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. 2012 Jun 15;125(Pt 12):2986-94.
doi: 10.1242/jcs.102632. Epub 2012 Mar 5.

Spatiotemporal resolution of mast cell granule exocytosis reveals correlation with Ca2+ wave initiation

Affiliations

Spatiotemporal resolution of mast cell granule exocytosis reveals correlation with Ca2+ wave initiation

Roy Cohen et al. J Cell Sci. .

Abstract

Mast cell activation initiated by antigen-mediated crosslinking of IgE receptors results in stimulated exocytosis of secretory lysosomes in the process known as degranulation. Much has been learned about the molecular mechanisms important for this process, including the crucial role of Ca(2+) mobilization, but spatio-temporal relationships between stimulated Ca(2+) mobilization and granule exocytosis are incompletely understood. Here we use a novel imaging-based method that uses fluorescein isothiocyanate (FITC)-dextran as a reporter for granule exocytosis in RBL mast cells and takes advantage of the pH sensitivity of FITC. We demonstrate the selectivity of FITC-dextran, accumulated by fluid-phase uptake, as a marker for secretory lysosomes, and we characterize its capacity to delineate different exocytotic events, including full fusion, kiss-and-run transient fusion and compound exocytosis. Using this method, we find strong dependence of degranulation kinetics on the duration of cell to substrate attachment. We combine imaging of degranulation and Ca(2+) dynamics to demonstrate a spatial relationship between the sites of Ca(2+) wave initiation in extended cell protrusions and exocytosis under conditions of limited antigen stimulation. In addition, we find that the spatially proximal Ca(2+) signaling and secretory events correlate with participation of TRPC1 channels in Ca(2+) mobilization.

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Figures

Fig. 1.
Fig. 1.
TxRed–dextran accumulates in secretory granules in RBL-2H3 cells. TxRed–dextran accumulates in secretory granules labeled with CD63–GFP (A) and EGFP-VAMP7 (B) in RBL-2H3 cells. Representative confocal images show each component separately, as well as merged image with bright-field illumination. Insets show high magnification of regions marked by yellow boxes. Cells in A are labeled with TxRed–dextran in the presence of 5-HT; cells in B are labeled without 5-HT and were fixed before imaging. Analysis of multiple cells demonstrates high colocalization of EGFP–VAMP7 and TxRed–dextran (Pearson's coefficient >0.7; n = 8).
Fig. 2.
Fig. 2.
Individual antigen-stimulated granule exocytosis events are imaged using FITC–dextran to label secretory granules. (A,B) ‘Full Fusion’ exocytosis event occurring proximal to the cell body (indicated by yellow box in right panel of B). Note release of FITC–dextran that diffuses away in the external medium. (C,D) ‘Kiss-and-Run’ exocytosis, where an initial transient granule fusion is reversed then followed by a prolonged secretion of FITC–dextran. (E,F) ‘Compound Exocytosis’, where three adjacent granules fuse with the plasma membrane in a continuous sequential manner. A,C,E show the progression in time-resolved frames. B,D,F show the time trace of fluorescence for depicted events. Kymographs of granule secretion are shown on the right in D and F, where x-axis represents time and y-axis represents distance along the arrow labeled ‘d’ in C and E.
Fig. 3.
Fig. 3.
Stimulation by antigen causes simultaneous release of FITC–dextran and TxRed–dextran from individual granules in co-loaded RBL cells. Antigen (1.7 µg/ml in the pipette) was puffed at t = 0, and fluorescence changes in the red and green channels were monitored. (A) Individual red and green frames showing the time course of granule fusion at the specified ROI (yellow circle) on the same cell. (B) Time traces of integrated fluorescence for ROI highlighted in A demonstrate concurrent FITC–dextran dequenching and TxRed–dextran release, followed by diffusion or dilution of both probes in the external medium.
Fig. 4.
Fig. 4.
Simultaneous amperometry and FITC–dextran imaging of an individual cell after antigen stimulation. RBL cells, which were incubated overnight with IgE, FITC–dextran and 5-HT, were approached individually with an amperometric carbon fiber and imaged after puffing antigen (1.7 µg/ml in micropipette) at t = 0. Amperometric signal (black trace) is due to 5-HT oxidation on the carbon fiber, and FITC fluorescence increase (color traces) is due to dequenching; both of these events occur upon granule release. Fluorescence can be visualized from individual granules and are represented by different colors (see inset; the imaging frame rate was 4 Hz). The amperometric signal does not distinguish among granules. Inset is a micrograph showing cell outlined in yellow and carbon fiber outlined in black; location of individual granule exocytosis events monitored by fluorescence increases are indicated by corresponding colors in the time trace.
Fig. 5.
Fig. 5.
Degranulation of FITC–dextran in suspended RBL cells. Degranulation of FITC–dextran in suspended RBL cells, monitored by fluorimetry, depends on extracellular Ca2+. Cells (∼106/ml) in a cuvette were stimulated by addition of antigen (1.7 µg/ml) in the presence (black trace) or absence (gray trace) of extracellular Ca2+ as indicated. Subsequently, A23187 (1 µM) was added to the sample with extracellular Ca2+ as indicated (top panel). Fluorescence for both samples is normalized to total FITC–dextran fluorescence detected following addition of 0.1% Triton X-100 at the end of each experiment (not shown). Initiation of degranulation responses of attached cells depends on time of adherence (bottom panel). Cells were allowed to adhere for indicated times, then stimulated with delivery of antigen (1.7 µg/ml) from a micropipette, and exocytotic events were monitored in individual cells by enumerating bursts of FITC–dextran fluorescence.
Fig. 6.
Fig. 6.
Cellular Ca2+ and degranulation responses, monitored by independent fluorescent probes, can be compared in time and space. (A) Representative images of an RBL cell loaded with FITC–dextran and Fura Red. Left panel shows bright-field and FITC–dextran images 75 seconds after initiation of stimulation (1.7 µg/ml in pipette). Middle panel is Fura Red image at same time point and ROI (yellow line) evaluated in B. Right panel is overlay of Fura Red image and Z projection of FITC–dextran images and indicates degranulation events (green spots) over time. (B) Kymograph of Ca2+ dynamics and degranulation events for the cell in A. The x-axis represents the time dimension (time bar indicates the first 30 seconds of image acquisition); the y-axis corresponds to the distance along the yellow line specified in middle panel of A (wave direction indicated by arrowhead). Top panel shows Ca2+ changes along the cell as imaged with Fura Red; dashed yellow line indicates Ca2+ wave direction from protrusion toward cell body. Middle panel is FITC–dextran channel, showing individual degranulation events as detected in time and along cell length. Bottom panel is overlay of Fura Red and FITC channels, showing the spatial and temporal relationship between Ca2+ dynamics and degranulation events. (C) Timing of degranulation compared with Ca2+ oscillations in an individual cell. Degranulation events (green, blue, red), measured as bursts of dequenched FITC–dextran, are superimposed on traces of Ca2+ oscillations (black) that were imaged in the same cell using Fura Red and plotted as the inverse of the fluorescence intensity (b marks Ca2+ peak). (D) Histogram for multiple cells of timing for peaks in Ca2+ oscillations. The width of individual Ca2+ peaks, normalized by the distance between two troughs (e.g. a = 0, c = 1 in C), was used to quantify the relative time of the peak (e.g. b corresponds to ∼0.5 in C). Relative times for peak Ca2+ were calculated and plotted as a histogram in binned intervals for multiple events. (E) Histogram for multiple cells of timing for degranulation events. Using the same normalized time scale based on Ca2+ peaks as for D, the relative times of exocytosis bursts in respective cells were calculated, binned, and plotted as a histogram. The histograms in D and E were derived from 20 stimulated cells showing oscillatory Ca2+ with a total of 51 exocytosis events. The smooth curves are normal distribution fits. The analysis of D and E confirms visual impression for the representative cell in C that degranulation events usually follow just after the peak of the Ca2+ oscillations.
Fig. 7.
Fig. 7.
Spatial distributions of exocytotic events stimulated by high (1.7 µg/ml) and low (1.7 ng/ml) doses of antigen. Cells loaded with FITC–dextran and Fura Red were stimulated by puffs of designated doses of antigen in the micropipette, and images were collected as in Fig. 6. (A) Summary of the numbers and relative distributions of exocytosis events in protrusions (PRTS) compared with the cell body as functions of antigen dose and initiation site for Ca2+ waves (B-waves originate in cell body; P-waves originate in extended cell protrusions; **P = 0.008 refers to the total number of events). For 1.7 µg/ml DNP-BSA, n = 30 cells: 19 cells had P-waves (80 P-events and 350 B-events), and 11 cells had B-waves (29 P-events and 208 B-events). For 1.7 ng/ml DNP-BSA, n = 25 cells: 19 cells had P-waves (169 P-events and 143 B-events) and 6 cells had B-waves (13 P-events and 22 B-events). (B) Time courses for distributions of degranulation events in protrusions, following P-waves, for high and low antigen doses. (C) Number of degranulation events per cell occurring in protrusions as a function of the number of preceding Ca2+ transients in that location for stimulation with 1.7 ng/ml. Error bars show s.e.m.
Fig. 8.
Fig. 8.
TRPC1 knockdown reduces the frequency and delays the onset of exocytotic events. (AC) Cells were transfected with shTRPC1 or control vector, loaded with FITC–dextran, and stimulated with 7 ng/ml antigen in the micropipette. Time courses show average numbers of exocytotic events per cell: total events (A), events in cell body (B), and events along protrusions (C). (D) Cumulative exocytotic events over 300 seconds from A–C. Error bars show s.e.m. (n = 33 for shTRPC1; n = 26 for shRNA control). Percentage values above shTRPC1 columns represent the decrease of exocytosis due to TRPC1 knockdown. Differences between shTRPC1 and control: total events, P = 0.0099; cell body events, P = 0.051; protrusion events, P = 0.050.

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