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. 1997 Sep 8;138(5):999-1008.
doi: 10.1083/jcb.138.5.999.

Kinesin- and myosin-driven steps of vesicle recruitment for Ca2+-regulated exocytosis

G Q Bi  1 , R L Morris, G Liao, J M Alderton, J M Scholey, R A Steinhardt
Affiliations

Kinesin- and myosin-driven steps of vesicle recruitment for Ca2+-regulated exocytosis

G Q Bi et al. J Cell Biol. .

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.

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Figures

Figure 1
Figure 1
Primary sequence of KHC. Arrows indicate approximate sites for antibody recognition. Numbers refer to KHC amino acid sequence number starting from the amino terminus. The “Stalk-Tail” and “Stalk” are the two KHC fragments used in this experiment.
Figure 2
Figure 2
Effect of reagents targeted against motor proteins on Ca2+-regulated exocytosis induced by laser wounding. The pseudocolor pictures are confocal fluorescence images of extracellular rhodamine dextran showing the exocytotic pockets into confocal focal plane just under the plasma membrane (Bi et al., 1995). Exocytotic events are visualized as the appearance of bright disks (0.5–1 μm diameter) against the dark intracellular background indicated by arrows. Blue arrows indicate early events, while green arrows indicate later events. (A) The exocytotic pattern of a sea urchin embryonic cell in natural sea water. In other series, the embryos were previously injected with (B) SUK4, (C) SUK2 antikinesin, (D) stalk-tail fragment, and (E) stalk fragment of KHC and were kept in natural sea water. In series F, the embryo was treated with 50 mM BDM. In series G, the embryo was injected with CaMK(273-302), an autoinhibitory peptide from the regulatory domain of CaM kinase type II. The large central stain in G is dye entering the unhealed wound. The first frame in each series was collected before wounding. Time 0 is defined as the moment immediately after wounding. The unit for time labels is seconds. Arrows indicate new exocytotic events that occurred since the previous frame. Bar, 5 μm.
Figure 3
Figure 3
Specific inhibition of a slow phase of Ca2+-regulated exocytosis by function-blocking antikinesin antibody SUK4. A quantifies cumulative number of exocytotic events in individual cells from typical experiments. The control cell (Ctrl) was not injected with any reagent. B summarizes the average number of exocytotic events that occurred within different time ranges from n experiments. n = 37 for Ctrl, 17 for SUK4, and 23 for SUK2. A biphasic temporal pattern of exocytosis is seen by comparing the SUK4 injection data with the Ctrl or SUK2 injection data. The slow phase (after 16 s), but not the fast phase (0–15 s) of exocytosis is inhibited by SUK4 antikinesin. Error bars are standard errors.
Figure 4
Figure 4
Specific inhibition of the slow phase of Ca2+-regulated exocytosis by KHC stalk-tail fragment. (A) Typical examples of the cumulative number of exocytotic events in individual cells. (B) The average number of exocytotic events that occurred within different time ranges from n experiments. n = 37 for Ctrl, 27 for Stalk-tail, and 33 for Stalk.
Figure 5
Figure 5
Reversible inhibition of both the slow and the fast phases of exocytosis by BDM. The cells were in 50 mM BDM for 10–60 min. For “Wash” experiments, cells were in 50 mM BDM for at least 45 min and were then transferred to BDM-free sea water for at least 15 min before imaging. (A) Quantified examples of individual experiments under different conditions. (B) Average number of exocytotic events that occurred within different time ranges from n experiments. n = 37 for Ctrl, 17 for BDM, and 8 for Wash.
Figure 6
Figure 6
Inhibition of both the slow and the fast phases of exocytosis by CaMK(273-302), an autoinhibitory peptide from the regulatory domain of CaM kinase type II. Control peptide CaMK(284-302) did not inhibit exocytosis. (A) Typical examples of cumulative number of exocytotic events in individual cells. (B) The average number of exocytotic events that occurred within different time ranges from n experiments. n = 18 for Ctrl, 39 for CaMK(273-302), and 22 for CaMK(284-302).
Figure 7
Figure 7
Three distinct vesicle pools and two-step vesicular recruitment for Ca2+-regulated exocytosis. (A) Three vesicle pools and their temporal distribution of exocytosis rates (number of exocytotic events per unit time) based on the data shown in Figs. 3–5. The “Immediate” pool of vesicles (squares) are BDM insensitive (and also kinesin reagents insensitive) and are therefore not dependent of either kinesin or myosin transport mechanism. The “Fast” pool (diamonds) is myosin dependent but kinesin independent. It was calculated by subtracting the myosin-independent (BDM-insensitive) component from the kinesin-independent exocytosis (average of SUK4 and Stalk-tail data). The “Slow” pool (circles) is kinesin dependent and was obtained by subtracting the kinesin-independent exocytosis from the average of control, SUK2, and Stalk data. (B) The proposed relative distribution of different vesicle pools and the kinesin- and myosin-mediated transport mechanisms.
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