Figure 4.

Astroglial lysosomes undergo Ca²⁺-dependent complete exocytosis. A, Spontaneous Ca²⁺ oscillations did not trigger exocytosis of FM4-64-labeled puncta (n = 92 Ca²⁺ transients in 12 cells). Scale bar, 3 μm. B, Typical signature of a fusion event in response to m-Stim on sequential TIRF images of a 488 nm excited 10 Hz ministack and kymograph. The central FM4-64-loaded vesicle (arrowhead) fuses at a time 0. Note the rapid destaining and spread of FM4-64 fluorescence (asterisk). Right, Fluorescence changes with time, measured in a small subregion centered on the same vesicle (3 × 3 pixels; circles) and in several concentric regions (0.2 μm wide) with increasing distance d. Scale bar, 2 μm. C, Left, Epifluorescence (EPI) and TIRF images of the same region of an FM4-64-labeled astrocyte before (Pre-Sti) and after (Post-Sti) stimulation. Epifluorescence FM4-64 and TIRF OGB-1 fluorescence from 1 × 1 μm single-spot regions in response to m-Stim (black arrowhead). Systematic and simultaneous Ca²⁺-dependent loss of the vesicle from EPI and TIRF images (supplemental Movie S6, available at www.jneurosci.org as supplemental material) indicates dye release in the extracellular space and not movement from the evanescent field into deeper cytoplasmic regions. Scale bar, 5 μm. D, Top, Dual-color 488-nm TIRF images of a EGFP-sialin/FM4-64 double-labeled astrocyte. Inset, Zoom near arrowhead. Bottom, Kymographs of the same vesicle (10 Hz) undergoing exocytosis in response to m-Stim (supplemental Movie S7, available at www.jneurosci.org as supplemental material). The white arrowhead identifies FM4-64 release at t = 0 s. The initiation of exocytosis was defined as the first point of FM4-64 fluorescence falling below the 2 s prestimulus average minus three times its SD. Evolution with time of FM4-64 (top) and EGFP (bottom) fluorescence of n = 13 double-labeled organelles undergoing Ca²⁺ triggered exocytosis after m-Stim. The gray traces are superimposed individual normalized and temporally aligned events. The traces were corrected for local background. Scale bar, 10 μm.