Unexpected mobility variation among individual secretory vesicles produces an apparent refractory neuropeptide pool

Abstract

Most stored neuropeptide cannot be released from nerve terminals suggesting the existence of a refractory pool of dense core vesicles (DCVs). Past fluorescence photobleaching recovery, single particle tracking and release experiments suggested that the refractory neuropeptide pool corresponds to a distinct immobile fraction of cytoplasmic DCVs. However, tracking of hundreds of individual green fluorescent protein-labeled neuropeptidergic vesicles by wide-field or evanescent-wave microscopy shows that a separate immobile fraction is not evident. Instead, the DCV diffusion coefficient (D) distribution is unusually broad and asymmetric. Furthermore, the distribution shifts with a release facilitator. This unexpected variation, which could reflect heterogeneity among vesicles or in their medium, is shown to generate the appearance of a regulated refractory neuropeptide pool.

FIGURE 1

Expected distribution of diffusion coefficients for two populations of secretory vesicles. (A) Two populations with D values that vary by 10-fold with K = 20 measurements per vesicle. Plots are shown for different fractions (0, thin line; 1/3, short dashed line; 2/3, long dashed line; 1, thick line) of the fast population. The left graph is on a linear scale whereas the right graph is semilogarithmic. (B) Same as A except that D values for the two populations differed by threefold. The distributions f of log D_obs were obtained by transforming Eq. 2 to give f (log D_obs) = D_obs p(D_obs) (Stuart and Ord, 1994).

FIGURE 2

Distributions of DCV diffusion coefficients deduced by SPT performed with wide-field epifluorescence microscopy. The left plot uses linear binning whereas the right plot uses logarithmic binning. Note that wide-field microscopy samples a thick optical section.

FIGURE 3

Distributions of DCV diffusion coefficients deduced by SPT measured with total internal fluorescence microscopy. The left plot uses linear binning whereas the right plot uses logarithmic binning. Note that total internal reflection microscopy (also called evanescent-wave microscopy) samples a thin optical section near the cell surface.

FIGURE 4

Vesicle mobility is maintained until release. (A) Wide-field fluorescence images of secretory vesicles at the end of a process outlined in white. Numbers in corners show the period in seconds since the onset of stimulation by depolarization in the presence of Ba²⁺. Bar equals 1 μm. The vesicle indicated by the arrow in the first panel moves prior to release. (B) Rate of peptide release measured as the change in total peptide fluorescence (dGFP/dt) in the region shown in A. The last 4 points correspond to the images in A. Note that the vesicle disappearance is associated with a sudden drop in peptide content indicative of release. (C) Trajectory of the DCV indicated in A. (D) Open bars show the comparison of the diffusion coefficient derived from the final trajectory step prior to release (D_f) to the mean diffusion coefficient () and to the diffusion coefficient for the first 14 s of tracking (D_i). The closed bars show the comparisons after normalization. N = 8. No statistically significant differences are evident.

FIGURE 5

Model of neuropeptide release based on diffusion of secretory vesicles. The open circles show the release time course produced by using the one-term model with the data shown in Fig. 2 A. The solid line shows a fit of the open circles to a single exponential plus a constant. The dashed line shows the time course produced by the first term model for the mean D from the data in Fig. 2 A. The asterisks show the time course produced by using the three-term model.

FIGURE 6

A shift in the vesicle D distribution accounts for the change in release induced by Ba²⁺. (A) Comparison of observed neuropeptide release evoked by depolarization in the presence of Ca²⁺ (open circles) and Ba²⁺ (closed diamonds). N ≥ 8. (B) Diffusion coefficient histograms for vesicles under control conditions (a) and after stimulation with Ba²⁺ for 15 min (b). Secretory vesicles were tracked for 20 s at a rate of 0.5 Hz. C. Output of one-term model using the data shown in B. Note that modeled release is similar to the experimental data in A.