Annexin2 coating the surface of enlargeosomes is needed for their regulated exocytosis

Enlargeosomes are small cytoplasmic vesicles that undergo rapid, Ca2+-dependent exo/endocytosis. The role of the cytoskeleton in these processes was unknown. In PC12-27 cells, microtubule disassembly had little effect on enlargeosomes, whereas microfilament disassembly increased markedly both their resting and stimulated exocytosis, and inhibited their endocytosis. Even at rest enlargeosomes are coated at their cytosolic surface by an actin-associated protein, annexin2, bound by a dual, Ca2+-dependent and Ca2+-independent mechanism. In contrast, the other enlargeosome marker, desmoyokin/Ahnak, is transported across the organelle membrane, apparently by an ABC transporter, and binds to its lumenal face. Annexin2-GFP expression revealed that, upon stimulation, the slow and random enlargeosome movement increases markedly and becomes oriented toward the plasma membrane. After annexin2 downregulation enlargeosome exocytosis induced by both [Ca2+]i rise and cytoskeleton disruption is inhibited, and the NGF-induced differentiation is blocked. Binding of annexin2 to the enlargeosome membrane, the most extensive ever reported (>50% annexin2 bound to approximately 3% of total membrane area), seems therefore to participate in the regulation of their exocytosis.     

Translocation of annexin B1 in response to the stimulation of PMA and ionomycin in cervical cancer cells

Annexin B1 is a novel member of the annexin superfamily which was isolated from a Cysticercus cellulosae cDNA library. To investigate the physiological roles of annexin B1, we firstly performed immunohistochemical analysis on frozen Cysticercus cellulosae sections and found that annexin B1 was present not only in the tegument of the bladder wall, but also in the host-derived inflammatory layer; In addition, ELISA analysis revealed that annexin B1 could be detected in the cystic fluid of Cysticercus cellulosae and the sera of pigs with cysticercosis. These findings indicated that annexin B1 might be a secretary protein. We further constructed a pEGFP-annexin B1 plasmid and transfected it into SiHa cells. We found that GFP-annexin B1 was stimulated to translocate to the plasma membrane by phorbol 12-myristate 13-acetate (PMA). By contrast, it was induced to distribute at the plasma and nuclear membranes by treatment with calcium ionophore ionomycin. PMA increased annexin B1 membrane binding, which might facilitate exocytosis. Moreover, translocation of the protein to the plasma and nuclear membranes after stimulated by ionomycin, was predicted to be related to an additional function.     

The interaction of protein kinase C and other specific cytoplasmic proteins with phospholipid bilayers

The role of lipid composition in the interaction of purified protein kinase C with large unilamellar vesicles was determined by the extent of photolabelling of the enzyme with 5-[125I]iodonaphthalene-I-azide. The protein kinase C was only slightly labelled when exposed to phosphatidylcholine (PC) liposomes. The addition of phorbol 12-myristate 13-acetate (PMA) or of diacylglycerol to the PC liposomes enhanced significantly the labelling of the protein kinase C at low calcium concentrations. A further enhancement in the photolabelling of the protein kinase C was observed in liposomes containing 2% phosphatidylserine (PS). At low calcium concentrations, the binding of the enzyme to these liposomes increased in the presence of added PMA or diacylglycerol. Raising the levels of PS beyond 2% in the liposomes did not enhance the binding of the protein kinase C. However, when the enzymatic activity of the protein kinase C was measured using basic histones as substrates, maximum phosphorylation was obtained in liposomes with a PC to PS ratio of 1. The fact that the translocation of the protein kinase C from solution to the surface of the liposomes could be monitored by its labelling with 5-iodonaphthalene 1-azide prompted us to determine whether other cytoplasmic proteins might share this property. The interaction of cytoplasmic proteins from HeLa cells with PC liposomes gave trace labelling irrespective of whether calcium was added. When the HeLa cell cytoplasmic proteins were allowed to interact with liposomes containing PS, selective 5-iodonaphthalene-1-azide photolabelling was observed in distinct proteins. Addition of calcium and of PMA or diacylglycerol modified the labelling of some but not all of these proteins. These results suggest that the methodology developed might serve to identify proteins that move to the membrane during stimulation of cells by phorbol esters or by growth factors which induce the generation of diacylglycerol. These results also suggest a role for the phospholipid composition of the plasma membrane (or any intracellular membrane) in the modulation of the activation processes of specific phospholipid-dependent proteins, in particular protein kinase C.     

Proteins are secreted by both constitutive and regulated secretory pathways in lactating mouse mammary epithelial cells

Lactating mammary epithelial cells secrete high levels of caseins and other milk proteins. The extent to which protein secretion from these cells occurs in a regulated fashion was examined in experiments on secretory acini isolated from the mammary glands of lactating mice at 10 d postpartum. Protein synthesis and secretion were assayed by following the incorporation or release, respectively, of [35S]methionine-labeled TCA-precipitable protein. The isolated cells incorporated [35S]methionine into protein linearly for at least 5 h with no discernible lag period. In contrast, protein secretion was only detectable after a lag of approximately 1 h, consistent with exocytotic secretion of proteins immediately after passage through the secretory pathway and package into secretory vesicles. The extent of protein secretion was unaffected by the phorbol ester PMA, 8-bromo-cAMP, or 8-bromo-cGMP but was doubled by the Ca2+ ionophore ionomycin. In a pulse-label protocol in which proteins were prelabeled for 1 h before a chase period, constitutive secretion was unaffected by depletion of cytosolic Ca2+ but ionomycin was found to give a twofold stimulation of the secretion of presynthesized protein in a Ca(2+)-dependent manner. Ionomycin was still able to stimulate protein secretion after constitutive secretion had terminated. These results suggest that lactating mammary cells possess both a Ca(2+)-independent constitutive pathway and a Ca(2+)-activated regulatory pathway for protein secretion. The same proteins were secreted by both pathways. No ultrastructural evidence for apocrine secretion was seen in response to ionomycin and so it appears that regulated casein release involves exocytosis. Ionomycin was unlikely to be acting by disassembling the cortical actin network since cytochalasin D did not mimic its effects on secretion. The regulated pathway may be controlled by Ca2+ acting at a late step such as exocytotic membrane fusion.     

The Slp4-a linker domain controls exocytosis through interaction with Munc18-1.syntaxin-1a complex

Synaptotagmin-like protein 4-a (Slp4-a)/granuphilin-a is specifically localized on dense-core vesicles in certain neuroendocrine cells and negatively controls dense-core vesicle exocytosis through specific interaction with Rab27A. However, the precise molecular mechanism of its inhibitory effect on exocytosis has never been elucidated and is still a matter of controversy. Here we show by deletion and chimeric analyses that the linker domain of Slp4-a interacts with the Munc18-1.syntaxin-1a complex by directly binding to Munc18-1 and that this interaction promotes docking of dense-core vesicles to the plasma membrane in PC12 cells. Despite increasing the number of plasma membrane docked vesicles, expression of Slp4-a strongly inhibited high-KCl-induced dense-core vesicle exocytosis. The inhibitory effect by Slp4-a is absolutely dependent on the linker domain of Slp4-a, because substitution of the linker domain of Slp4-a by that of Slp5 (the closest isoform of Slp4-a that cannot bind the Munc18-1.syntaxin-1a complex) completely abrogated the inhibitory effect. Our findings reveal a novel docking machinery for dense-core vesicle exocytosis: Slp4-a simultaneously interacts with Rab27A and Munc18-1 on the dense-core vesicle and with syntaxin-1a in the plasma membrane.     

A Highlights from MBoC Selection: Age-dependent Preferential Dense-Core Vesicle Exocytosis in Neuroendocrine Cells Revealed by Newly Developed Monomeric Fluorescent Timer Protein

Although it is evident that only a few secretory vesicles accumulating in neuroendocrine cells are qualified to fuse with the plasma membrane and release their contents to the extracellular space, the molecular mechanisms that regulate their exocytosis are poorly understood. For example, it has been controversial whether secretory vesicles are exocytosed randomly or preferentially according to their age. Using a newly developed protein-based fluorescent timer, monomeric Kusabira Green Orange (mK-GO), which changes color with a predictable time course, here we show that small GTPase Rab27A effectors regulate age-dependent exocytosis of secretory vesicles in PC12 cells. When the vesicles were labeled with mK-GO–tagged neuropeptide Y or tissue-type plasminogen activator, punctate structures with green or red fluorescence were observed. Application of high [K⁺] stimulation induced exocytosis of new (green) fluorescent secretory vesicles but not of old (red) vesicles. Overexpression or depletion of rabphilin and synaptotagmin-like protein4-a (Slp4-a), which regulate exocytosis positively and negatively, respectively, disturbed the age-dependent exocytosis of the secretory vesicles in different manners. Our results suggest that coordinate functions of the two effectors of Rab27A, rabphilin and Slp4-a, are required for regulated secretory pathway.     

Resident CAPS on dense-core vesicles docks and primes vesicles for fusion

The Ca²⁺-dependent exocytosis of dense-core vesicles in neuroendocrine cells requires a priming step during which SNARE protein complexes assemble. CAPS (aka CADPS) is one of several factors required for vesicle priming; however, the localization and dynamics of CAPS at sites of exocytosis in live neuroendocrine cells has not been determined. We imaged CAPS before, during, and after single-vesicle fusion events in PC12 cells by TIRF micro­scopy. In addition to being a resident on cytoplasmic dense-core vesicles, CAPS was present in clusters of approximately nine molecules near the plasma membrane that corresponded to docked/tethered vesicles. CAPS accompanied vesicles to the plasma membrane and was present at all vesicle exocytic events. The knockdown of CAPS by shRNA eliminated the VAMP-2–dependent docking and evoked exocytosis of fusion-competent vesicles. A CAPS(ΔC135) protein that does not localize to vesicles failed to rescue vesicle docking and evoked exocytosis in CAPS-depleted cells, showing that CAPS residence on vesicles is essential. Our results indicate that dense-core vesicles carry CAPS to sites of exocytosis, where CAPS promotes vesicle docking and fusion competence, probably by initiating SNARE complex assembly.     

High mobility of vesicles supports continuous exocytosis at a ribbon synapse

BACKGROUND: Most synapses release neurotransmitter as transient pulses, but ribbon synapses of sensory neurons support continuous exocytosis in response to maintained stimulation. We have investigated how the movement and retrieval of vesicles might contribute to continuous exocytosis at the ribbon synapse of retinal bipolar cells. RESULTS: Using a combination of total internal reflection fluorescence microscopy and fluorescence recovery after photobleaching, we found that the great majority of vesicles within 50-120 nm of the plasma membrane move in a random fashion with an effective diffusion coefficient of approximately 1.5 x 10(-2) microm(2) s(-1). Using confocal microscopy, we found that vesicles are similarly mobile across the whole terminal and that this motion is not altered by calcium influx or the actin cytoskeleton. We calculated that the cytoplasmic reservoir of approximately 300,000 vesicles would generate about 900 vesicle collisions/s against ribbons and 28,000 collisions/s against the surface membrane. The efficient resupply of vesicles to ribbons was confirmed by electron microscopy. A 1 min depolarization, releasing 500-1000 vesicles/s, caused a 70% reduction in the number of vesicles docked at the active zone without reducing the number of vesicles attached to ribbons or remote areas of the plasma membrane. These sites were not repopulated by retrieved vesicles because 80-90% of the recycled membrane was taken up into cisternae that pinched off from the surface. CONCLUSIONS: These results indicate that the random motion of cytoplasmic vesicles provides an efficient supply to the ribbon and plasma membrane and allows the maintenance of high rates of exocytosis without an equally rapid recycling of vesicles. The selective depletion of vesicles docked under ribbons suggests that the transfer of vesicles to the active zone limits the rate of exocytosis during maintained stimulation.     

Ahnak protein activates protein kinase C (PKC) through dissociation of the PKC-protein phosphatase 2A complex

We have previously reported that central repeated units (CRUs) of Ahnak act as a scaffolding protein networking phospholipase Cgamma and protein kinase C (PKC). Here, we demonstrate that an Ahnak derivative consisting of four central repeated units binds and activates PKC-alpha in a phosphatidylserine/1,2-dioleoyl-sn-glycerol-independent manner. Moreover, NIH3T3 cells expressing the 4 CRUs of Ahnak showed enhanced c-Raf, MEK, and Erk phosphorylation in response to phorbol 12-myristate 13-acetate (PMA) compared with parental cells. To evaluate the effect of loss-of-function of Ahnak in cell signaling, we investigated PKC activation and Raf phosphorylation in embryonic fibroblast cells (MEFs) of the Ahnak knock-out (Ahnak(-/-)) mouse. Membrane translocation of PKC-alpha and phosphorylation of Raf in response to PMA or platelet-derived growth factor were decreased in Ahnak null MEF cells compared with wild type MEFs. Several lines of evidence suggest that PKC-alpha activity is regulated through association with protein phosphatase 2A (PP2A). A co-immunoprecipitation assay indicated that the association of PKC-alpha with PP2A was disrupted in NIH3T3 cells expressing 4 CRUs of Ahnak in response to PMA. Consistently, Ahnak null MEF cells stimulated by PMA showed enhanced PKC-PP2A complex formation, and add-back expression of Ahnak into Ahnak null MEF cells abolished the PKC-PP2A complex formation in response to PMA. These data indicate that Ahnak potentiates PKC activation through inhibiting the interaction of PKC with PP2A.     

Phospholipid metabolism in bradykinin-stimulated human fibroblasts. II. Phosphatidylcholine breakdown by phospholipases C and D; involvement of protein kinase C

Bradykinin (BK) and phorbol 12-myristate 13-acetate (PMA) both stimulate the hydrolysis of phosphatidylcholine (PC) in human fibroblasts, resulting in the formation of phosphatidic acid (PA) and diacylglycerol (DG) (Van Blitterswijk, W.J., Hilkmann, H., de Widt, J., and Van der Bend, R.L. (1990) J. Biol. Chem. 266, 10337-10343). Stimulation with BK resulted in the rapid and synchronous formation of [3H]choline and [3H]myristoyl-PA from the correspondingly prelabeled PC, indicative of phospholipase D (PLD) activity. In the presence of ethanol or n-butanol, transphosphatidylation by PLD resulted in the formation of [3H]phosphatidylethanol or - butanol, respectively, at the cost of PA and DG formation. This suggests that PC-derived DG is generated via a PLD/PA phosphohydrolase pathway. A more pronounced but delayed formation of these products was observed by PMA stimulation. The Ca2+ ionophore ionomycin also activated PLD and accelerated (synergized) the response to PMA. Both [3H] choline and [3H]phosphocholine were released into the extracellular medium in a time- and stimulus-dependent fashion, without apparent changes in the high intracellular levels of [3H]phosphocholine. The protein kinase C (PKC) inhibitors staurosporin and 1-O-hexadecyl-2-O-methylglycerol inhibited BK- and PMA-induced activation of PLD. Down-regulation of PKC by long-term pretreatment of cells with phorbol ester caused a dramatic drop in background [3H]choline levels, while subsequent stimulation with BK, ionomycin, or PMA failed to increase these levels and failed to induce transphosphatidylation. From these results we conclude that PLD activation is entirely mediated by (downstream of) PKC. Unexpectedly, however, BK stimulation of these PKC-depleted cells caused a marked generation of DG from PC within 15 s, which was not seen in BK-stimulated control cells, suggesting PC breakdown by a phospholipase C (PLCc). We conclude that cells stimulated with BK generate DG via both the PLCc and the PLD/PA hydrolase pathway, whereas PMA stimulates mainly the latter pathway. BK stimulation of normal cells leads to activation of PKC and, by consequence, to attenuation of the level of PLCc-generated DG and to stimulation of the PLD pathway, whereas the reverse occurs in PKC-down-regulated cells.     

Characterization of the immature secretory granule, an intermediate in granule biogenesis

The events in the biogenesis of secretory granules after the budding of a dense-cored vesicle from the trans-Golgi network (TGN) were investigated in the neuroendocrine cell line PC12, using sulfate-labeled secretogranin II as a marker. The TGN-derived dense-cored vesicles, which we refer to as immature secretory granules, were found to be obligatory organellar intermediates in the biogenesis of the mature secretory granules which accumulate in the cell. Immature secretory granules were converted to mature secretory granules with a half-time of approximately 45 min. This conversion entailed an increase in their size, implying that the maturation of secretory granules includes a fusion event involving immature secretory granules. Pulse-chase labelling of PC12 cells followed by stimulation with high K+, which causes the release of secretogranin II, showed that not only mature, but also immature secretory granules were capable of undergoing regulated exocytosis. The kinetics of secretion of secretogranin II, as well as those of a constitutively secreted heparan sulfate proteoglycan, were reduced by treatment of PC12 cells with nocodazole, suggesting that both secretory granules and constitutive secretory vesicles are transported to the plasma membrane along microtubules. Our results imply that certain membrane proteins, e.g., those involved in the fusion of post-TGN vesicles with the plasma membrane, are sorted upon exit from the TGN, whereas other membrane proteins, e.g., those involved in the interaction of post-TGN vesicles with the cytoskeleton, may not be sorted.     

Inhibition of interleukin-2 receptor (CD25) expression induced on T cells from children with acute lymphoblastic leukemia

 Peripheral blood lymphocytes obtained from children with acute lymphoblastic leukemia (ALL) at onset were studied for the expression of interleukin-2 (IL-2) receptor α-chain (CD25) by two-color flow-cytometric analysis. Stimulated with anti-CD3 monoclonal antibody (mAb) alone, CD25 expression was significantly suppressed in CD4⁺ T cells from 27 of 48 (56.3%) cases and in CD8⁺ T cells from 29 of 48 (60.4%) cases. When stimulated with anti-CD3 mAb plus phorbol 12-myristate 13-acetate (PMA), CD25 expression was clearly restored in certain cases of ALL. When PMA plus ionomycin were used for stimulation of T cells, CD25 was inducible in a majority of cases. Interestingly CD25 expression upon anti-CD3 mAb stimulation was recovered after complete remission had been achieved. These observations suggest the presence in ALL children at onset of an in vitro defect in the signal transduction pathway of the T-cell-receptor/CD3 complex, resulting in inefficient CD25 expression. However, immune-staining analysis indicated that protein kinase C was normally translocated from the cytosol fraction to the cell membrane fraction. The mobilization of cytoplasmic free calcium is also normal. Received: 27 March 1996 / Accepted: 23 December 1996     

Structural modifications induced by TPA (12-O-tetradecanoyl phorbol-13-acetate) in sea urchin eggs

We investigated the effect of the phorbol ester TPA (12-O-tetradecanoyl phorbol 13-acetate) on the egg morphology of the sea urchin Arbacia lixula. Our study indicates that TPA alters the cortical region of the egg: the pigment granules migrate toward the surface, while cortical granules detach from the plasma membrane. Cortical granule exocytosis did not occur but the endocytosis process was turned on. Prolonged treatment of the eggs by TPA partially inhibits the cortical granule exocytosis normally triggered by fertilization. We discuss the effects of TPA in terms of its interaction with the Ca2+ pool and cytoskeletal structures. In order to discern the respective roles of pHi and protein kinase C activity in endocytosis process activation, we compared the ultrastructural effects of TPA and ammonia. Finally, the role of pigment vesicles in egg metabolism activation is discussed.     

Translocation of the classic protein kinase C isoforms in porcine oocytes: implications of protein kinase C involvement in the regulation of nuclear activity and cortical granule exocytosis

Protein kinase C (PKC) is a family of Ser/Thr protein kinases categorized into three subfamilies: classical, novel, and atypical. The subcellular localization of classical PKCalpha, -betaI, and -gamma in the process of porcine oocyte maturation, fertilization, and parthenogenetic activation and their involvement in cortical granule (CG) exocytosis were investigated. The results of Western blot showed that PKCalpha, -betaI, and -gamma were expressed in the oocytes at the germinal vesicle (GV) and metaphase II (MII) stages. Confocal microscopy revealed that the three PKC isoforms were concentrated in the GV but evenly distributed in the cytoplasm of MII eggs. PKCalpha and -gamma were translocated to the plasma membrane soon after sperm penetration. cPKCs migrated into the pronucleus in fertilized eggs. Following treatment with a PKC activator, phorbol 12-myristate 13-acetate (PMA), CGs were released and PKCalpha and -gamma were translocated to the membrane. The CG exocytosis and PKC redistribution induced by PMA could be blocked by the PKC inhibitor staurosporine. Parthenogenetic stimulation with ionophore A23187 or electrical pulse also induced cPKC translocation and CG exocytosis. Eggs injected with PKCalpha isoform-specific antibody failed to undergo CG exocytosis after PMA treatment or fertilization. The results suggest that cPKCs, especially the alpha-isotype, regulate nuclear function and CG exocytosis in porcine eggs.     

Differential phosphorylation of SNAP-25 in vivo by protein kinase C and protein kinase A

SNAP-25 is a key protein required for the fusion of synaptic vesicles with the plasma membrane during exocytosis. This study establishes that SNAP-25 is differentially phosphorylated by protein kinase C and protein kinase A in neuroendocrine PC12 cells. Using phosphopeptide mapping and site-directed mutagenesis we identified both Thr138 and Ser187 as the targets of SNAP-25 phosphorylation by protein kinase C and Thr138 as the exclusive site of SNAP-25 phosphorylation by protein kinase A in vivo. Finally, despite published data to the contrary, we demonstrate that stimulation of regulated exocytosis under physiological conditions is independent of a measurable increase in SNAP-25 phosphorylation in PC12 cells.     

Establishment of variant PC12 subclones deficient in stimulation–secretion coupling

Clonal rat pheochromocytoma (PC12) cells have been widely used to study the molecular mechanism of exocytosis. We have isolated variant PC12 subclones with deficiencies in stimulation–secretion coupling, by a single cell recloning, and investigated the defects. PC12-1G2 hardly released dopamine following high-K⁺-induced depolarization, but normal release was evoked by the Ca²⁺-ionophore, ionomycin. Fura-2 fluorometry indicated that a nicardipine-sensitive component of Ca²⁺ influx was missing, suggesting that PC12-1G2 has defects in L-type Ca²⁺ channel function. PC12-2B3 was not responsive to high-K⁺-induced depolarization and ionomycin, and voltage-dependent Ca²⁺ entry was identical to that of the normal clone. Electron microscopy revealed that the number of vesicles adjacent or directly attached to the plasma membrane was decreased in PC12-2B3. The expression of presynaptic proteins was analyzed by immunoblotting using a panel of antibodies. Syntaxin 1, VAMP-2, SNAP-25, Munc18, Rab3C and Sec-6 were decreased compared to the control clone and that of synaptophysin was extremely low. PC12-D60 synthesized and released dopamine normally, but had almost lost its catecholamine-uptake activity. These results show that multiple PC12 cells variants are spontaneously generated, and that recloning can select PC12 subclones useful for the study of the molecular mechanisms of neurotransmitter release.     

Increased endocytosis and formation of multivesicular bodies in phorbol ester-stimulated human monoblastic U-937 cells

The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) is known to arrest mitotic activity and induce macrophage differentiation in the U-937 monoblastic cell line. The acute effect of TPA on ultrastructural morphology and endocytic activity of U-937 cells was studied. TPA induced within 15 min a marked enlargement of multivesicular bodies (MVBs), comprising both volume and number of inclusion vesicles (other organelles appeared unchanged). At this stage the MBVs frequently showed tubular cytoplasmic extensions. Inclusion vesicles accumulated in MBVs with prolonged incubation (60 min). Horseradish peroxidase (HRP) and cationized ferritin (CF) added to the medium were routed preferentially to MVBs in TPA-stimulated cells. In contrast to MVBs of unstimulated cells many of the TPA-induced MVBs showed a positive cytochemical reaction to acid phosphatase. The MVBs in cells incubated with ionomycin, a calcium ionophore, did not differ from those of unstimulated cells. Cellular uptake of 125I-HRP was increased five times the control values already after 5 min of TPA stimulation. The uptake increased further with prolonged incubation (60 min), but at a slower rate. Together these indicate a TPA-induced transfer by endocytosis of portions of the plasma membrane to the lysosomal system via MVBs. Consideration of MVBs as part of the receptor-mediated endocytic pathway suggests that this effect of TPA might involve down-regulation of cell-surface receptors. The possibility of MVBs as a proton-sequestrating compartment, responsible for the cytoplasmic alkalinization previously reported for TPA-stimulated U-937 monoblastic cells, is discussed.     

Change in Membrane Fluidity of Sand Dollar Egg Cortices Caused by Ca2+-Induced Exocytosis: Microscopic Analysis with Fluorescence Anisotropy

The surface membrane fluidity of sand dollar eggs after exocytosis was investigated by using a specially designed video-microscope system to measure the fluorescence depolarization of isolated cortices stained with hexadecanoylaminofluorescein. When unfertilized eggs were stained before isolation, the plasma membranes became labeled with fluorescent dye, but cortical vesicles did not. The fluorescence anisotropy of the isolated cortices increased from 0.256 to 0.285 during exocytosis induced by Ca²⁺. The increased anisotropy was not changed by lowering the Ca²⁺concentration after exocytosis. Dislodging of cortical vesicles by shearing with a stream of solution had no affect on the anisotropy. These results suggest that the fluidity of the plasma membrane decreases after exocytosis. When cortices were stained after isolation, both plasma membranes and cortical membrane organelles became labeled. These cortices possessed an anisotropy of 0.215. After dislogding the cortical organelles the anisotropy increased to 0.232. These results indicate that the fluidity of the cytoplasmic membrane leaflets of cortical organelles is higher than that of the plasma membrane. Therefore, it was suggested that only the outer leaflet of the plasma membrane becomes less fluid after exocytosis.     

Phorbol ester induces transient focal concentrations of functional, newly expressed CR3 in neutrophils at sites of specific granule exocytosis

Treatment of neutrophils with phorbol myristate acetate (PMA) increases surface expression of CR3 (iC3b-receptor; CD11b/CD18). This up-regulation was examined in whole-mount preparations of adherent neutrophils by stereo high voltage immunoelectron microscopy. In the absence of PMA, immunogold-labeled CR3 was uniformly distributed in the plasma membrane. After 5 to 15-min incubations with PMA, when the highest rates of specific granule exocytosis occurred, the average density of CR3 in the membrane doubled. During this time, dense foci of CR3 were observed in addition to the uniform distribution of CR3. These dense concentrations of CR3 colocalized with secreted lactoferrin (LF), a specific granule marker, above assemblies of cytoplasmic vesicles that were morphologically similar to specific granules and contained LF. After longer incubations in PMA, LF secretion ceased, LF staining became rare, and the high density areas of CR3 were no longer present. These data demonstrate that incipient CR3 appear on the cell surface in high concentration at sites of specific granule exocytosis and then diffuse out into the plasmalemma. PMA also induced shedding of CR3 from the cell surface at the cell margins on structures which also contained LF. Shed CR3 was immunoprecipitated from incubation supernatants and shown to be of identical subunit composition to surface CR3. Although others have shown that the mobile, incipient surface CR3 do not mediate neutrophil adherence to endothelium or homotypic aggregation, the current experiments demonstrated that such CR3 do mediate iC3b-dependent adhesion. The rapid appearance and subsequent dissipation of high concentrations of CR3 on the neutrophil surface caused by specific granule fusion may be essential for neutrophil function at sites of complement deposition.     

The polybasic sequence in the C2B domain of rabphilin is required for the vesicle docking step in PC12 cells

Rabphilin is generally thought to be involved in the regulation of secretory vesicle exocytosis in neurons and neuroendocrine cells, and it has recently been hypothesized that the C2B domain of rabphilin promotes the docking of dense-core vesicles to the plasma membrane through simultaneous interaction with a vesicle protein, Rab3A/27A, and a plasma membrane protein, SNAP-25 (synaptosome-associated protein of 25 kDa). However, the physiological significance of the rabphilin-SNAP-25 interaction in the vesicle-docking step has never been elucidated. In this study we demonstrated by a mutation analysis that the polybasic sequence (587 KKAKHKTQIKKK 598) in the C2B domain of rabphilin is required for SNAP-25 binding, and that the Asp residues in the Ca(2+)-binding loop 3 (D628 and D630) of the C2B domain are not required. We also investigated the effect of Lys-->Gln (KQ) mutations in the polybasic sequence of the C2B domain on vesicle dynamics by total internal reflection fluorescence microscopy in individual PC12 cells. A rabphilin(KQ) mutant that completely lacks SNAP-25-binding activity significantly decreased the number of plasma-membrane-docked vesicles and strongly inhibited high-KCl-induced dense-core vesicle exocytosis. These results indicate that the polybasic sequence in the C2B domain functions as an effector domain for SNAP-25 and controls the number of 'releasable' vesicles docked to the plasma membrane.