Secretory granules are recaptured largely intact after stimulated exocytosis in cultured endocrine cells

Classical cell biology teaches that exocytosis causes the membrane of exocytic vesicles to disperse into the cell surface and that a cell must later retrieve by molecular sorting whatever membrane components it wishes to keep inside. We have tested whether this view applies to secretory granules in intact PC-12 cells. Three granule proteins were labeled with fluorescent proteins in different colors, and two-color evanescent-field microscopy was used to view single granules during and after exocytosis. Whereas neuro-peptide Y was lost from granules in seconds, tissue plasminogen activator (tPA) and the membrane protein phogrin remained at the granule site for over 1 min, thus providing markers for postexocytic granules. When tPA was imaged simultaneously with cyan fluorescent protein (CFP) as a cytosolic marker, the volume occupied by the granule appeared as a dark spot where it excluded CFP. The spot remained even after tPA reported exocytosis, indicating that granules failed to flatten into the cell surface. Phogrin was labeled with GFP at its luminal end and used to sense the pH in granules. When exocytosis caused the acidic granule interior to neutralize, GFP-phogrin at first brightened and later dimmed again as the interior separated from the extracellular space and reacidified. Reacidification and dimming could be reversed by application of NH(4)Cl. We conclude that most granules reseal in <10 s after releasing cargo, and that these empty or partially empty granules are recaptured otherwise intact.     

FM dyes enter via a store-operated calcium channel and modify calcium signaling of cultured astrocytes

The amphiphilic fluorescent styryl pyridinium dyes FM1-43 and FM4-64 are used to probe activity-dependent synaptic vesicle cycling in neurons. Cultured astrocytes can internalize FM1-43 and FM4-64 inside vesicles but their uptake is insensitive to the elevation of cytosolic calcium (Ca²⁺) concentration and the underlying mechanism remains unclear. Here we used total internal reflection fluorescence microscopy and pharmacological tools to study the mechanisms of FM4-64 uptake into cultured astrocytes from mouse neocortex. Our data show that: (i) endocytosis is not a major route for FM4-64 uptake into astrocytes; (ii) FM4-64 enters astrocytes through an aqueous pore and strongly affects Ca²⁺ homeostasis; (iii) partitioning of FM4-64 into the outer leaflet of the plasma membrane results in a facilitation of store-operated Ca²⁺ entry (SOCE) channel gating; (iv) FM4-64 permeates and competes with Ca²⁺ for entry through a SOCE channel; (v) intracellular FM4-64 mobilizes Ca²⁺ from the endoplasmic reticulum stores, conveying a positive feedback to activate SOCE and to sustain dye uptake into astrocytes. Our study demonstrates that FM dyes are not markers of cycling vesicles in astrocytes and calls for a careful interpretation of FM fluorescence.     

Imaging direct,dynamin-dependent recapture of fusing secretory granules on plasma membrane lawns from PC12 cells

During exocytosis, secretory granules fuse with the plasma membrane and discharge their content into the extracellular space. The exocytosed membrane is then reinternalized in a coordinated fashion. A role of clathrin-coated vesicles in this process is well established, whereas the involvement of a direct retrieval mechanism (often called kiss and run) is still debated. Here we report that a significant population of docked secretory granules in the neuroendocrine cell line PC12 fuses with the plasma membrane, takes up fluid-phase markers, and is retrieved at the same position. Fusion allows for complete discharge of small molecules, whereas GFP-labeled neuropeptide Y (molecular mass approximately equal 35 kDa) is only partially released. Retrieved granules were preferentially associated with dynamin. Furthermore, recapture is inhibited by guanosine 5'-[gamma-thio]triphosphate and peptides known to block dynamin function. We conclude that secretory granules can be recaptured immediately after formation of an exocytotic opening by an endocytic reaction that is spatially and temporally coupled to soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-dependent fusion, but is not a reversal of the fusion reaction.     

Alcohol-induced protein kinase Calpha phosphorylation of Munc18c in carbachol-stimulated acini causes basolateral exocytosis

BACKGROUND & AIMS: Acute or chronic alcohol treatment does little to the exocrine pancreas but predisposes the pancreas to postprandial cholinergic stimulation that triggers cellular events leading to pancreatitis. This alcohol-induced susceptibility mechanism of pancreatitis is unknown. METHODS: We employed alcohol-treated dispersed rat pancreatic acini and alcohol diet-fed rats to examine the effects of submaximal carbachol-induced changes in exocytosis (FM1-43 epifluorescence imaging and electron microscopy), Munc18c cellular translocation (confocal microscopy and subcellular fractionation), and protein kinase C (PKC) alpha-induced phosphorylation in relation to pancreatitis. RESULTS: Acute low-dose alcohol (20 mmol/L) in vitro exposure or chronic alcohol diet reduces postprandial cholinergic-stimulated amylase secretion from rat pancreatic acinar cells by blocking apical exocytosis and redirecting exocytosis to less efficient basolateral plasma membrane sites. This ectopic exocytosis is mediated by PKCalpha-induced phosphorylation of Munc18c, causing Munc18c displacement from the basolateral plasma membrane into the cytosol in which it undergoes proteolytic degradation; these processes can be blocked by PKCalpha inhibition. CONCLUSIONS: We conclude that sequential low-dose alcohol and postprandial cholinergic stimulation can induce PKCalpha-mediated Munc18c plasma membrane displacement. This relieves cognate SNARE proteins on zymogen granules and basolateral membrane to complex and consummate pathologic ectopic exocytosis at the basolateral surface. This change in vesicle trafficking may be related to the pathogenesis of pancreatitis.     

Ultrastructural observations on the maturation and secretion of granules in atrial myocardium.

Specific granules in the atrial myocardium of the rat, monkey and baboon are formed in the Golgi vesicles and saccules of these cells. During maturation of the granules, the membranes of coated dense vesicles fuse with the granule limiting membrane and enzyme proteins such as adenosine triphosphatase are transferred from these vesicles to the granules. The Golgi-derived membranes of mature atrial granules appear fragmented and granular core material is apparently released into the cytoplasm of atrial cells. There is no structural evidence for secretion of granular content by exocytosis.     

Isolation and characterization of gelatinase granules from human neutrophils

We recently confirmed the existence of gelatinase granules as a subpopulation of peroxidase-negative granules by double-labeling immunogold electron microscopy on intact cells and by subcellular fractionation. Further characterization of gelatinase granules has been hampered by poor separation of specific and gelatinase granules on both two-layer Percoll gradients and sucrose gradients. We have developed a three-layer Percoll density gradient that allows separation of the different granules and vesicles from human neutrophils; in particular, it allows separation of specific and gelatinase granules. This allows us to characterize these two granule populations with regard to their content of membrane proteins, which become incorporated into the plasma membrane during exocytosis. We found that gelatinase granules, defined as peroxidase-negative granules containing gelatinase but lacking lactoferrin, contain 50% of total cell gelatinase, with the remaining residing in specific granules. Furthermore, we found that 20% to 25% of both the adhesion protein Mac-1 and the NADPH-oxidase component cytochrome b558 is localized in gelatinase granules. Although no qualitative difference was observed between specific granules and gelatinase granules with respect to cytochrome b558 and Mac-1, stimulation of the neutrophil with FMLP resulted in a selective mobilization of the least dense peroxidase-negative granules, ie, gelatinase granules, which, in concert with secretory vesicles, furnish the plasma membrane with Mac-1 and cytochrome b558. This shows that gelatinase granules are functionally important relative to specific granules in mediating early inflammatory responses.     

Transmembranous incorporation of photoelectrically active bacteriorhodopsin in planar lipid bilayers.

Various methods to incorporate bacteriorhodopsin in black lipid membranes are reported. Both purple membrane patches and monomeric bacteriorhodopsin were used as starting material. The incorporation of bacteriorhodopsin into planar lipid bilayers was achieved by the following methods. (i) Purple membrane patches were transferred from water to solutions of lipids in n-alkanes. Black membranes were formed from such organic suspensions. (ii) Lipid layers containing solvent and purple membranes were spread on an air/water interface. These layers were used to form planar bilayers. (iii) Vesicles containing purple membranes or monomeric bacteriorhodopsin were spread on an air/water interface and, from the resulting layer, bilayers were formed. On illumination, steady-state photocurrents were observed in all three cases, indicating that these methods lead to functional transmembranous integration of the protein in the planar black lipid membrane. The influence of an applied electric field on the pumping process was studied on membranes formed by using method i. At approximately 200 mV, the photocurrent tends to zero. Furthermore, it was possible to make planar lipid bilayers photoelectrically active by adding vesicles containing monomeric bacteriorhodopsin to the bathing solution. Because, in this case, only transient photocurrents were observed, it can be concluded that the vesicles are attached to but not fused with the black lipid membrane.     

Reconstitution of purified acetylcholine receptors with functional ion channels in planar lipid bilayers.

Acetylcholine receptor, solubilized and purified from Torpedo californica electric organ under conditions that preserve the activity of its ion channel, was reconstituted into vesicles of soybean lipid by the cholate-dialysis technique. The reconstituted vesicles were then spread into monolayers at an air-water interface and planar bilayers were subsequently formed by apposition of two monolayers. Addition of carbamoylcholine caused an increase in membrane conductance that was transient and relaxed spontaneously to the base level (i.e., became desensitized). The response to carbamoylcholine was dose dependent and competitively inhibited by curare. Fluctuations of membrane conductance corresponding to the opening and closing of receptor channels were observed. Fluctuation analysis indicated a single-channel conductance of 16 +/- 3 pS (in 0.1 M NaCl) with a mean channel open time estimated to be 35 +/- 5 ms. Thus, purified acetylcholine receptor reconstituted into lipid bilayers exhibited the pharmacological specificity, activation, and desensitization properties expected of this receptor in native membranes.     

Syntaxin clusters assemble reversibly at sites of secretory granules in live cells

Syntaxin resides in the plasma membrane, where it helps to catalyze membrane fusion during exocytosis. The protein also forms clusters in cell-free and granule-free plasma-membrane sheets. We imaged the interaction between syntaxin and single secretory granules by two-color total internal reflection microscopy in PC12 cells. Syntaxin-GFP assembled in clusters at sites where single granules had docked at the plasma membrane. Clusters were intermittently present at granule sites, as syntaxin molecules assembled and disassembled in a coordinated fashion. Recruitment to granules required the N-terminal domain of syntaxin, but not the entry of syntaxin into SNARE complexes. Clusters facilitated exocytosis and disassembled once exocytosis was complete. Syntaxin cluster formation defines an intermediate step in exocytosis.     

Defective lysosomal exocytosis and plasma membrane repair in Chediak-Higashi/beige cells

Plasma membrane resealing is a Ca(2+)-dependent process that involves the exocytosis of intracellular vesicles next to the wound site. Recent studies revealed that conventional lysosomes behave as Ca(2+)-regulated secretory compartments and play a central role in membrane resealing. These findings raised the possibility that the complex pathology of lysosomal diseases might also include defects in plasma membrane repair. Here, we investigated the capacity for lysosomal exocytosis and membrane resealing of fibroblasts derived from Chediak-Higashi syndrome (CHS) patients, or from beige-J mice. By using a sensitive electroporation/fluorescence-activated cell sorter-based assay, we show that lysosomal exocytosis triggered by membrane wounding is impaired in both human Chediak-Higashi and mouse beige-J fibroblasts. Lysosomal exocytosis increased when the normal size of lysosomes was restored in beige-J cells by expression of the CHS/Beige protein. A similar effect was seen when the lysosomal enlargement in beige-J cells was reversed by treatment with E64d. In addition, the survival of Chediak-Higashi and beige-J fibroblasts after wounding was reduced, indicating that impaired lysosomal exocytosis inhibits membrane resealing in these mutant cells. Thus, the severe symptoms exhibited by CHS patients may also include defects in the ability of cells to repair plasma membrane lesions.     

Simultaneous electrical and optical measurements show that membrane fusion precedes secretory granule swelling during exocytosis of beige mouse mast cells.

Mast cells show dramatic morphological changes when undergoing exocytosis. We have investigated whether the first of those morphological changes, swelling of the secretory granule, precedes--and therefore possibly initiates--secretion or whether it occurs after fusion of the granule and plasma membranes. We used cell membrane capacitance to detect the moment when granule and plasma membrane become continuous. We measured large capacitance increases, often preceded by transients in capacitance. The rise-times of the capacitance increases were half-maximal at 2-59 msec. We observed cells with high-resolution video microscopy while these measurements were done. The capacitance increase always preceded the granular swelling that leads to exocytosis. To rule out the possibility that fusion was induced by a mechanical stress imparted by the internal pressure of a taut granule, we performed control experiments using cells in which vesicles were shrunken with hyperosmotic solutions. With these flaccid granules, again, the capacitance rise always preceded the swelling of the granules. We conclude that swelling cannot be the driving force for membrane fusion in this system.     

Taurocholate transport by liver plasma membrane vesicles is not altered in cirrhotic rats

Recently, we have shown that taurocholate transport is impaired in hepatocytes isolated from CCl4 cirrhotic rats. Na+,K+-ATPase activity depends on the lipid composition of the surrounding membrane. Therefore, we performed this study in order to detect differences in plasma membrane composition and membrane functions between livers of CCl4 cirrhotic (n = 17) and of control rats (n = 15). After biochemical characterization of the animals we isolated basolateral and canalicular membrane vesicles and determined membrane enzyme activities, transport functions and lipid composition. We found no differences in the isolation characteristics of the plasma membranes between the two groups. The lipid composition of the membrane fractions was not altered, except for a lower cholesterol content in the canalicular membranes of the cirrhotic group (200 +/- 15 vs. 246 +/- 18 micrograms/mg protein, P less than 0.05). Taurocholate transport into basolateral membrane vesicles and marker enzyme activities of the membrane fractions were also equal in control and cirrhotic animals. We conclude that the plasma membrane composition and membrane enzyme/transport activities have returned to normal in CCl4 cirrhotic rats 14 days after cessation of exposure to CCl4. Thus, a disturbed transport system is not the cause for the observed decreased taurocholate transport into hepatocytes from cirrhotic rats. Even a cirrhotic liver has a high potential for recovery after acute CCl4 intoxication.     

Effect of sulfhydryl reagents on pancreatic islet secretion granule-plasma membrane interaction

To determine what role, if any, sulfhydryl groups may play in the fusion of islet secretion granules with the plasma membrane that takes place during exocytosis, we have studied the effect of several sulfhydryl-binding reagents, reducing agents, and oxidizing agents on the binding of 125I-labeled inside-out plasma membrane vesicles to isolated secretion granules. Three sulfhydryl-binding reagents, p-hydroxymercuribenzoate, Hg++, and N-ethyl maleimide, stimulated this binding, and the stimulation was greater in the absence of Ca++ than in its presence. In contrast, the three reducing agents used, glutathione, cysteine, and sodium bisulfite, inhibited the binding. Of the oxidizing agents, oxidized glutathione inhibited binding, whereas menadione and o-iodosobenzoate stimulated. The actions of Hg++ and glutathione were found to be on the secretion granules rather than the plasma membrane vesicles. It is concluded that the presence of a preponderance of sulfhydryl groups on the secretion granule membranes tends to limit their interaction with the plasma membrane and that these must be removed or masked for maximum fusion to occur.     

A highly Ca2+-sensitive pool of vesicles is regulated by protein kinase C in adrenal chromaffin cells

We have used flash photolysis of caged Ca2+ and membrane capacitance measurements to probe exocytosis in chromaffin cells at low concentrations of intracellular Ca2+ ([Ca2+]i) (<10 microM). We observed a small pool of granules that is more sensitive to [Ca2+]i than the previously described "readily releasable pool." Upon activation of PKC, this "highly Ca2+-sensitive pool" is enhanced in size to a greater extent than the readily releasable pool but is eliminated upon expression of a C-terminal deletion mutant (Delta9) of synaptosome-associated protein of 25 kDa (SNAP-25). Thus, in chromaffin cells, PKC enhances exocytosis both by increasing the number of readily releasable vesicles and by shifting vesicles to a highly Ca2+-sensitive state, enabling exocytosis at sites relatively distant from Ca2+ channels.     

Essential role of Epac2/Rap1 signaling in regulation of insulin granule dynamics by cAMP

cAMP is well known to regulate exocytosis in various secretory cells, but the precise mechanism of its action remains unknown. Here, we examine the role of cAMP signaling in the exocytotic process of insulin granules in pancreatic beta cells. Although activation of cAMP signaling alone does not cause fusion of the granules to the plasma membrane, it clearly potentiates both the first phase (a prompt, marked, and transient increase) and the second phase (a moderate and sustained increase) of glucose-induced fusion events. Interestingly, all granules responsible for this potentiation are newly recruited and immediately fused to the plasma membrane without docking (restless newcomer). Importantly, cAMP-potentiated fusion events in the first phase of glucose-induced exocytosis are markedly reduced in mice lacking the cAMP-binding protein Epac2 (Epac2(ko/ko)). In addition, the small GTPase Rap1, which is activated by cAMP specifically through Epac2 in pancreatic beta cells, mediates cAMP-induced insulin secretion in a protein kinase A-independent manner. We also have developed a simulation model of insulin granule movement in which potentiation of the first phase is associated with an increase in the insulin granule density near the plasma membrane. Taken together, these data indicate that Epac2/Rap1 signaling is essential in regulation of insulin granule dynamics by cAMP, most likely by controlling granule density near the plasma membrane.     

Quantum dots provide an optical signal specific to full collapse fusion of synaptic vesicles

Synaptic vesicles are responsible for releasing neurotransmitters and are thus essential to brain function. The classical mode of vesicle recycling includes full collapse of the vesicle into the plasma membrane and clathrin-mediated regeneration of a new vesicle. In contrast, a nonclassical mode known as "kiss-and-run" features fusion by a transient fusion pore without complete loss of vesicle identity and offers possible advantages for increasing the throughput of neurotransmission. Studies of vesicular traffic have benefited greatly from fluorescent probes like FM dyes and synaptopHluorin. However, intrinsic properties of these probes limit their ability to provide a simple and precise distinction between classical and nonclassical modes. Here we report a novel optical probe specific to full collapse fusion, capitalizing on the size and superior photo-properties of photoluminescent quantum dots (Qdots). Qdots with exposed carboxyl groups were readily taken up by synaptic vesicles in an activity-, Ca(2+)-, and clathrin-dependent manner. Electron microscopy showed that Qdots were harbored within individual vesicles in a 1:1 ratio. The release of Qdots was activity- and Ca(2+)-dependent, similar to FM dyes. As artificial cargo, approximately 15 nm in diameter, Qdots will not escape vesicles during kiss-and-run but only with full collapse fusion. Strikingly, Qdots unloaded with kinetics substantially slower than destaining of FM dye, indicating that full-collapse fusion contributed only a fraction of all fusion events. As a full-collapse-fusion-responsive reporter, Qdots will likely promote better understanding of vesicle recycling at small CNS nerve terminals.     

Final steps in exocytosis observed in a cell with giant secretory granules.

Secretion by single mast cells was studied in normal and beige mice, a mutant with grossly enlarged secretory vesicles or granules. During degranulation, the membrane capacitance increased in steps, as single secretory vesicles fused with the cell membrane. The average step size was 10 times larger in beige than in normal mice, in agreement with the different granule sizes measured microscopically in the two preparations. Following individual capacitance steps in beige mice, individual granules of the appropriate size were observed to swell rapidly. Capacitance steps are frequently followed by the stepwise loss of a fluorescent dye loaded into the vesicles. Stepwise capacitance increases were occasionally intermittent before they became permanent, indicating the existence of an early, reversible, and incomplete state of vesicle fusion. During such "capacitance flicker," loss of fluorescent dye from vesicles did not occur, suggesting that the earliest aqueous connection between vesicle interior and cell exterior is a narrow channel. Our results support the view that the reversible formation of such a channel, which we term the fusion pore, is an early step in exocytosis.     

The cromolyn binding protein constitutes the Ca2+ channel of basophils opening upon immunological stimulus.

Ca2+ channel opening has been proposed to be induced in the plasma membrane of mast cells and basophils upon crosslinking their Fc epsilon receptors. Here we report direct conductance measurements on planar lipid bilayers containing membrane components of rat basophils (RBL-2H3 line). These studies identify the Ca2+ channel-forming membrane component as the cromolyn binding protein [CBP, in which cromolyn is the anti-asthmatic drug 1,3-bis(2-carboxy-chromon-5-yloxy)-2-hydroxypropane]. Planar membranes were first formed from lipid vesicles containing unfractionated plasma membrane components prepared from RBL-2H3 cells. Conductance of these bilayers was induced by crosslinking IgE bound to the Fc epsilon receptors of this membrane by a specific polyvalent antigen. Channel conductance in the presence of only Ca2+ ions (2 mM) was 2 pS. When only sodium ions were present (150 mM), conductance was 10 pS. Upon addition of Ca2+ (2 mM) to the Na+ ion-containing solution, the conductance decreased from 10 pS to that of the Ca2+ ions--namely, 2pS. Open channel times were in the range of several hundred milliseconds. Conductance amplitudes and time characteristics were independent of the applied voltage. As our earlier studies revealed the essential role of the CBP in Ca2+ conductance of basophil membranes, we formed planar bilayers containing this isolated protein alone. Crosslinking of the CBP by a monoclonal antibody specific to it resulted in the appearance of channel conductances. All characteristics of these channels exhibited great similarity to those observed in planar membranes containing unfractionated RBL-2H3 membrane components. Moreover, in the latter membranes, the monoclonal anti-CBP antibody induced channel conductances that display an even closer similarity to those observed in membranes containing CBP alone. Conductances of both types of planar membranes, irrespective of the mode of activation used, were inhibited by cromolyn. Furthermore, the conductance induced in RBL membranes by polyvalent antigen was inhibited on dissociation of the crosslinked aggregates by a monovalent hapten. The detailed resemblance in channel behavior observed in experiments with the two types of planar bilayers provides compelling evidence that the CBP is the essential and sufficient component forming Ca2+ channels in basophil plasma membranes.     

A coupled proton-transfer and twisting-motion fluorescence probe for lipid bilayers

A new and sensitive molecular probe, 2-(2′-hydroxyphenyl)imidazo[1,2-a]pyridine (HPIP), for monitoring structural changes in lipid bilayers is presented. Migration of HPIP from water into vesicles involves rupture of hydrogen (H) bonds with water and formation of an internal H bond once the probe is inside the vesicle. These structural changes of the dye allow the occurrence of a photoinduced intramolecular proton-transfer reaction and a subsequent twisting/rotational process upon electronic excitation of the probe. The resulting large Stokes-shifted fluorescence band depends on the twisting motion of the zwitterionic phototautomer and is characterized in vesicles of dimyristoyl-phosphatidylcholine and in dipalmitoyl-phosphatidylcholine at the temperature range of interest and in the presence of cholesterol. Because the fluorescence of aqueous HPIP does not interfere in the emission of the probe within the vesicles, HPIP proton-transfer/twisting motion fluorescence directly allows us to monitor and quantify structural changes within bilayers. The static and dynamic fluorescence parameters are sensitive enough to such changes to suggest this photostable dye as a potential molecular probe of the physical properties of lipid bilayers.     

Phosphatidylinositol 4-kinase serves as a metabolic sensor and regulates priming of secretory granules in pancreatic beta cells

Insulin secretion is controlled by the beta cell's metabolic state, and the ability of the secretory granules to undergo exocytosis increases during glucose stimulation in a membrane potential-independent fashion. Here, we demonstrate that exocytosis of insulin-containing secretory granules depends on phosphatidylinositol 4-kinase (PI 4-kinase) activity and that inhibition of this enzyme suppresses glucose-stimulated insulin secretion. Intracellular application of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)] stimulated exocytosis by promoting the priming of secretory granules for release and increasing the number of granules residing in a readily releasable pool. Reducing the cytoplasmic ADP concentration in a way mimicking the effects of glucose stimulation activated PI 4-kinase and increased exocytosis whereas changes of the ATP concentration in the physiological range had little effect. The PI(4,5)P(2)-binding protein Ca(2+)-dependent activator protein for secretion (CAPS) is present in beta cells, and neutralization of the protein abolished both Ca(2+)- and PI(4,5)P(2)-induced exocytosis. We conclude that ADP-induced changes in PI 4-kinase activity, via generation of PI(4,5)P(2), represents a metabolic sensor in the beta cell by virtue of its capacity to regulate the release competence of the secretory granules.