Physical mobilization of secretory vesicles facilitates neuropeptide release by nerve growth factor-differentiated PC12 cells

Abstract

It has been speculated that neurosecretion can be enhanced by increasing the motion, and hence, the availability of cytoplasmic secretory vesicles. However, facilitator-induced physical mobilization of secretory vesicles has not been observed directly in living cells, and recent experimental results call this hypothesis into question. Here, high resolution green fluorescent protein (GFP)-based measurements in nerve growth factor-differentiated PC12 cells are used to test whether altering dense core vesicle (DCV) motion affects neuropeptide release. Experiments with mycalolide B and jasplakinolide demonstrate that neuropeptidergic DCV motion at the ends of processes is proportional to F-actin. Furthermore, Ba2+ increases DCV mobility without detectably modifying F-actin. Finally, we show that altering DCV motion by changing F-actin or stimulating with Ba2+ proportionally changes sustained neuropeptide release. Therefore, increasing DCV mobility facilitates prolonged neuropeptide release.

Figure 5

F-actin is unaffected by Ba²⁺ Confocal images of representative processes that were fixed and stained for F-actin after exposure to control saline or depolarized in the presence of Ba²⁺. Left panels show Texas Red-X phalloidin labelling of F-actin while right panels show GFP fluorescence from DCVs. Scale bars indicate 2μm.

Figure 1

Actin depolymerization increases DCV motion A, wide field epifluorescence images of Texas Red-X phalloidin labelling of F-actin. Left, treated with vehicle. Right, treated with 2 μm mycalolide B for 30 min at room temperature. Scale bars indicate 2 μm. B, top, peptidergic vesicles remain nearly immobile for long periods in the presence of vehicle. Three images were acquired every 6 s and RGB colour-coded red, green and blue, respectively, as described in the Methods. With this scheme, stationary vesicles appear white and moving vesicles produce multicoloured spots. Bottom, DCV motion increases after actin depolymerization. Note that mycalolide B treatment stimulates secretory vesicle movement as indicated by the replacement of white vesicle images with coloured vesicle images. Scale bars indicate 2 μm. C, diffusion coefficients of secretory vesicles before (open bars) and after 15 min of treatment (black bars) with vehicle (four cells) or mycalolide B (MB, five cells). Number of vesicle trajectories shown in parentheses. ***P < 0.0005.

Figure 2

Jasplakinolide reduces DCV motion A, sets of images acquired every 6 s were RGB colour-coded as in Fig. 1. Note that DCV movement decreases after exposure to 10 μm jasplakinolide for 10 min as indicated by the increase in white vesicle images in the right panel. Scale bar indicates 2 μm. B, jasplakinolide (Jpk) decreases DCV diffusion coefficients. Number of vesicle trajectories for each measurement shown in parentheses. Data was collected from four cells. ***P < 0.0001.

Figure 3

F-actin limits sustained neuropeptide release Peptide content at the ends of processes is normalized to initial fluorescence (100 % F₀) so that release is evident as a decrease in this quantity. A, F-actin depolymerization with mycalolide B (•) enhances sustained neuropeptide release evoked by continual depolarization (○). n > 10. B, promoting actin polymerization with jasplakinolide (•) inhibits sustained neuropeptide release evoked by continual depolarization (○). n > 10.

Figure 4

Facilitation of neuropeptide release by Ba²⁺ A, continual depolarization in the presence Ba²⁺ (▵) causes more neuropeptide release than in the presence of Ca²⁺ (○). Note that while the initial rate of release is comparable, sustained release is enhanced by depolarization with Ba²⁺. n > 8. B, 2 μm mycalolide B (▴) does not facilitate Ba²⁺-evoked release (▵). n > 12. C, 10 μm jasplakinolide (▴) inhibits Ba²⁺-facilitated release (▵). n > 8.

Figure 6

Ba²⁺ increases secretory vesicle motion A, sets of images acquired over 6 s in NEM-treated cells either before (left) or after depolarization with Ba²⁺ for 15 min (right) were RGB colour-coded. Scale bar indicates 2 μm. B, change in DCV diffusion coefficients in NEM-treated cells that were exposed for 15 min to control saline (six cells) or depolarized in the presence of Ba²⁺ (eight cells). Number of vesicle trajectories is shown in parentheses. ***P < 0.0001.