Munc13-1 acts as a priming factor for large dense-core vesicles in bovine chromaffin cells - PubMed (original) (raw)

Munc13-1 acts as a priming factor for large dense-core vesicles in bovine chromaffin cells

U Ashery et al. EMBO J. 2000.

Abstract

In chromaffin cells the number of large dense-core vesicles (LDCVs) which can be released by brief, intense stimuli represents only a small fraction of the 'morphologically docked' vesicles at the plasma membrane. Recently, it was shown that Munc13-1 is essential for a post-docking step of synaptic vesicle fusion. To investigate the role of Munc13-1 in LDCV exocytosis, we overexpressed Munc13-1 in chromaffin cells and stimulated secretion by flash photolysis of caged calcium. Both components of the exocytotic burst, which represent the fusion of release-competent vesicles, were increased by a factor of three. The sustained component, which represents vesicle maturation and subsequent fusion, was increased by the same factor. The response to a second flash, however, was greatly reduced, indicating a depletion of release-competent vesicles. Since there was no apparent change in the number of docked vesicles, we conclude that Munc13-1 acts as a priming factor by accelerating the rate constant of vesicle transfer from a pool of docked, but unprimed vesicles to a pool of release-competent, primed vesicles.

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Figures

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Fig. 1. Endogenous and overexpressed Munc13 isoforms in chromaffin cells. (A) Analysis of endogenously expressed Munc13 isoforms in chromaffin cells. Protein samples of indicated origin (20 µg per lane) were separated by SDS–PAGE and blotted onto nitrocellulose. Munc13 isoforms and Munc18 were detected by immunoblotting using specific antibodies. Note that low levels of Munc13-1, Munc13-3 and Munc18 are expressed in bovine chromaffin cells. The asterisk indicates a cross-reactive band of unknown origin present in bovine tissues. (B) Overexpression of Munc13-1 in bovine chromaffin cells. Chromaffin cells were infected with SFV–Munc13-1–GFP and harvested 16 h after infection. Protein samples of uninfected (Chr.C, no) and infected (Chr.C, yes) chromaffin cells (20 µg per lane) as well as rat brain synaptosomes (Brain, 20 µg per lane) were separated by SDS–PAGE and blotted on to nitrocelulose. Endogenous Munc13-1 and overexpressed Munc13-1–GFP were detected using a specific antibody directed against the C-terminus of Munc13-1. Note the massive overexpression of Munc13-1–GFP in chromaffin cells that had been infected with SFV–Munc13-1–GFP. (C) Distribution of overexpressed Munc13-1–GFP in adrenal chromaffin cells before and after application of 100 nM PMA. Scale bar represents 5 µm. (D) Ratio of fluorescence intensities of plasma membrane and cytoplasm before and after PMA application. Before PMA application, Munc13-1–GFP is distributed equally between the plasma membrane and the cytoplasm (_f_R = 0.86) while translocation of Munc13-1–GFP to the plasma membrane after PMA application caused an increase of more than 2-fold in the ratio (_f_R = 1.89). Error bars represent SEM.

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Fig. 1. Endogenous and overexpressed Munc13 isoforms in chromaffin cells. (A) Analysis of endogenously expressed Munc13 isoforms in chromaffin cells. Protein samples of indicated origin (20 µg per lane) were separated by SDS–PAGE and blotted onto nitrocellulose. Munc13 isoforms and Munc18 were detected by immunoblotting using specific antibodies. Note that low levels of Munc13-1, Munc13-3 and Munc18 are expressed in bovine chromaffin cells. The asterisk indicates a cross-reactive band of unknown origin present in bovine tissues. (B) Overexpression of Munc13-1 in bovine chromaffin cells. Chromaffin cells were infected with SFV–Munc13-1–GFP and harvested 16 h after infection. Protein samples of uninfected (Chr.C, no) and infected (Chr.C, yes) chromaffin cells (20 µg per lane) as well as rat brain synaptosomes (Brain, 20 µg per lane) were separated by SDS–PAGE and blotted on to nitrocelulose. Endogenous Munc13-1 and overexpressed Munc13-1–GFP were detected using a specific antibody directed against the C-terminus of Munc13-1. Note the massive overexpression of Munc13-1–GFP in chromaffin cells that had been infected with SFV–Munc13-1–GFP. (C) Distribution of overexpressed Munc13-1–GFP in adrenal chromaffin cells before and after application of 100 nM PMA. Scale bar represents 5 µm. (D) Ratio of fluorescence intensities of plasma membrane and cytoplasm before and after PMA application. Before PMA application, Munc13-1–GFP is distributed equally between the plasma membrane and the cytoplasm (_f_R = 0.86) while translocation of Munc13-1–GFP to the plasma membrane after PMA application caused an increase of more than 2-fold in the ratio (_f_R = 1.89). Error bars represent SEM.

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Fig. 2. Munc13-1 overexpression causes a 300% increase in catecholamine secretion in response to flash photolysis of caged calcium. (A) Averaged high time resolution recordings of membrane capacitance (Cm; middle trace) and amperometric current (IAMP; lower trace) in response to flash photolysis of caged calcium (indicated by an arrow) from control (black; n = 25), Munc13-1 (red; n = 20) and Munc13-1H567K (blue; n = 15) cells. For all cells, [Ca2+]i was kept at ∼20 µM for 5 s (upper trace). (B) The average increase in capacitance during the exocytotic burst (0–1 s) and during the sustained component (1–5 s) were about three times larger in Munc13-1 cells. The integral of the amperometric currents was increased by the same factor in Munc13-1 cells. (C) Detailed analysis of the exocytotic burst revealed that the fast (RRP) and the slow burst components (SRP) both increased in Munc13-1 cells (upper panels). In contrast, time constants for secretion were similar for control and Munc13-1 cells (lower panels). Error bars represent SEM. *p <0.02; **p <0.001 (_t_-test).

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Fig. 3. The number of morphologically docked vesicles is unchanged in Munc13-1 cells. Representative electron micrographs of control (A) and Munc13-1-overexpressing (B) bovine chromaffin cells. The overall distribution of organelles within a Munc13-1-expressing cell is similar to that of control cell. Dense-core vesicles are distributed homogeneously in the cell interior. (C) Quantitative analysis revealed that the fraction of ‘morphologically docked’ vesicles (≤200 nm from the plasma membrane) and the overall relative frequency (≤1300 nm from the plasma membrane) is similar in control (open circles) and Munc13-1 cells (closed circles). Furthermore, the density of vesicles is similar between control (3.8 ± 0.37 vesicles/µm2) and Munc13-1 cells (3.7 ± 0.46 vesicles/µm2). Bar represents 2 µm.

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Fig. 4. Munc13-1 cells secrete more vesicles, which contain a similar amount of catecholamine to control cells. (A) Examples of amperometric spike activity recorded from control (upper) and Munc13-1 cells (lower trace). Bars represent 10 pA, 1 s. (B) A plot of the amperometric charge against the number of spikes for control (open circles) and Munc13-1 cells (closed circles) shows a similar, linear relation. The difference in the number of spikes is statistically significant (p <0.02; _t_-test). (C) The average charge per vesicle was also similar in control and Munc13-1 cells [1.72 ± 0.19 pC (n = 12) and 1.86 ± 0.33 pC (n = 10), respectively]. Error bars represent SEM.

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Fig. 5. Catecholamine secretion in response to a second flash is significantly smaller in Munc13-1 cells. (A) The average response to a second flash in control cells (gray) was similar to the response to the first flash (black). (B) In contrast, secretion in response to the second flash in Munc13-1 cells (gray) was three to four times smaller than the response to the first flash (black). The burst amplitude of the second flash response was reduced to 65% of the first flash response in Munc13-1 cells (D), while it was similar in control cells (C). The amplitudes of the sustained component of the second flash response were reduced to 65% and 45% of the first flash response in Munc13-1 (D) and control cells (C), respectively. Error bars represent SEM.

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Fig. 6. Control cells contain a limited number of releasable vesicles. (A) Average membrane capacitance increase in control cells for four successive flashes given at 2 min intervals. The responses to the first two flashes were similar, but the third and fourth flashes led to significantly smaller responses. (B) The sum of the four flash responses from control cells (gray) was still smaller than the averaged response to the first flash in Munc13-1 cells (black).

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Fig. 7. A refined model for secretion in chromaffin cells. (A) According to this model, vesicles can reside in four separate states or pools. The estimated number of vesicles in each pool at steady state is indicated in brackets (gray for control; black for Munc13-1). The model predicts that Munc13-1 increases the forward rate constant (_k_1) from the unprimed pool (UPP) to the SRP by a factor of 20–30. (B) Simulation of secretion for Munc13-1 cells (dashed line) according to the parameters given in (A) yields an excellent fit for the experimental data (gray line). PM, plasma membrane; DV, docked vesicles.

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