Single secretory granules of live cells recruit syntaxin-1 and synaptosomal associated protein 25 (SNAP-25) in large copy numbers

Abstract

Before secretory vesicles undergo exocytosis, they must recruit the proteins syntaxin-1 and synaptosomal associated protein 25 (SNAP-25) in the plasma membrane. GFP-labeled versions of both proteins cluster at sites where secretory granules have docked. Single-particle tracking shows that minority populations of both molecules are strongly hindered in their mobility, consistent with their confinement in nanodomains. We measured the fluorescence of granule-associated clusters, the fluorescence of single molecules, and the numbers of unlabeled syntaxin-1 and SNAP-25 molecules per cell. There was a more than 10-fold excess of SNAP-25 over syntaxin-1. Fifty to seventy copies each of syntaxin-1 and SNAP-25 molecules were associated with a single docked granule, many more than have been reported to be required for fusion.

Fig. 1.

Clusters vary with expression levels. (A) Red (Left) and green fluorescence (Right) of a cell transfected with NPY-mCherry and with Syx-GFP using the CMV promoter. Strong expression of Syx makes S = 15,700. (B) (Left) Averages of ΔF and S were determined for each cell. The averages were sorted by S and binned at 15 cells per point. Next, ΔF was determined in regions placed randomly onto the same cells, plotted against S, and fitted by a straight line (slope ΔF/ S = 0.011). Finally, the difference was formed. ●, Measurements with a crippled CMV promoter (dCMV, n = 224 cells); ○, measurements with an intact CMV promoter (n = 122 cells). Curve is the best fit of ΔF = B_max x S/(k + S) to the combined data, with B_max = 297 CU and k = 2,225 CU. Coordinates on the top and right are based on the conversion factors in rows 5 and 6 of Table 1. (Right) Syx clusters at expression levels corresponding to S = 200–1000 CU (mean, 485 CU, 63 cells, dCMV) and S = 12,400–23,000 CU (mean 17,543 CU, 16 cells, CMV). Gaussian fits gave essentially identical widths. For clarity, most of the plasma membrane fluorescence was subtracted. (Scale bar: 0.5 μM.) (C) Percentage of granules associated with Syx-GFP. Pairs of images centered on single granules (2) were viewed with auto-scaling and scored visually by two independent viewers as to whether they contained a spot centered to within 89 nm. The fraction scoring positive was determined for each cell. Each point averages multiple cells with similar S values. ●, Measurements with the dCMV promoter (n = 224 cells); ○, measurements with an intact CMV promoter (n = 122 cells; ▲, association of Syx-GFP with random locations. (D) Schematic with two granules (shaded), one associated with a Syx cluster in the plasma membrane (blue). Vertical lines represent GFP-labeled (red) and endogenous Syx molecules (black). (E) As in B but for cells expressing GFP-SNAP-25 (dCMV promoter). (Left) ΔF as a function of S as in B (Left), and in bins of 20. (Right) Average of granules as in B (Left) and at S = 200–380 CU (mean 290 CU). ΔF represents 0.8 fluorescent molecules.

Fig. 2.

Photobleaching of Syx-GFP clusters and single molecules. (A). Averages of five consecutive 20-ms exposures (0.1 mJ at 488 nm). Time starts on illumination. (Cell MK2316) (B) C vs. time for the spot at the center. (C) Average of nine traces as in B. Exponential time constant is 1.62 s. (D) A stationary Syx-GFP molecule in a cell with extremely low Syx-GFP expression. Consecutive 20-ms exposures at 10-fold stronger illumination (1 mJ per exposure at 488 nm). (Cell SB2823) (E) C vs. time for the spot at the center. (F) Average of 50 traces as in E (four cells). Time constant is 0.17 s.

Fig. 3.

Single molecules in paraformaldehyde-fixed cells. (A) Average of the first 10 frames in a 20-Hz movie (1 mJ per exposure). (Cell MK3012) (B) Fluorescence at six locations in one cell. Excitation (488 nm) started at the vertical red line; vertical black lines mark the moment of bleaching, and dashed horizontal lines indicate the background. Bleaching is detected as a downward step followed by an abrupt lessening of noise. (Cell SB2450) (C) Mean time to bleaching (latency) as function of step size. Spots are located in 10-frame averages as in A. Step size (ΔC) is the fluorescence before bleaching minus fluorescence in the first 1.5 s thereafter, in traces as in B. Data are sorted by step amplitude and binned into packets of 10 points. The plot overestimates the latencies of the brightest molecules because we excluded spots bleaching in <200 ms. The plot underestimates the latencies of the dimmest molecules because our observation time was <7.5 s. Red line is taken from simulation in D. (D) Histogram of step amplitudes. Red line indicates a Monte-Carlo simulation assuming a normal distribution for both the brightness of molecules (coefficient of variation 0.3) and their distances from the coverslip (SI Text and Fig. S2). (Inset) Region near the origin on an expanded abscissa. Red line indicates fraction of detected synthetic spots plotted as a function of their brightness.

Fig. 4.

An excess of SNAP-25 over Syx. (A) Twofold serial dilutions of cell lysate (Left) and of recombinant Syx-1A (Right), both probed with antibody SC-12736. Numbers of cells (Left) and amounts of protein (Right) are given. We calculated 321,000 molecules per cell from this example. (B) Twofold serial dilutions of cell lysate (Left) and of recombinant SNAP-25 (Right) probed for SNAP-25 with antibody SySy 111002. We calculated 5,190,000 molecules per cell in this example. (C) Data in Fig. 1B (Left) are replotted, including all results with S > 50/μm². E = 540/μm² from Table 1. B = ΔF/R is the total amount of Syx bound per granule. Curve is the best fit to B = B_max R/(R + k) with B_max = 50 per granule and k = 467/μm². Results are weighted by the reciprocals of their variance. ●, Measurements with the dCMV promoter; ○, measurements with the intact CMV promoter.

Fig. 5.

Movement of single Syx-GFP molecules. (A–C) Image in a 50-Hz movie. The area outlined in red in A is magnified in B and bandpass-filtered in C. (D) The area outlined in red in C is shown at 20-ms intervals. A Syx-GFP molecule first moves downward and then moves toward the right; red points indicate its center of mass. The dot vanished temporarily in the penultimate frame as the GFP entered a dark state (“blinked”). Blinking terminated a track. (E) Histograms of distances traveled in 200 ms were fitted (red line) by Eq. [1] (22):Here r is the distance traveled during the time t (200 ms in this case), D₁ and D₂ are the diffusion coefficients, and A₁ and A₂ are proportional to the fractions of fast and slow molecules, respectively. With a single diffusion coefficient and A₂ forced to zero, the fit is poor for both Syx-GFP (black line) and for SNAP-25. These calculations are based on 8,811 trajectories in 17 cells (Syx-GFP) and 5,799 trajectories in 10 cells (GFP-SNAP-25). (F) Overlay of 50-Hz movies (0.14 mJ per frame at 488 nm) of granules (red) and Syx-GFP molecules (green). Granules were stationary during the observation period, and their images are averages. Green movies were low-pass filtered. (Upper) Syx-GFP was captured at the granule site (*) and bleached there about 0.44 s later. (**) was recorded 20 ms after the preceding image to show abrupt bleaching. (Lower) Syx-GFP was present during the first 0.24 s and then released. Times are relative to the beginning of the sequences (cell SB2278).