Neuropeptide release by efficient recruitment of diffusing cytoplasmic secretory vesicles

Abstract

Neuropeptides are slowly released from a limited pool of secretory vesicles. Despite decades of research, the composition of this pool has remained unknown. Endocrine cell studies support the hypothesis that a population of docked vesicles supports the first minutes of hormone release. However, it has been proposed that mobile cytoplasmic vesicles dominate the releasable neuropeptide pool. Here, to determine the cellular basis of the releasable pool, single green fluorescent protein-labeled secretory vesicles were visualized in neuronal growth cones with the use of an inducible construct or total internal reflection fluorescence microscopy. We report that vesicle movement follows the diffusion equation. Furthermore, rapidly moving secretory vesicles are used more efficiently than stationary vesicles near the plasma membrane to support stimulated release. Thus, randomly moving cytoplasmic vesicles participate in the first minutes of neuropeptide release. Importantly, the preferential recruitment of diffusing cytoplasmic secretory vesicles contributes to the characteristic slow kinetics and limited extent of sustained neuropeptide release.

Figure 1

Single GFP-labeled secretory vesicles in neuronal growth cones. (A) Cells were treated with 1 μM muristerone to activate the inducible expression construct before imaging. (Upper) Bright-field view of neurites illuminated with a Hoffman condenser. (Lower) Epifluorescence image shows that single secretory vesicles labeled with proANF-Emd are evident in one growth cone. Outline shows transfected neurite. (B Upper) Conventional epifluorescence image of a growth cone expressing the constitutive proANF-Emd construct. Note that it is difficult to resolve individual vesicles. (Lower) Single secretory vesicles are evident in the TIRFM image of the same growth cone. (Bar = 2 μm.)

Figure 2

Secretory vesicle trajectories within growth cones. (A) Examples from vesicles labeled with the inducible construct. (B) Examples detected by TIRFM. The xy trajectory and the simultaneous movement over time perpendicular to the substrate are shown for two vesicles. Images were acquired at 1 Hz. Note that motion in all dimensions appears to be random. Furthermore, a great range of vesicle speeds is evident.

Figure 3

Secretory vesicles move by diffusion. (A) d² vs. time plots for rapid, mobile, and slow, immobile vesicles. Note that both sets of data can be fit with straight lines yielding diffusion coefficients of 6.1 × 10⁻¹¹ cm²/s for the mobile vesicles and 3.1 × 10⁻¹² cm²/s for the immobile vesicles. n ≥ 15 for each point. (B) Cumulative plots of number of trajectory steps vs. distance traveled. Note that both sets of data can be fit by the relationship predicted by diffusion (see text). Data included 152 points from 6 representative immobile vesicles and 145 points from 7 representative mobile vesicles labeled with the inducible construct sampled at 0.9 Hz. Calculated diffusion coefficients were 4.6 × 10⁻¹¹ cm²/s for the mobile vesicles and 3.3 × 10⁻¹² cm²/s for the immobile vesicles.

Figure 4

Secretion depletes mobile secretory vesicles viewed by TIRFM in growth cones. (A) Image processing was used to display vesicles that took large steps (“mobile”) or that were stationary. Note that after depolarization, mobile vesicles are depleted whereas stationary vesicles show little change. (Bar = 2 μm.) (B) Quantitation from five experiments. Sustained depolarization started at 0 min. Mobile vesicle depletion was greater than stationary vesicle depletion. Furthermore, mobile vesicle depletion was at least as rapid as for stationary vesicles. Finally, although the measurement of mobile vesicles is not prone to contamination by slow, “immobile” vesicles, mobile vesicles occasionally stop and, hence, contribute to the measurement of stationary vesicles.

Figure 5

Mobile vesicles release their contents. (A) Normalized time course of peptide release from growth cones with mostly mobile vesicles labeled with the inducible construct. Data derived from epifluorescence measurements from four growth cones. Note that residual fluorescence present after depolarization because of cell autofluorescence, light scattering, and unreleased peptide is not shown in this graph. Sustained depolarization began at 0 min. (B–F) Color-coded feature-extracted images of vesicles in a stimulated growth cone. Two images were acquired 5 s apart and then color coded as described in the text. Mobile vesicles appear red or green and stationary vesicles are yellow. The yellow vesicle in B subsequently moved, indicating that it was a mobile vesicle that had paused randomly. Note that sustained depolarization causes red and green vesicle images to disappear, indicating that mobile vesicles undergo exocytosis. Similar results were obtained in three other experiments.