We have used quick-freezing and freeze-fracture to study early stages of exocytosis in rat peritoneal mast cells. Mast cells briefly stimulated with 48/80 (a synthetic polycation and well-known histamine-releasing agent) at 22°C displayed single, narrow-necked pores (some as small as 0.05 pm in diameter) joining single granules with the plasma membrane . Pores that had become as large as 0.1 lm in diameter were clearly etchable and thus represented aqueous channels connecting the granule interior with the extracellular space. Granules exhibiting pores usually did not have wide areas of contact with the plasma membrane, and clearings of intramembrane particles, seen in chemically fixed mast cells undergoing exocytosis, were not present on either plasma or granule membranes. Fusion of interior granules later in the secretory process also appeared to involve pores; granules were often joined by one pore or a group of 2-4 pores . Also found were groups of extremely small, etchable pores on granule membranes that may represent the earliest aqueous communication between fusing granules .Ultrastructural studies have shown that histamine secretion by mast cells, in response to either antigens or synthetic polycations, is mediated by a rapid, compound exocytosis (I, 3, 15, 16, 18, 19, 28). Early in the secretory process, single granules fuse with the plasma membrane, whereas at later stages membranes of adjacent granules in the cell interior fuse in succession to produce deep invaginations in the cell surface (1,19,28). These morphological observations correlate well with chemical measurements of release showing that 40-80% of the total cell histamine is secreted within 2 min (3, 28). There is good evidence that secretion is initiated by a rise in intracellular calcium activity (9,10,16,17,22), and for this reason the mast cell represents an important model of calcium-mediated stimulus-secretion coupling (8,12).This rapid exocytotic response of the mast cell has proven extremely useful for studying membrane fusion because numerous fusion events occur within a short period ; electron microscopy studies have already demonstrated what appear to be preliminary stages in this process. In thin sections of stimulated mast cells, granule membranes often contact the plasma membrane at sites where the intervening cytoplasm has been expressed (7,21), forming what Palade and Bruns (25) termed a "pentalaminar figure ." The occurrence of these structures in many types of secretory cells during exocytosis has suggested that they are a preliminary stage in membrane fusion (2,24,29,30) . In mast cells these contacts can be extensive and are often seen where the granule bulges against the plasma membrane. In freeze-fracture, these bulges frequently have plasma membrane domes that have been cleared of intramembrane particles (IMP) (7, 21). Furthermore, Lawson et al. (21) have shown in thin sections that a variety of ferritin-conjugated surface ligands will not bind to the plasma membrane where it contacts an underlying secretory...

Previously, we demonstrated that a protein from Xenopus egg jelly exhibits sperm chemoattractant activity when assayed by either video microscopy or by sperm passage across a porous filter. Here we describe the isolation and purification of allurin, the protein responsible for this activity. Freshly oviposited jellied eggs were soaked in buffer, and the conditioned medium was loaded onto an anion exchange column and eluted with an NaCl gradient. The active fraction was purified further by RP-HPLC, the chemoattractant protein appearing as a single sharp peak. The amino acid sequence of the protein, determined by direct sequencing and cloning of cDNAs coding for the protein, consisted of 184 amino acids having a molecular mass of 21,073 Da. The protein shares homology with the mammalian cysteine-rich secretory protein (CRISP) family that includes testes-specific spermatocyte protein 1, a cell adhesion protein which links spermatocytes to Seritoli cells, and acidic epididymal glycoproteins that bind to sperm and have been implicated in sperm-egg fusion. Phylogenetic analysis suggests that allurin evolved from the ancestral protein that gave rise to the mammalian CRISP family. Addition of allurin to this family portends that the CRISP family represents a group of ''sperm escort'' proteins, which bind to sperm at various steps in their life history, facilitating passage from one functional stage to the next. Allurin stands out in this regard, representing both the first vertebrate sperm chemoattractant to be purified and sequenced and the first member of the CRISP family to be found in the female reproductive tract. sperm chemotaxis ͉ fertilization ͉ TPX-1 ͉ AEG ͉ CRISP

Rapid-freezing/freeze-fracture electron microscopy and whole-cell capacitance techniques were used to study degranulation in peritoneal mast cells of the rat and the mutant beige mouse. These studies allowed us to create a time-resolved picture for fusion pore formation. After stimulation, a dimple in the plasma membrane formed a small contact area with the secretory granule membrane. Within this zone of apposition no ordered proteinaceous specializations were seen. Electrophysiological technique measured a small fusion pore which widened rapidly to 1 nS. Thereafter, the fusion pore remained at semi-stable conductances between 1 and 20 nS for a wide range of times, between 10 and 15,000 msec. These conductances correspond to pore diameters 25-36 nm. Ultrastructural data confirmed small pores of hourglass morphology, composed of biological membrane coplanar with both the plasma and granular membranes. Later, the fusion pore rapidly increased in conductance, consistent with the observed morphology of omega-figures. The hallmarks of channel-like behavior, instantaneous jumps in pore conductance between defined levels, and sharp peaks in histograms of conductance dwell-time, were not seen. Since the morphology of small pores shows contiguous fracture planes, the electrical data represent pores that contain lipid. These combined morphological and electrophysiological data are consistent with a lipid/protein complex mediating both the initial and later stages of membrane fusion.

Exocytosis of cortical granules was observed in sea urchin eggs, either quickfrozen or chemically fixed after exposure to sperm . Fertilization produced a wave of exocytosis that began within 20 s and swept across the egg surface in the following 30 s. The front of this wave was marked by fusion of single granules at well-separated sites . Toward the rear of the wave, granule fusion became so abundant that the egg surface was left with confluent patches of granule membrane . The resulting redundancy of the egg surface was accommodated by elaboration of characteristic branching microvilli, and by an intense burst of coated vesicle formation at -2 min after insemination.Freeze-fracture replicas of eggs fixed with glutaraldehyde and soaked in glycerol before freezing displayed forms of granule membrane interaction with the plasma membrane which looked like what other investigators have considered to be intermediates in exocytosis . These were small disks of membrane contact or membrane fusion, which often occurred in multiple sites on one granule and also between adjacent granules . However, such membrane interactions were never found in eggs that were quick-frozen without fixation, or in eggs fixed and frozen without exposure to glycerol . Glycerination of fixed material appeared to be the important variable; more concentrated glycerol produced a greater abundance of such "intermediates ." Thus, these structures may be artifacts produced by dehydrating chemically fixed membranes, and may not be directly relevant to the mechanism by which membranes naturally fuse.KEY WORDS exocytosis -membrane fusionquick-freezing -fixation and glycerination artifacts -fertilization Sea urchin eggs contain a single layer of cortical granules just under the plasma membrane, which undergo exocytosis during fertilization (1,3,7,8,22,30,38) . This so-called "cortical reaction" occurs in a wave that originates at the point of sperm J. CELL BIOLOGY

Stimulus-secretion coupling in pancreatic exocrine cells was studied using dissociated acini, prepared from mouse pancreas, and chlorotetracycline (CTC), a fluorescent probe which forms highly fluorescent complexes with Ca ~+ and Mg ~+ ions bound to membranes. Acini, preloaded by incubation with CTC (100/xM), displayed a fluorescence having spectral properties like that of CTC complexed to calcium (excitation and emission maxima at 398 and 527 nm, respectively). Stimulation with either bethanechol or caerulein resulted in a rapid loss of fluorescence intensity and an increase in outflux of CTC from the acini. After 5 rain of stimulation, acini fluorescence had been reduced by 40% and appeared to be that of CTC complexed to Mg 2+ (excitation and emission maxima at 393 and 521 nm, respectively). The fluorescence loss induced by bethanechol was blocked by atropine and was seen at all agonist concentrations that elicited amylase release. Maximal fluorescence loss, however, required a bethanechol concentration three times greater than that needed for maximal amylase release. In contrast, acini preloaded with ANS or oxytetracycline, probes that are relatively insensitive to membrane-bound divalent cations, displayed no secret~ agogue-induced fluorescence changes. These results are consistent with the hypothesis that CTC is able to probe some set of intracellular membranes which release calcium during secretory stimulation and that this release results in dissociation of Ca~+-complexed CTC.KEY WORDS exocrine pancreas stimulussecretion coupling calcium fluorescent probe chlorotetracyclinePancreatic acinar cells are thought to use calcium as a second messenger because the divalent cation ionophore, A23187, is able to increase enzyme release from these cells in the presence of extracellular calcium (10,16,32,39). However, the manner in which physiological secretagogues initiate a rise in cytosol calcium is not known.