+-regulated Exocytosis

Abstract

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.

Membrane damage-induced vesicle–vesicle fusion of dysferlin-containing vesicles in muscle cells requires microtubules and kinesin

“…Dysferlincontaining vesicles are thought to play a critical role in muscle resealing, but there have been very few studies attempting to directly examine the behavior of dysferlin-containing vesicles in livemuscle cells in the context of cellular wounding. Studies from non-muscle model systems suggest that microtubule-based transport of intracellular vesicles is critical for resealing (15,21), but the role of microtubules and kinesin motors in dysferlin-mediated membrane repair in muscle remains unexplored. Therefore, the aim of this study was to examine the behavior of dysferlincontaining vesicles under normal conditions and following cellular wounding in muscle cells, and test the hypothesis that microtubules and kinesin are required for dysferlin-containing vesicle function in muscle cells.…”

“…Membrane resealing is a conserved process by which cells are able to survive mechanical disruption of the plasma membrane (14)(15)(16)(17)(18)(19). Dysferlin-null skeletal and cardiac muscle show enhanced uptake of membrane impermeable dye following laser-induced wounding, suggesting that dysferlin may play a role in membrane resealing (3,20).…”

“…Current knowledge of membrane resealing is largely derived from studies in the sea urchin egg and fibroblast model systems, which demonstrated that fusion of intracellular vesicles with the plasma membrane is critical for resealing (15)(16)(17)21). Dysferlin localizes to the plasma membrane and intracellular vesicles in developing myotubes, and interacts with numerous proteins involved in membrane transport, including caveolin-3 (22,23), annexin-4 (5), annexin-6 (24), enlargeosomal marker AHNAK (25) and tubulin (26), but the exact contribution of dysferlincontaining vesicles to resealing following wounding remains elusive, as few studies have examined the behavior of dysferlincontaining vesicles in live cells following cellular wounding.…”

Mechanisms of Exocytosis in Insulin‐Secreting B‐Cells and Glucagon‐Secreting A‐Cells

“…The previous finding that exogenous Ca 2π -chelators inhibit exocytosis in endocrine cells (Ämmälä et al 1993;Chow et al 1996) (Chen et al 1999), the Ca 2π -dependent phosphatase calcineurin (Renström et al 1996b), CAPS (Ca 2π -dependent activator protein for secretion; Ann et al 1997), and Ca 2π /calmodulin kinase II (Ämmälä et al 1993;Gromada et al 1999). Blockage of the latter inhibited the slow phase, and suggested a myosin-actin-dependent step in the granule recruitment (Bi et al 1997). The basal concentration of cytosolic Ca 2π depends to a great extent on intracellular Ca 2π -handling, and there is evidence for Ca 2π -release from the ER (Tengholm et al 1998;Holz et al 1999), for Ca 2π -uptake by the mitochondria (Rutter et al 1993), and for accumulation of Ca 2π in the granules (Scheenen et al 1998;Mitchell et al 2001).…”

Clonal Tests of Conventional Kinesin Function during Cell Proliferation and Differentiation

“…It is also thought to be important for vesicle transport from the Golgi to the plasma membrane, one of the late steps in the secretion pathway, and in Golgi-to-ER membrane recycling, which is an indirect but essential part of the early secretion pathway (reviewed by Goodson et al, 1997;Lane and Allan, 1998;Goldstein and Philp, 1999). For example, based on the effects of anti-kinesin heavy chain (KHC) antibodies or a KHC tail fragment microinjected into sea urchin embryos, kinesin has been proposed to be important for outward transport, to the cortex, of a class of vesicles used for rapid membrane repair (Bi et al, 1997). Furthermore, based on immunolocalization and on the effects of anti-KHC antibodies on brefeldin A-induced Golgi-to-ER membrane transport in vertebrate cultured cells, kinesin has been proposed to be the motor for Golgito-ER membrane recycling (Lippincott-Schwartz et al, 1995).…”