Membrane hemifusion is a stable intermediate of exocytosis

Abstract

Membrane fusion during exocytosis requires that two initially distinct bilayers pass through a hemifused intermediate in which the proximal monolayers are shared. Passage through this intermediate is an essential step in the process of secretion, but is difficult to observe directly in vivo. Here we study membrane fusion in the sea urchin egg, in which thousands of homogeneous cortical granules are associated with the plasma membrane prior to fertilization. Using fluorescence redistribution after photobleaching, we find that these granules are stably hemifused to the plasma membrane, sharing a cytoplasmic-facing monolayer. Furthermore, we find that the proteins implicated in the fusion process-the vesicle-associated proteins VAMP/synaptobrevin, synaptotagmin, and Rab3-are each immobile within the granule membrane. Thus, these secretory granules are tethered to their target plasma membrane by a static, catalytic fusion complex that maintains a hemifused membrane intermediate.

FIGURE 1. The sea urchin egg as a model for studying membrane fusion

(A-C) Sequential snapshots of cortical granule exocytosis 0 (A), 20 (B), and 60 (C) seconds after fertilization. Note the polarized origin of secretion (arrow), originating at the site of sperm fusion. Scale bar equals 50μm. (A’) Detail of the egg cortical granules and their proximity to the plasma membrane. Scale bar equals 500 nm. (D) Differential interference contrast image of a cortical lawn preparation made from eggs. The edge of the plasma membrane is indicated (dashed line). Scale bar equals 5 μm. (E) Models of the membrane status of cortical granules at their closest proximity to the plasma membrane. Members of the SNARE complex used in this study are included for reference.

FIGURE 2. Cytoplasmically exposed, vesicle-associated proteins are immobile in the plane of the vesicle membrane

(A) Mean fluorescence recovery curves are shown for vesicle protein (AF488; reagent labeled all free amines) and Fab-labeled vesicle-associated proteins (Rab, VAMP/synaptobrevin, synaptotagmin) into one half of a bleached cortical granule attached to cortical lawns (see FIG 1D). Decay in fluorescence during the recovery period (Rab, synaptotagmin fluorescence at 270 sec) is a consequence of sample photobleaching during the time course. ^†VAMP/synaptobrevin recovery is calculated to be nearly zero, with a diffusion rate of (0.006 ± 0.008)μm² sec^-1. (Inset) Magnification of mean recovery of total protein associated with cortical granules. Diffusion rate of mobile protein population is listed. Note that total protein on unattached granules do not redistribute, and are thus immobile when attached to charged glass (data not shown). The time of bleaching was arbitrarily set as the origin of the time axis. All time-series data per replicate were normalized between the background (0%) and maximum fluorescence (100%) of the first pre-bleach frame. Numbers in brackets equal replicates per Fab. Colored dots correspond with images in ‘B’. (B) Snapshots of representative time-series using total protein labeling (left), anti-VAMP/synaptobrevin (middle), and anti-synaptotagmin Fabs (right). Prebleach and two post-bleach images are shown, with corresponding time during the recovery. The bleach area is bracketed to emphasize any retention of post-bleach asymmetry over time. Images are pseudocolored according to the colorized scale, showing low (black) to high (white)fluorescenceintensity. (C) Fluorescence intensity plots averaged across synaptotagmin-probed granules. Per replicate, intensity totals were calculated for each pixel of a line drawn perpendicular to the bleach boundary (snapshot y-axis; see ‘B’), summing the values for each pixel parallel to the bleach boundary (snapshot x-axis; see ‘B’). Data represent the mean (line) and standard deviation (shading) across all anti-synaptotagmin replicates. Curves correspond to snapshot images in ‘B’. Total width of each granule is 15 pixels (y-axis); fluorescence intensity is normalized to the first prebleach data set, as in ‘A’.

FIGURE 3. Cortical granules are hemifused to the plasma membrane

(A) List of specific diffusion rates and recovery times calculated for the 5 lipid probes used (see Methods). Numbers in brackets equal replicates per treatment. “nmr” = no measurable recovery. (B) Representative pseudocolor image of the types of vesicles analyzed. Attached and isolated cortical granules are found in the background image, with the edge of the plasma membrane indicated (dashed line). Colorized scale indicates lowest (black) to highest (white) fluorescence intensity. (C) Mean fluorescence recovery curves are shown for diffusion of lipid probes into fully bleached, attached cortical granules (solid lines) and re-associated cortical granules (dashed lines; di-4-ANEPPS n=3, di-8-ANEPPS n=4). (Inset) Mean recovery of di-8-ANEPPS in isolated, half-bleached granules. The time of bleaching was arbitrarily set as the origin of the time axis. All time-series data per replicate were normalized between the background (0%) and maximum fluorescence (100%) of the first pre-bleach frame. (D) Sequential snapshots from a representative fully bleached, attached and re-attached cortical granule stained with di-8-ANEPPS. Images are pseudocolored (as in ‘A’), with each separated from its predecessor by 25 seconds within the time-series. Arrowhead indicates a protrusion from a re-attached granule indicative of a hemifusion stalk (Zampighi et al., 2006). None of the preparations contained free probe in the media that could account for the recovery (see Methods).