Roles of microfilaments in exocytosis: a new hypothesis

We observed the dynamic changes in the localization of microfilaments during the exocytic secretion of rat parotid and submandibular gland acinar cells, and obtained results which led us to propose a new concept of microfilament function in exocytosis. With the electron microscopy, NBD-Phallacidin (NBD-PL) fluorescence technique and immunohistochemistry for myosin, microfilaments consisting of F-actin and myosin were localized mainly underneath the luminal plasma membrane. Microfilaments were not detectable around the secretory granules which were stored in the cytoplasm, but were clearly observed around them whose membranes were continuous with the luminal plasma membrane. When viewed with NBD-PL and myosin fluorescence, the area of fused granule membranes revealed bright fluorescence in association with the luminal border, so that the luminal membrane undergoing exocytosis appeared like a 'bunch of grapes'. When excess exocytosis was stimulated by isoproterenol (IPR), the number of individual 'grapes' increased dramatically, indicating that the secretory granules are surrounded by microfilaments after the fusion with the luminal membrane. Microfilaments thus continuously undercoat the luminal membrane during exocytosis although the exocytic process involves the dilation and subsequent reduction of the luminal membrane due to the addition and removal of secretory granule membranes. This reduction of the dilated luminal membrane following exocytosis was, however, inhibited when the microfilaments were disrupted by cytochalasin D. Following this treatment, the lumina was expanded extraordinarily and the secretory products remained in the enlarged lumina, showing that the release of secretory products is inhibited when the microfilament function is disturbed. These results indicate that 1) microfilaments are localized mainly underneath the luminal plasma membrane and act as an obstacle to exocytosis when cells are at the resting phase and 2) at the secretory phase microfilaments allow exocytosis by disorganizing their barrier system and then, by encircling the discharged secretory granule membranes, provide forces for the extrusion of secretory products through the action of the acto-myosin contractile system.     

Fluorescence microscopy of viable mast cells stained with different concentrations of acridine orange.

Freshly harvested rat peritoneal mast cells were stained with different concentrations of acridine orange, a metachromatic fluorochrome known to form complexes with chromatin and muscopolysaccharides. Fluorescence metachromasia was observed in cytoplasmic granules in cell populations with intracelluar dye contents as low as 5 X 10(-16) mole per cell, one-half decade lower than required to produce metachromatic staining of the nucleus. Cytoplasmic granules did not stain uniformly throughout the cell; some granules exhibited red fluorescence and others green. As the amount of acridine orange uptake per cell was increased, cytoplasmic fluorescence became uniformly red and nuclear fluorescence gradually changed from green to yellow.     

Identification of P-glycoprotein at the membrane of mast cell secretory granules. An immunofluorescence and protein A-gold electron microscopical investigation

The presence of P-glycoprotein has been investigated in rat peritoneal mast cells by means of immunofluorescence and immunogold electron microscopy, using the specific monoclonal antibody JSB-1. Immunofluorescence studi es showed that the glycoprotein is primarily concentrated in mast cell granules, and little is localized at the plasma membrane. Electron microscope observations revealed a marked accumulation of colloidal gold particles at the granule-coating membranes, whereas decoration of the plasma membrane is much less intense. When mast cells are stimulated to exocytate with compound 48/80, both immunofluorescence and electron microscopy showed concentration of P-glycoprotein reactivity at the plasma membrane level. Indeed, fusion of the granule with the plasma membrane allowed transfer of immunoreactive P-glycoprotein material from the granule-coating membrane to the cell surface membrane. These findings confirmed the presence of P-glycoprotein in mast cells; it is predominantly localized in the granules and is exposed on the cell surface only after exocytosis, suggesting, therefore, a possible physiological role for P-glycoprotein in the secretion of certain mediators. This revised version was published online in November 2006 with corrections to the Cover Date.     

Identification of P-glycoprotein at the membrane of mast cell secretory granules. An immunofluorescence and protein A-gold electron microscopical investigation

The presence of P-glycoprotein has been investigated in rat peritoneal mast cells by means of immunofluorescence and immunogold electron microscopy, using the specific monoclonal antibody JSB-1. Immunofluorescence studi es showed that the glycoprotein is primarily concentrated in mast cell granules, and little is localized at the plasma membrane. Electron microscope observations revealed a marked accumulation of colloidal gold particles at the granule-coating membranes, whereas decoration of the plasma membrane is much less intense. When mast cells are stimulated to exocytate with compound 48/80, both immunofluorescence and electron microscopy showed concentration of P-glycoprotein reactivity at the plasma membrane level. Indeed, fusion of the granule with the plasma membrane allowed transfer of immunoreactive P-glycoprotein material from the granule-coating membrane to the cell surface membrane. These findings confirmed the presence of P-glycoprotein in mast cells; it is predominantly localized in the granules and is exposed on the cell surface only after exocytosis, suggesting, therefore, a possible physiological role for P-glycoprotein in the secretion of certain mediators. This revised version was published online in November 2006 with corrections to the Cover Date.     

A cell-free system for regulated exocytosis in PC12 cells

We have developed a cell-free system for regulated exocytosis in the PC12 neuroendocrine cell line. Secretory vesicles were preloaded with acridine orange in intact cells, and the cells were sonicated to produce flat, carrier-supported plasma membrane patches with attached vesicles. Exocytosis resulted in the release of acridine orange which was visible as a disappearance of labeled vesicles and, under optimal conditions, produced light flashes by fluorescence dequenching. Exocytosis in vitro requires cytosol and Ca(2+) at concentrations in the micromolar range, and is sensitive to Tetanus toxin. Imaging of membrane patches at diffraction- limited resolution revealed that 42% of docked granules were released in a Ca(2+)-dependent manner during 1 min of stimulation. Electron microscopy of membrane patches confirmed the presence of dense-core vesicles. Imaging of membrane patches by atomic force microscopy revealed the presence of numerous particles attached to the membrane patches which decreased in number upon stimulation. Thus, exocytotic membrane fusion of single vesicles can be monitored with high temporal and spatial resolution, while providing access to the site of exocytosis for biochemical and molecular tools.     

Fluorescence microscopy of viable mast cells stained with different concentrations of acridine orange

Summary Freshly harvested rat peritoneal mast cells were stained with different concentrations of acridine orange, a metachromatic fluorochrome known to form complexes with chromatin and mucopolysaccharides. Fluorescence metachromasia was observed in cytoplasmic granules in cell populations with intracellular dye contents as low as 5×10^–16 mole per cell, one-half decade lower than required to produce metachromatic staining of the nucleus. Cytoplasmic granules did not stain uniformly throughout the cell; some granules exhibited red fluorescence and others green. As the amount of acridine orange uptake per cell was increased, cytoplasmic fluorescence became uniformly red and nuclear fluorescence gradually changed from green to yellow.     

Digitonin-Permeabilized Cells Are Exocytosis Competent

Release of norepinephrine from PC12 cells can be stimulated by free Ca2+ in micromolar concentrations after permeabilization with 10 micrograms/ml of digitonin. This release is time and temperature dependent, half-maximal at 0.3 microM Ca2+, and, after washing out of endogenous ATP, half-maximal at about 0.5 mM MgATP when exogenously added. Similar results were obtained with bovine adrenal chromaffin cells using the same protocol. Support for the idea that the mechanism of release from both permeabilized cell types is still exocytosis is demonstrated at the electron microscopic level by immunolabeling chromaffin granule membrane antigens that were introduced into the plasma membrane following stimulation. Electron micrographs furthermore demonstrate that chromaffin granules retain typical dense cores after permeabilization, indicating that leakiness of catecholamines from the granules was not a major factor. Pores, formed by digitonin in the plasma membranes, were utilized to introduce antibodies into such exocytosis-competent cells. Anti-actin and anti-chromaffin granule membrane antibodies show a staining pattern similar to conventionally fixed and stained preparations. Our results demonstrate that pores formed by digitonin do not impair the process of exocytosis although they are big enough to allow macromolecules to pass in both directions. The digitonin-permeabilized cell is therefore an ideal in vitro system with which to study the fusion process between chromaffin granules and the plasma membrane.     

Simultaneous evanescent wave imaging of insulin vesicle membrane and cargo during a single exocytotic event

The classical model of secretory vesicle recycling after exocytosis involves the retrieval of membrane (the omega figure) at a different site. An alternative model involves secretory vesicles transiently fusing with the plasma membrane (the 'kiss and run' mechanism) [1,2]. No continuous observation of the fate of a single secretory vesicle after exocytosis has been made to date. To study the dynamics of fusion immediately following exocytosis of insulin-containing vesicles, enhanced green fluorescent protein (EGFP) fused to the vesicle membrane protein phogrin [3] was delivered to the secretory vesicle membrane of INS-1 beta-cells using an adenoviral vector. The behaviour of the vesicle membrane during single exocytotic events was then examined using evanescent wave microscopy [4-6]. In unstimulated cells, secretory vesicles showed only slow Brownian movement. After a depolarizing pulse, most vesicles showed a small decrease in phogrin-EGFP fluorescence, and some moved laterally over the plasma membrane for approximately 1 microm. In contrast, secretory vesicles loaded with acridine orange all showed a transient (33-100 ms) increase in fluorescence intensity followed by rapid disappearance. Simultaneous observations of phogrin-EGFP and acridine orange indicated that the decrease in EGFP fluorescence occurred at the time of the acridine orange release, and that the lateral movement of EGFP-expressing vesicles occurred after this. Post-exocytotic retrieval of the vesicle membrane in INS-1 cells is thus slow, and can involve the movement of empty vesicles under the plasma membrane ('kiss and glide').     

Arrest of membrane fusion events in mast cells by quick-freezing

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 degrees C displayed single, narrow-necked pores (some as small as 0.05 micrometer in diameter) joining single granules with the plasma membrane. Pores that had become as large as 0.1 micrometer 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.     

Imaging exocytosis of single insulin secretory granules with evanescent wave microscopy: distinct behavior of granule motion in biphasic insulin release.

To study insulin exocytosis by monitoring the single insulin secretory granule motion, evanescent wave microscopy was used to quantitatively analyze the final stage of insulin exocytosis with biphasic release. Green fluorescent protein-tagged insulin transfected in MIN6 beta cells was packed in insulin secretory granules, which appeared to preferentially dock to the plasma membrane. Upon fusion evoked by secretagogues, evanescent wave microscopy revealed that fluorescence of green fluorescent protein-tagged insulin brightened, spread (within 300 ms), and then vanished. Under KCl stimulation, which represents the 1st phase of release, the successive fusion events were seen mostly from previously docked granules for the first minute, followed by the recruitment of new granules to the plasmalemmal docking sites. Stimulation with glucose, in contrast, caused the fusion events from previously docked granules for the first 120 s, thereafter a continuous fusion (2nd phase of release) was observed over 10 min mostly from newly recruited granules that progressively accumulated on the plasma membrane. Thus, our data revealed the distinct behavior of the insulin granule motion during the 1st and 2nd phase of release.     

Membrane recycling after exocytosis: An ultrastructural study of cultured chromaffin cells

When exocytosis of granule contents is induced by nicotine stimulation, glycoprotein III (a chromaffin granule membrane constituent) is exposed on the surface of cultured chromaffin cells, where it may be labeled with an immunocytochemical tracer. The subsequent fate of this glycoprotein after endocytosis was followed at the ultrastructural level using immunogold methods and was analyzed by morphometry. After stimulation exocytosis membranes newly inserted into the plasma membrane labeled with gold particles for glycoprotein III were found to be endocytosed via coated vesicles and finally found in organelles devoid of chromogranin A, the major secretory granule protein. At intervals between 30 min and 24 h after cell stimulation and immunolabeling, most labeled structures were identified by two different morphological approaches as prelysosomes and lysosomes. In contrast with results obtained on freshly isolated chromaffin cells, it is thus concluded that in cultured cells granule membrane recycling into new granules does not occur. It is suggested that the fate of granule membrane endocytosed after cell stimulation may be influenced by the external conditions to which cells are previously exposed.     

Early membrane retrieval following exocytosis in rat mast cells

The fate of the surplus membrane following exocytosis of mast cell granules was studied by the extracellular tracer Ruthenium red (Ru red). Isolated rat peritoneal mast cells were stimulated with 4 micrograms/ml polylysine, washed and maintained in a culture medium for 80 min. Mast cells were observed both with the light microscope after adding Ru red in physiological solution and with the electron microscope after fixation in Ru red-containing fixatives. Whereas all exocytotic cavities were found to be stained with Ru red immediately after stimulation, a gradual lack of staining was observed in the subsequent period. The exits of the cavities were sealed by membrane fusions which resulted in closed vacuoles containing exocytosed granule remnants. These vacuoles often fused with each other to form a few giant vacuoles. The overwhelming majority of the vacuoles were observed to be closed 30 to 80 min after stimulation. In one experiment a quantitative analysis was performed to assess the degree of membrane recapture by sealing of the exocytotic cavities. A considerable portion of the plasma membrane area was retrieved in this way as early as between 15 and 30 min after stimulation. We conclude that the dominant mechanism of membrane retrieval in the early period following exocytosis is the recapture of large membrane areas by sealing of exocytotic cavities.     

Soluble NSF attachment protein receptors (SNAREs) in RBL-2H3 mast cells: functional role of syntaxin 4 in exocytosis and identification of a vesicle-associated membrane protein 8-containing secretory compartment

Mast cells upon stimulation through high affinity IgE receptors massively release inflammatory mediators by the fusion of specialized secretory granules (related to lysosomes) with the plasma membrane. Using the RBL-2H3 rat mast cell line, we investigated whether granule secretion involves components of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) machinery. Several isoforms of each family of SNARE proteins were expressed. Among those, synaptosome-associated protein of 23 kDa (SNAP23) was central in SNARE complex formation. Within the syntaxin family, syntaxin 4 interacted with SNAP23 and all vesicle-associated membrane proteins (VAMPs) examined, except tetanus neurotoxin insensitive VAMP (TI-VAMP). Overexpression of syntaxin 4, but not of syntaxin 2 nor syntaxin 3, caused inhibition of FcepsilonRI-dependent exocytosis. Four VAMP proteins, i.e., VAMP2, cellubrevin, TI-VAMP, and VAMP8, were present on intracellular membrane structures, with VAMP8 residing mainly on mediator-containing secretory granules. We suggest that syntaxin 4, SNAP23, and VAMP8 may be involved in regulation of mast cell exocytosis. Furthermore, these results are the first demonstration that the nonneuronal VAMP8 isoform, originally localized on early endosomes, is present in a regulated secretory compartment.     

Phosphorylation of SNAP-23 regulates exocytosis from mast cells

Regulated exocytosis is a process in which a physiological trigger initiates the translocation, docking, and fusion of secretory granules with the plasma membrane. A class of proteins termed SNAREs (including SNAP-23, syntaxins, and VAMPs) are known regulators of secretory granule/plasma membrane fusion events. We have investigated the molecular mechanisms of regulated exocytosis in mast cells and find that SNAP-23 is phosphorylated when rat basophilic leukemia mast cells are triggered to degranulate. The kinetics of SNAP-23 phosphorylation mirror the kinetics of exocytosis. We have identified amino acid residues Ser(95) and Ser(120) as the major phosphorylation sites in SNAP-23 in rodent mast cells. Quantitative analysis revealed that approximately 10% of SNAP-23 was phosphorylated when mast cell degranulation was induced. These same residues were phosphorylated when mouse platelet degranulation was induced with thrombin, demonstrating that phosphorylation of SNAP-23 Ser(95) and Ser(120) is not restricted to mast cells. Although triggering exocytosis did not alter the absolute amount of SNAP-23 bound to SNAREs, after stimulation essentially all of the SNAP-23 bound to the plasma membrane SNARE syntaxin 4 and the vesicle SNARE VAMP-2 was phosphorylated. Regulated exocytosis studies revealed that overexpression of SNAP-23 phosphorylation mutants inhibited exocytosis from rat basophilic leukemia mast cells, demonstrating that phosphorylation of SNAP-23 on Ser(120) and Ser(95) modulates regulated exocytosis by mast cells.     

Compound exocytosis and cumulative fusion in eosinophils

Focal release of cytotoxic proteins by eosinophils onto the target surface plays an important role in parasite killing. Degranulation was stimulated by intracellular application of calcium and guanosine 5'-3-O-(thio)triphosphate via the recording patch pipette or via streptolysin-O permeabilization. Exocytotic fusion was monitored by capacitance measurements, whereas release of fluorescent weak bases, which accumulate selectively within eosinophil granules, was followed by fluorescence imaging. Several distinct types of granule fusion events were directly observed by simultaneous capacitance and fluorescence measurements. These are fusion of a single granule with the plasma membrane, intracellular granule-granule fusion, fusion of large compounds of pre-fused granules with the plasma membrane (compound exocytosis), and sequential fusion of granules to granules previously fused to the plasma membrane. Extensive granule-granule fusion was also observed by electron microscopy of permeabilized cells. All these fusion mechanisms contribute to focal release. The coexistence of distinct modes of exocytosis suggests that their regulation may modulate effector functions of eosinophils during helminth infection and allergic response.     

Mast cell degranulation requires N-ethylmaleimide-sensitive factor-mediated SNARE disassembly

Mast cells possess specialized granules that, upon stimulation of surface FcR with IgE, fuse with the plasma membrane, thereby releasing inflammatory mediators. A family of membrane fusion proteins called SNAREs, which are present on both the granule and the plasma membrane, plays a role in the fusion of these granules with the plasma membrane of mast cells. In addition to the SNAREs themselves, it is likely that the SNARE accessory protein, N-ethylmaleimide-sensitive factor (NSF), affects the composition and structure of the SNARE complex. NSF is a cytoplasmic ATPase that disassembles the SNARE complexes. To investigate the role of NSF in mast cell degranulation, we developed an assay to measure secretion from transiently transfected RBL (rat basophilic leukemia)-2H3 mast cells (a tumor analog of mucosal mast cells). RBL-2H3 cells were cotransfected with a plasmid encoding a human growth hormone secretion reporter along with either wild-type NSF or an NSF mutant that lacks ATPase activity. Human growth hormone was targeted to and released from secretory granules in RBL-2H3 cells, and coexpression with mutant NSF dramatically inhibited regulated exocytosis from the transfected cells. Biochemical analysis of SNARE complexes in these cells revealed that overexpression of the NSF mutant decreased disassembly and resulted in an accumulation of SNARE complexes. These data reveal a role for NSF in mast cell exocytosis and highlight the importance of SNARE disassembly, or priming, in regulated exocytosis from mast cells.     

Morphological study of rat mast cells stimulated with compound 48/80 at different temperatures

Changes of mast cells stimulated with compound 48/80 were morphologically investigated at different temperatures. Peritoneal mast cells of male rats were stimulated in vitro at 4 or 17° C. At 17° C, mast cells stimulated for 10 s gave decreased fluorescent reactions for phalloidin. At 30 s stimulation, they showed typical exocytosis initiated by fusions of peripherally located secretory granules to the plasma membrane. In contrast, mast cells stimulated at 4° C exhibited neither decrease of phalloidin reactions nor typical excytosis even after 30 s. It was inferred that the fusions were mediated by cytoplasmic elements, probably the actin filaments previously suggested to prevent release of secretory granules. Furthermore, the space between the perigranular membrane and granular contents was enlarged in some mast cells stimulated at 4° C. The morphological changes suggested that equivocal events occurred also in the cytoplasm of these cells. The mast cells showed no typical exocytosis at 4° C.     

Docking and fusion of insulin secretory granules in SUR1 knock out mouse beta-cells observed by total internal reflection fluorescence microscopy

To explore how the sulfonylurea receptor (SUR1) is involved in docking and fusion of insulin granules, dynamic motion of single insulin secretory granules near the plasma membrane was examined in SUR1 knock-out (Sur1KO) beta-cells by total internal reflection fluorescence microscopy. Sur1KO beta-cells exhibited a marked reduction in the number of fusion events from previously docked granules. However, the number of docked granules declined during stimulation as a consequence of the release of docked granules into the cytoplasm vs. fusion with the plasma membrane. Thus, the impaired docking and fusion results in decreased insulin exocytosis from Sur1KO beta-cells.     

Molecular probes for sensing the cholesterol composition of subcellular organelle membranes

Neuroendocrine cells contain two types of secretagogue-regulated acidic compartments: secretory granules (SGs) and synaptic-like microvesicles (SLMVs), which can be identified by acidotropic probes such as acridine orange (AO) and DAMP. We investigated the accumulation of these probes in SGs and SLMVs as a function of glucose levels in the culture media using a pancreatic beta-cell line MIN6. AO was accumulated in the low-glucose condition, but not in the high-glucose condition. The AO accumulation correlated well with the SLMV dynamics by glucose and DAMP was localized in the SGs. Because SG membranes are reportedly high in cholesterol, we prepared liposomes with increasing cholesterol levels. AO is well incorporated into liposomes having a 20 to 40 mol% cholesterol composition, whereas DAMP was so in those having over 40 mol% cholesterol levels. Indeed, when cholesterol was depleted from MIN6 SG membranes, DAMP incorporation decreased, instead AO was incorporated. In PC12 cells, AO incorporation into SGs was significant but DAMP incorporation was limited. Consistently, the cholesterol composition was found 37 to 39 mol% in the SG membrane of PC12 cells. We suggest that cholesterol-sensing probes, AO and DAMP, are useful tools for investigating cholesterol compositions in acidic organelle membranes.     

Low molecular weight GTP-binding proteins in human neutrophil granule membranes

Degranulation of neutrophils involves the differential regulation of the exocytosis of at least two populations of granules. Low molecular weight GTP-binding proteins (LMW-GBPs) have been implicated in the regulation of vesicular traffic in the secretory pathways of several types of cells. In the present study we identify distinct subsets of LMW-GBPs associated with the membranes of neutrophil-specific and azurophilic granules. Ninety-four percent of total [35S]guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) binding activity was equally distributed between the plasma membrane and cytosol with the remaining 6% localized in the granules. In contrast, the cytosol contained only 10% of the total GTPase activity while the specific granules accounted for 13%. [alpha-32P]GTP binding to proteins transferred to nitrocellulose revealed LMW-GBPs in all fractions except the azurophilic granules. The specific granules contained three out of four bands which were found in the plasma membrane; these ranged from 20 to 23 kDa and all were resistant to alkaline extraction. Photoaffinity labeling with [alpha-32P]8-azido-GTP in the presence of micromolar Al3+ identified proteins of 25 and 26 kDa unique to azurophilic granules; these could not be labeled with [alpha-32P]8-azido-ATP and could be extracted by acidic but not alkaline pH. Botulinum C3-mediated [32P]ADP-ribosylation identified proteins of 16, 20, and 24 kDa both in plasma membranes and those of specific granules. An anti-ras monoclonal antibody, 142-24E5, recognized a 20-kDa protein localized to the plasma and specific granule membranes which could not be extracted by alkaline pH, was not a substrate for botulinum C3 ADP-ribosyltransferase, and was translocated from specific granules to plasma membrane after exposure of neutrophils to phorbol myristate acetate. We conclude that neutrophil-specific and azurophilic granules contain distinct subsets of LMW-GBPs which are uniquely situated to regulate the differential exocytosis of these two compartments.