Visualization of exocytotic secretory processes of mast cells by fluorescence techniques

Secretory processes via exocytosis in rat peritoneal mast cells were visualized by two complementary fluorescence techniques; one staining pre-exocytotic granules with a basic probe and the other staining post-exocytotic granules with acidic probes. Granules within mast cells were selectively stained with acridine orange and emitted orange yellow fluorescence. Upon stimulation with compound 48/80, release of acridine orange from granules was observed both in population and single cell measurements. This release was seen in some localized area of mast cells. Opening of pores between plasma membranes and granule membranes was monitored using acidic fluorescence probes such as 6-carboxyfluorescein or lucifer yellow CH. Not only granules located at peripheral region, but also granules near the core region participated in exocytosis. The existence of junctions between these granules was suggested. TMA-DPH, a lipophilic membrane probe, which was localized at plasma membrane before stimulation, diffused into granule membranes after stimulation. This shows that after stimulation, some constituents of plasma and granule membranes were mixed. Even after extensive degranulation, mast cells extruded acidic probes, indicating the plasma membranes still play a role of barrier. Activation of lateral motion of granules preceding to exocytosis was not observed. It was concluded that the visualization of secretory processes by fluorescence and image processing techniques will be useful for the study of molecular mechanisms underlying exocytosis.     

Formation and contraction of a microfilamentous shell in saponin- permeabilized platelets

To study the mechanism of granule centralization in platelets, we permeabilized with saponin in either EGTA (5 mM) or calcium (1 or 10 microM). Under all conditions, platelets retained 40-50% of their total actin and greater than 70% of their actin-binding protein (ABP) but lost greater than 80% of talin and myosin to the supernatant. Thin sections of platelets permeabilized in EGTA showed a microfilament network under the residual plasma membrane and throughout the cytoplasm. Platelets permeabilized in calcium contained a microfilament shell partly separated from the residual membrane. The shell stained brightly for F-actin. A less dense microfilament shell was also seen in sections of ADP-stimulated intact platelets subsequently permeabilized in EGTA. In the presence of 1 mM ATP gamma S and calcium, myosin was retained (70%) and was localized by indirect immunofluorescence in bright central spots that also stained intensely for F-actin. Electron micrographs showed centralized granules surrounded by a closely packed mass of microfilaments much like the structures seen in thrombin-stimulated intact platelets subsequently permeabilized in EGTA. Permeabilization in calcium, ATP, and okadaic acid, produced the same configuration of centralized granules and packed microfilaments; myosin was retained and the myosin regulatory light chain became phosphorylated. Microtubule coil disassembly before permeabilization did not inhibit granule centralization. These results suggest a possible mechanism for granule centralization in these models. The cytoskeletal network first separates from some of its connections to the plasma membrane by a calcium-dependent mechanism not involving ABP proteolysis. Phosphorylated myosin interacts with the microfilaments to contract the shell moving the granules to the platelet's center.     

Exocytosis in bovine chromaffin cells: studies with patch-clamp capacitance and FM1-43 fluorescence

In response to physiological stimuli, neuroendocrine cells secrete neurotransmitters through a Ca(2+)-dependent fusion of secretory granules with the plasma membrane. We studied insertion of granules in bovine chromaffin cells using capacitance as a measure of plasma membrane area and fluorescence of a membrane marker FM1-43 as a measure of exocytosis. Intracellular dialysis with [Ca(2+)] (1.5-100 microM) evoked massive exocytosis that was sufficient to double plasma membrane area but did not swell cells. In principle, in the absence of endocytosis, the addition of granule membrane would be anticipated to produce similar increases in the capacitance and FM1-43 fluorescence responses. However, when endocytosis was minimal, the changes in capacitance were markedly larger than the corresponding changes in FM1-43 fluorescence. Moreover, the apparent differences between capacitance and FM1-43 fluorescence changes increased with larger exocytic responses, as more granules fused with the plasma membrane. In experiments in which exocytosis was suppressed, increasing membrane tension by osmotically induced cell swelling increased FM1-43 fluorescence, suggesting that FM1-43 fluorescence is sensitive to changes in the membrane tension. Thus, increasing membrane area through exocytosis does not swell chromaffin cells but may decrease membrane tension.     

Microfilament-associated proteins in tissue culture cells viewed by stereo immunofluorescence microscopy.

Stereo immunofluorescence microscopy avoids the problem of juxtaposition of structures often encountered in normal fluorescence microscopy. The procedure has been used in conjunction with antibodies against microfilament associated proteins to reveal the arrangement of microfilaments in a rat mammary cell line both in the fully spread state and in cells during the process of spreading on the substratum. use of antibodies to myosin, tropomyosin, alpha-actinin and filamin emphasizes that at early times during the spreading process these proteins are abundantly present underneath the upper plasma membrane, suggesting that the cortical layer present underneath this membrane may be contractile. In addition the results emphasize that even in well spread cells microfilament bundles are expressed both above and below the nucleus, in agreement with the assumption that microfilaments may form a supporting layer underneath the plasma membrane.     

Calcium-dependent exocytosis in an in vitro secretory granule plasma membrane preparation from sea urchin eggs and the effects of some inhibitors of cytoskeletal function

Egg cortical granules remain attached to the egg plasma membrane when the egg is ruptured. We present evidence that demonstrates that, when the cytoplasmic face of the egg plasma membrane is exposed to micromolar calcium concentrations, an exocytosis of the cortical granules occurs which corresponds to the cortical granule exocytosis seen when the egg is fertilized. The calcium sensitivity of the preparation is decreased by an increase in magnesium concentration and increased by a decrease in magnesium concentration. Exocytosis is inhibited by trifluoperazine (half inhibition at 6 microM), a drug that inhibits the action of the calcium-dependent regulatory protein calmodulin. Colchicine, vinblastine, nocodazole, cytochalasin B, phalloidin, N-ethylmaleimide-modified myosin subfragment 1, and antibody to actin are without effect on this in vitro exocytosis at concentrations that far exceed those required to disrupt microtubules and microfilaments. Conditions are such that penetration to the exocytotic site is optimal. It is unlikely, therefore, that either actin or tubulin participate intimately in exocytosis. Our data also exclude on quantitative grounds several other mechanisms postulated to account for the fusion of the secretory granule with the plasma membrane.     

Anchorage of secretion-competent dense granules on the plasma membrane of bovine platelets in the absence of secretory stimulation

The ultrastructural changes in electropermeabilized bovine platelets that accompany the Ca2(+)-induced secretion of serotonin were investigated in ultra-thin sections of chemically fixed cells. Such preparations permitted us to study both the localization of and the structures associated with serotonin-containing dense granules. Localization of dense granules within cells was examined by measuring the shortest distances between the granular membranes and the plasma membrane. About 40% of total granules were located close to the plasma membrane at an average distance of 10.8 +/- 1.6 nm. 71% of the total number of granules were localized at a similar average distance of 12.5 +/- 2.7 nm in intact platelets. The percentage of granules apposed to the plasma membrane corresponded closely to the percentage of total serotonin that was maximally secreted after stimulation of the permeabilized (38 +/- 4.9%) and the intact platelets (72 +/- 3.6%). Furthermore, the percentage of granules anchored to the membrane, but not of those in other regions of permeabilized cells, decreased markedly when cells were stimulated for 30 s by extracellularly added Ca2+. The decrease in the numbers of granules in the vicinity of the plasma membrane corresponded to approximately 22% of the total number of dense granules that were used for measurements of the distances between the two membranes and corresponded roughly to the overall decrease (15%) in the average number of the granules per cell. Most dense granules were found to be associated with meshwork structures of microfilaments. Upon secretory stimulation, nonfilamentous, amorphous structures found between the plasma membrane and the apposed granules formed a bridge-like structure that connected both membranes without any obvious accompanying changes in the microfilament structures. These results suggest that the dense granules that are susceptible to secretory stimulation are anchored to the plasma membrane before stimulation, and that the formation of the bridge-like structure may participate in the Ca2(+)-regulated exocytosis.     

Docking is not a prerequisite but a temporal constraint for fusion of secretory granules

We examined secretory granule dynamics using total internal reflection fluorescence microscopy in normal pancreatic β cells and their mutants devoid of Rab27a and/or its effector, granuphilin, which play critical roles in the docking and recruitment of insulin granules to the plasma membrane. In the early phase of glucose stimulation in wild-type cells, we observed marked fusion of granules recruited from a relatively distant area, in parallel with that from granules located underneath the plasma membrane. Furthermore, despite a lack of granules directly attached to the plasma membrane, both spontaneous and evoked fusion was increased in granuphilin-null cells. In addition to these granuphilin-null phenotypes, Rab27a/granuphilin doubly deficient cells showed the decreases in granules located next to the docked area and in fusion from granules near the plasma membrane in the early phase of glucose-stimulated secretion, similar to Rab27a-mutated cells. Thus, the two proteins play nonoverlapping roles in insulin exocytosis: granuphilin acts on the granules underneath the plasma membrane, whereas Rab27a acts on those in a more distal area. These findings demonstrate that, in contrast to our conventional understanding, stable attachment of secretory granules to the plasma membrane is not prerequisite but temporally inhibitory for both spontaneous and evoked fusion.     

A role for myosin 1e in cortical granule exocytosis in Xenopus oocytes

Xenopus oocytes undergo dynamic structural changes during maturation and fertilization. Among these, cortical granule exocytosis and compensatory endocytosis provide effective models to study membrane trafficking. This study documents an important role for myosin 1e in cortical granule exocytosis. Myosin 1e is expressed at the earliest stage that cortical granule exocytosis can be detected in oocytes. Prior to exocytosis, myosin 1e relocates to the surface of cortical granules. Overexpression of myosin 1e augments the kinetics of cortical granule exocytosis, whereas tail-derived fragments of myosin 1e inhibit this secretory event (but not constitutive exocytosis). Finally, intracellular injection of myosin 1e antibody inhibits cortical granule exocytosis. Further experiments identified cysteine string proteins as interacting partners for myosin 1e. As constituents of the membrane of cortical granules, cysteine string proteins are also essential for cortical granule exocytosis. Future investigation of the link between myosin 1e and cysteine string proteins should help to clarify basic mechanisms of regulated exocytosis.     

Control of local actin assembly by membrane fusion-dependent compartment mixing

Local actin assembly is associated with sites of exocytosis in processes ranging from phagocytosis to compensatory endocytosis. Here, we examine whether the trigger for actin-coat assembly around exocytosing Xenopus egg cortical granules is 'compartment mixing'--the union of the contents of the plasma membrane with that of the secretory granule membrane. Consistent with this model, compartment mixing occurs on cortical granule-plasma membrane fusion and is required for actin assembly. Compartment mixing triggers actin assembly, at least in part, through diacylglycerol (DAG), which incorporates into the cortical granule membranes from the plasma membrane after cortical granule-plasma membrane fusion. DAG, in turn, directs long-term recruitment of protein kinase Cbeta (PKCbeta) to exocytosing cortical granules, where it is required for activation of Cdc42 localized on the cortical granules. The results demonstrate that mixing of two membrane compartments can direct local actin assembly and indicate that this process is harnessed during Xenopus egg cortical granule exocytosis to drive compensatory endocytosis.     

Widespread association of annexin II with intracellular membrane systems and involvement of annexin II in granule--granule fusion in addition to granule--plasma membrane fusion in anterior pituitary cells

The subcellular localization in anterior pituitary secretory cells of annexin II, one of the Ca2+-dependent phospholipid-binding proteins, was examined by immunohistochemistry and immunoelectron microscopy. Annexin II was associated with the plasma membrane, the membranes of secretory granules and cytoplasmic organelles, such as rough endoplasmic reticulum, mitochondria and vesicles, and with the nuclear envelope. Annexin II was frequently detected at the contact sites of secretory granules with other granules and with the plasma membrane. The anterior pituitary and adrenal medulla were treated with Clostridium perfringens enterotoxin, which induces Ca2+ influx, and examined under an electron microscope. The anterior pituitary cells showed multigranular exocytosis, i.e. multiple fusions of secretory granules with each other and with the plasma membrane, but adrenal chromaffin cells, which lack annexin II on the granule membranes, never showed granule--granule fusion and only single granule exocytosis. From these results, we conclude that, in anterior pituitary secretory cells, annexin II is involved in granule--granule fusion in addition to granule--plasma membrane fusion. © 1998 Chapman & Hall     

Cortical granule exocytosis in hamster eggs requires microfilaments

Earlier work has demonstrated that hamster eggs that do not release a second polar body after fertilization in vitro lack a block to polyspermy (Stewart-Savage and Bavister, 1987: Gamete Res 18:333–338). Since polar body release requires microfilaments, the involvement of microfilaments in cortical granule exocytosis was examined. When hamster eggs were treated with cytochalsin B (CB) for 1 hr and then coincubated with sperm for 90 min, there was a dose-dependent increase in both the percentage of eggs with more than one sperm penetrating the zona pellucida and the mean number of sperm that penetrated the zona, with a maximum effect at 20 μg CB/ml (100% polypenetration, 3.0 ± 0.3 sperm/egg). Cytochalasin-treated eggs retained 85% of their cortical granules 55 min after insemination, as compared to unfertilized eggs. Longer time periods did not result in any further reduction. As seen with the scanning confocal microscope, an extensive microfilament network was present in the cortex of untreated eggs, with the cortical granules located within this cortical network. The cortical microfilament network was highly reduced in CB-treated eggs. When viewed with the electron microscope, the same number of cortical granules were located next to the plasma membrane in both cytochalasin-treated and untreated, unfertilized eggs. These data indicate that intact microfilaments are required for normal cortical granule exocytosis in the hamster egg, but the role of the microfilaments in exocytosis is unresolved. Mol. Reprod. Dev. 47:334–340, 1997. © 1997 Wiley-Liss, Inc.     

Cytochemical studies on the localization and functional properties of calcium in anterior pituitary cells

The localization of calcium and its functional properties in anterior pituitary cells were studied using a potassium pyroantimonate technique. In all kinds of secretory cells, the precipitates of the calcium-pyroantimonate complex were distributed on the limiting membrane of the secretory granule. They were present also in the cytoplasmic matrix, the mitochondrial matrix, small smooth vesicles, coated vesicles, and in the nuclear euchromatin area. The precipitates were usually seen at the contact region between the limiting membranes of two adjacent secretory granules, or between the granule limiting membrane and the plasma membrane. When the tissues were incubated in the medium containing A23187 (10 microM) for 5 min, the deposits on the granule limiting membrane were increased in number and those on the mitochondrial matrix were decreased; the reaction products almost disappeared on the limiting membranes of the secretory granules after membrane fusion following single or multigranular exocytosis induced by A23187-treatment. In addition, small vesicles in the capillary endothelium contained reaction precipitates. Based on these results we propose a hypothetical model for the relationship between the localization of calcium and secretory activity.     

Secretory granule formation

A deeper understanding of the regulated exocytic pathway, and for that matter the constitutive exocytic pathway, will depend on our ability to characterize the proteins in the vesicle membranes. Characterizing the protein composition of secretory granule membrane has proven to be a formidable task, and as far as I know, the work done to date has not told us a great deal about the mechanisms involved in sorting the contents of regulated secretory granules, or bringing about constitutive or regulated fusion with the plasma membrane. Without knowing a great deal more about the membranes, there seems to be little prospect of real further progress in understanding the key properties of the regulated exocytic pathway.     

Monoclonal antibody to a common antigen of secretory granule membranes: intracellular localization and recycling of the antigen after secretion.

Monoclonal antibody (MAb) 170-5 was generated to the secretory granule membrane of rat parotid acinar cells. The MAb recognized integral membrane glycoproteins (SG 170 antigen) localized on the luminal side of the secretory granules with N-linked carbohydrates, molecular weights 92, 84, 76, 69, and 65 KD. Immunohistochemical studies indicated that the SG 170 antigen was found in the secretory granules of both exocrine and endocrine cells and in the lysosomes of various cells in the rat. Immunoelectron microscopy with immunogold revealed that the antigen was present on the membrane of the secretory granules, lysosomes, the Golgi vesicles, and condensing vacuoles in pancreatic and parotid acinar cells and in AR42J rat pancreatic tumor cells; the Golgi stacks exhibited no immunoreaction. The common localization of the antigen in the secretory granule membranes indicated that this antigen may play an essential role in regulated secretion. Employing HRP-labeled MAb 170-5, we followed the retrieval of the antigen after exocytosis in AR42J cells. The MAb was internalized specifically with antigen-mediated endocytosis. It was transported to endosomes, subsequently to the trans-Golgi network, and then packaged into secretory granules. However, the Golgi stacks revealed no uptake of the labeled antibody.     

Bovine chromaffin granule membranes undergo Ca(2+)-regulated exocytosis in frog oocytes

We have devised a new method that permits the investigation of exogenous secretory vesicle function using frog oocytes and bovine chromaffin granules, the secretory vesicles from adrenal chromaffin cells. Highly purified chromaffin granule membranes were injected into Xenopus laevis oocytes. Exocytosis was detected by the appearance of dopamine-beta-hydroxylase of the chromaffin granule membrane in the oocyte plasma membrane. The appearance of dopamine-beta-hydroxylase on the oocyte surface was strongly Ca(2+)-dependent and was stimulated by coinjection of the chromaffin granule membranes with InsP3 or Ca2+/EGTA buffer (18 microM free Ca2+) or by incubation of the injected oocytes in medium containing the Ca2+ ionophore ionomycin. Similar experiments were performed with a subcellular fraction from cultured chromaffin cells enriched with [3H]norepinephrine-containing chromaffin granules. Because the release of [3H]norepinephrine was strongly correlated with the appearance of dopamine-beta-hydroxylase on the oocyte surface, it is likely that intact chromaffin granules and chromaffin granule membranes undergo exocytosis in the oocyte. Thus, the secretory vesicle membrane without normal vesicle contents is competent to undergo the sequence of events leading to exocytosis. Furthermore, the interchangeability of mammalian and amphibian components suggests substantial biochemical conservation of the regulated exocytotic pathway during the evolutionary progression from amphibians to mammals.     

Localization of 5′-nucleotidase activity in the parotid acinar cells of a rat treated with isoproterenol

Summary Cytochemical localization of 5- nucleotidase (AMPase) has been investigated in the parotid acinar cells of rats at various stages of exocytic secretion induced by an administration of isoproterenol (IPR).In the resting stage, the acinar cells show AMPase activity located on the baso-lateral and luminal plasmalemma, and in the earliest secretory stage the luminal plasma membranes are devoid of the enzymatic activity. However, these particular regions exhibit AMPase activity during the advanced stages of secretion, and the AMPase positive membranes become absorbed into the cytoplasm by endocytic activity. The absorbed membrane components then seem to be degraded by the action of lysosomes.The intracellular fate of the endocytic vacuoles has been examined by the aid of ferritin particles introduced retrogradely through ductal lumina. Ferritin containing vacuoles are distributed in the cytoplasm, and these droplets change into secondary lysosomes. No tracer particles are recognized in the internal space of the Golgi lamella and its associated vesicles.The results suggested that in the exocytic secretion of parotid acinar cells, AMPase originating from plasma membrane intermingles with the membranes derived from secretion granules, and is translocated into cytoplasm by an endocytic mechanism. The internalized membrane components are, at least partly, degraded by lysosome action.     

Mechanisms of Granule Membrane Recapture following Exocytosis in Intact Mast Cells

In secretory cells, several exocytosis-coupled forms of endocytosis have been proposed including clathrin-mediated endocytosis, kiss-and-run endocytosis, cavicapture, and bulk endocytosis. These forms of endocytosis can be induced under different conditions, but their detailed molecular mechanisms and functions are largely unknown. We studied exocytosis and endocytosis in mast cells with both perforated-patch and whole-cell configurations of the patch clamp technique using cell capacitance measurements in combination with amperometric serotonin detection. We found that intact mast cells exhibit an early endocytosis that follows exocytosis induced by compound 48/80. Direct observation of individual exocytic and endocytic events showed a higher percentage of capacitance flickers (27.3%) and off-steps (11.4%) in intact mast cells than in dialyzed cells (5.4% and 2.9%, respectively). Moreover, we observed a type of endocytosis of large pieces of membrane that were likely formed by cumulative fusion of several secretory granules with the cell membrane. We also identified “large-capacitance flickers” that occur after large endocytosis events. Pore conductance analysis indicated that these transient events may represent “compound cavicapture,” most likely due to the flickering of a dilated fusion pore. Using fluorescence imaging of individual exocytic and endocytic events we observed that granules can fuse to granules already fused with the plasma membrane, and then the membranes and dense cores of fused granules are internalized. Altogether, our results suggest that stimulated exocytosis in intact mast cells is followed by several forms of compensatory endocytosis, including kiss-and-run endocytosis and a mechanism for efficient retrieval of the compound membrane of several secretory granules through a single membrane fission event.     

Live pancreatic acinar imaging of exocytosis using syncollin-pHluorin

In this report, a novel live acinar exocytosis imaging technique is described. An adenovirus was engineered, encoding for an endogenous zymogen granule (ZG) protein (syncollin) fused to pHluorin, a pH-dependent green fluorescent protein (GFP). Short-term culture of mouse acini infected with this virus permits exogenous adenoviral protein expression while retaining acinar secretory competence and cell polarity. The syncollin-pHluorin fusion protein was shown to be correctly localized to ZGs, and the pH-dependent fluorescence of pHluorin was retained. Coupled with the use of a spinning disk confocal microscope, the syncollin-pHluorin fusion protein exploits the ZG luminal pH changes that occur during exocytosis to visualize exocytic events of live acinar cells in real-time with high spatial resolution in three dimensions. Apical and basolateral exocytic events were observed on stimulation of acinar cells with maximal and supramaximal cholecystokinin concentrations, respectively. Sequential exocytic events were also observed. Coupled with the use of transgenic mice and/or adenovirus-mediated protein expression, this syncollin-pHluorin imaging method offers a superior approach to studying pancreatic acinar exocytosis. This assay can also be applied to acinar disease models to elucidate the mechanisms implicated in pancreatitis.     

Evidence that the ability to respond to a calcium stimulus in exocytosis is determined by the secretory granule membrane: Comparison of exocytosis of injected bovine chromaffin granule membranes and endogenous cortical granules inXenopus laevis oocytes

Summary 1. To understand better the mechanisms which govern the sensitivity of secretory vesicles to a calcium stimulus, we compared the abilities of injected chromaffin granule membranes and of endogenous cortical granules to undergo exocytosis inXenopus laevis oocytes and eggs in response to cytosolic Ca²⁺. Exocytosis of chromaffin granule membranes was detected by the appearance of dopamine--hydroxylase of the chromaffin granule membrane in the oocyte or egg plasma membrane. Cortical granule exocytosis was detected by release of cortical granule lectin, a soluble constituent of cortical granules, from individual cells.2. Injected chromaffin granule membranes undergo exocytosis equally well in frog oocytes and eggs in response to a rise in cytosolic Ca²⁺ induced by incubation with ionomycin.3. Elevated Ca²⁺ triggered cortical granule exocytosis in eggs but not in oocytes.4. Injected chromaffin granule membranes do not contribute factors to the oocyte that allow calcium-dependent exocytosis of the endogenous cortical granules.5. Protein kinase C activation by phorbol esters stimulates cortical granule exocytosis in bothXenopus laevis oocytes andX. laevis eggs (Bement, W. M., and Capco, D. G.,J. Cell Biol. 108, 885–892, 1989). Activation of protein kinase C by phorbol ester also stimulated chromaffin granule membrane exocytosis in oocytes, indicating that although cortical granules and chromaffin granule membranes differ in calcium responsiveness, PKC activation is an effective secretory stimulus for both.6. These results suggest that structural or biochemical characteristics of the chromaffin granule membrane result in its ability to respond to a Ca²⁺ stimulus. In the oocytes, cortical granule components necessary for Ca²⁺-dependent exocytosis may be missing, nonfunctional, or unable to couple to the Ca²⁺ stimulus and downstream events.     

Control of Granule Mobility and Exocytosis by Ca2+-Dependent Formation of F-Actin in Pancreatic Duct Epithelial Cells

Elevation of intracellular Ca²⁺ concentration ([Ca²⁺]_i) triggers exocytosis of secretory granules in pancreatic duct epithelia. In this study, we find that the signal also controls granule movement. Motions of fluorescently labeled granules stopped abruptly after a [Ca²⁺]_i increase, kinetically coincident with formation of filamentous actin (F-actin) in the whole cytoplasm. At high resolution, the new F-actin meshwork was so dense that cellular structures of granule size appeared physically trapped in it. Depolymerization of F-actin with latrunculin B blocked both the F-actin formation and the arrest of granules. Interestingly, when monitored with total internal reflection fluorescence microscopy, the immobilized granules still moved slowly and concertedly toward the plasma membrane. This group translocation was abolished by blockers of myosin. Exocytosis measured by microamperometry suggested that formation of a dense F-actin meshwork inhibited exocytosis at small Ca²⁺ rises <1 μ m . Larger [Ca²⁺]_i rises increased exocytosis because of the co-ordinate translocation of granules and fusion to the membrane. We propose that the Ca²⁺-dependent freezing of granules filters out weak inputs but allows exocytosis under stronger inputs by controlling granule movements.