Phosphatidylinositol 4,5-bisphosphate regulates SNARE-dependent membrane fusion

Phosphatidylinositol 4,5-bisphosphate (PI 4,5-P(2)) on the plasma membrane is essential for vesicle exocytosis but its role in membrane fusion has not been determined. Here, we quantify the concentration of PI 4,5-P(2) as approximately 6 mol% in the cytoplasmic leaflet of plasma membrane microdomains at sites of docked vesicles. At this concentration of PI 4,5-P(2) soluble NSF attachment protein receptor (SNARE)-dependent liposome fusion is inhibited. Inhibition by PI 4,5-P(2) likely results from its intrinsic positive curvature-promoting properties that inhibit formation of high negative curvature membrane fusion intermediates. Mutation of juxtamembrane basic residues in the plasma membrane SNARE syntaxin-1 increase inhibition by PI 4,5-P(2), suggesting that syntaxin sequesters PI 4,5-P(2) to alleviate inhibition. To define an essential rather than inhibitory role for PI 4,5-P(2), we test a PI 4,5-P(2)-binding priming factor required for vesicle exocytosis. Ca(2+)-dependent activator protein for secretion promotes increased rates of SNARE-dependent fusion that are PI 4,5-P(2) dependent. These results indicate that PI 4,5-P(2) regulates fusion both as a fusion restraint that syntaxin-1 alleviates and as an essential cofactor that recruits protein priming factors to facilitate SNARE-dependent fusion.     

Exo70 interacts with phospholipids and mediates the targeting of the exocyst to the plasma membrane

The exocyst is an octameric protein complex implicated in the tethering of post-Golgi secretory vesicles to the plasma membrane before fusion. The function of individual exocyst components and the mechanism by which this tethering complex is targeted to sites of secretion are not clear. In this study, we report that the exocyst subunit Exo70 functions in concert with Sec3 to anchor the exocyst to the plasma membrane. We found that the C-terminal Domain D of Exo70 directly interacts with phosphatidylinositol 4,5-bisphosphate. In addition, we have identified key residues on Exo70 that are critical for its interaction with phospholipids and the small GTPase Rho3. Further genetic and cell biological analyses suggest that the interaction of Exo70 with phospholipids, but not Rho3, is essential for the membrane association of the exocyst complex. We propose that Exo70 mediates the assembly of the exocyst complex at the plasma membrane, which is a crucial step in the tethering of post-Golgi secretory vesicles for exocytosis.     

Phosphatidylinositol 4,5-bisphosphate and Arf6-regulated membrane traffic

ADP-ribosylation factor (Arf) 6 regulates the movement of membrane between the plasma membrane (PM) and a nonclathrin-derived endosomal compartment and activates phosphatidylinositol 4-phosphate 5-kinase (PIP 5-kinase), an enzyme that generates phosphatidylinositol 4,5-bisphosphate (PIP2). Here, we show that PIP2 visualized by expressing a fusion protein of the pleckstrin homology domain from PLCdelta and green fluorescent protein (PH-GFP), colocalized with Arf6 at the PM and on tubular endosomal structures. Activation of Arf6 by expression of its exchange factor EFA6 stimulated protrusion formation, the uptake of PM into macropinosomes enriched in PIP2, and recycling of this membrane back to the PM. By contrast, expression of Arf6 Q67L, a GTP hydrolysis-resistant mutant, induced the formation of PIP2-positive actin-coated vacuoles that were unable to recycle membrane back to the PM. PM proteins, such as beta1-integrin, plakoglobin, and major histocompatibility complex class I, that normally traffic through the Arf6 endosomal compartment became trapped in this vacuolar compartment. Overexpression of human PIP 5-kinase alpha mimicked the effects seen with Arf6 Q67L. These results demonstrate that PIP 5-kinase activity and PIP2 turnover controlled by activation and inactivation of Arf6 is critical for trafficking through the Arf6 PM-endosomal recycling pathway.     

Phosphatidylinositol 4,5-bisphosphate hydrolysis mediates histamine-induced KCNQ/M current inhibition

The M-type potassium channel, of which its molecular basis is constituted by KCNQ2-5 homo- or heteromultimers, plays a key role in regulating neuronal excitability and is modulated by many G protein-coupled receptors. In this study, we demonstrate that histamine inhibits KCNQ2/Q3 currents in human embryonic kidney (HEK)293 cells via phosphatidylinositol 4,5-bisphosphate (PIP(2)) hydrolysis mediated by stimulation of H(1) receptor and phospholipase C (PLC). Histamine inhibited KCNQ2/Q3 currents in HEK293 cells coexpressing H(1) receptor, and this effect was totally abolished by H(1) receptor antagonist mepyramine but not altered by H(2) receptor antagonist cimetidine. The inhibition of KCNQ currents was significantly attenuated by a PLC inhibitor U-73122 but not affected by depletion of internal Ca(2+) stores or intracellular Ca(2+) concentration ([Ca(2+)](i)) buffering via pipette dialyzing BAPTA. Moreover, histamine also concentration dependently inhibited M current in rat superior cervical ganglion (SCG) neurons by a similar mechanism. The inhibitory effect of histamine on KCNQ2/Q3 currents was entirely reversible but became irreversible when the resynthesis of PIP(2) was impaired with phosphatidylinsitol-4-kinase inhibitors. Histamine was capable of producing a reversible translocation of the PIP(2) fluorescence probe PLC(delta1)-PH-GFP from membrane to cytosol in HEK293 cells by activation of H(1) receptor and PLC. We concluded that the inhibition of KCNQ/M currents by histamine in HEK293 cells and SCG neurons is due to the consumption of membrane PIP(2) by PLC.     

Phosphatidylinositol 4,5-bisphosphate induces actin stress-fiber formation and inhibits membrane ruffling in CV1 cells

Phosphatidylinositol 4,5 bisphosphate (PIP(2)) is widely implicated in cytoskeleton regulation, but the mechanisms by which PIP(2) effect cytoskeletal changes are not defined. We used recombinant adenovirus to infect CV1 cells with the mouse type I phosphatidylinositol phosphate 5-kinase alpha (PIP5KI), and identified the players that modulate the cytoskeleton in response to PIP(2) signaling. PIP5KI overexpression increased PIP(2) and reduced phosphatidylinositol 4 phosphate (PI4P) levels. It promoted robust stress-fiber formation in CV1 cells and blocked PDGF-induced membrane ruffling and nucleated actin assembly. Y-27632, a Rho-dependent serine/threonine protein kinase (ROCK) inhibitor, blocked stress-fiber formation and inhibited PIP(2) and PI4P synthesis in cells. However, Y-27632 had no effect on PIP(2) synthesis in lysates, although it inhibited PI4P synthesis. Thus, ROCK may regulate PIP(2) synthesis by controlling PI4P availability. PIP5KI overexpression decreased gelsolin, profilin, and capping protein binding to actin and increased that of ezrin. These changes can potentially account for the increased stress fiber and nonruffling phenotype. Our results establish the physiological role of PIP(2) in cytoskeletal regulation, clarify the relation between Rho, ROCK, and PIP(2) in the activation of stress-fiber formation, and identify the key players that modulate the actin cytoskeleton in response to PIP(2).     

Diffusion coefficient of fluorescent phosphatidylinositol 4,5-bisphosphate in the plasma membrane of cells

Phosphatidylinositol 4,5-bisphosphate (PIP₂) controls a surprisingly large number of processes in cells. Thus, many investigators have suggested that there might be different pools of PIP₂ on the inner leaflet of the plasma membrane. If a significant fraction of PIP₂ is bound electrostatically to unstructured clusters of basic residues on membrane proteins, the PIP₂ diffusion constant, D, should be reduced. We microinjected micelles of Bodipy TMR-PIP₂ into cells, and we measured D on the inner leaflet of fibroblasts and epithelial cells by using fluorescence correlation spectroscopy. The average ± SD value from all cell types was D = 0.8 ± 0.2 μm²/s (n = 218; 25°C). This is threefold lower than the D in blebs formed on Rat1 cells, D = 2.5 ± 0.8 μm²/s (n = 26). It is also significantly lower than the D in the outer leaflet or in giant unilamellar vesicles and the diffusion coefficient for other lipids on the inner leaflet of these cell membranes. The simplest interpretation is that approximately two thirds of the PIP₂ on inner leaflet of these plasma membranes is bound reversibly.     

Phosphatidylinositol 4,5-bisphosphate is important for stomatal opening

Previously, we demonstrated that a protein that binds phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] inhibits both light-induced stomatal opening and ABA-induced stomatal closing. The latter effect is due to a reduction in free PtdIns(4,5)P(2), decreasing production of inositol 1,4,5-trisphosphate and phosphatidic acid by phospholipases C and D. However, it is less clear how PtdIns(4,5)P(2) modulates stomatal opening. We found that in response to white light irradiation, the PtdIns(4,5)P(2)-binding domain GFP:PLCdelta1PH translocated from the cytosol into the plasma membrane. This suggests that the level of PtdIns(4,5)P(2) increases at the plasma membrane upon illumination. Exogenously administered PtdIns(4,5)P(2) substituted for light stimuli, inducing stomatal opening and swelling of guard cell protoplasts. To identify PtdIns(4,5)P(2) targets we performed patch-clamp experiments, and found that anion channel activity was inhibited by PtdIns(4,5)P(2). Genetic analyses using an Arabidopsis PIP5K4 mutant further supported the role of PtdIns(4,5)P(2) in stomatal opening. The reduced stomatal opening movements exhibited by a mutant of Arabidopsis PIP5K4 (At3g56960) was countered by exogenous application of PtdIns(4,5)P(2). The phenotype of reduced stomatal opening in the pip5k4 mutant was recovered in lines complemented with the full-length PIP5K4. Together, these data suggest that PIP5K4 produces PtdIns(4,5)P(2) in irradiated guard cells, inhibiting anion channels to allow full stomatal opening.     

Phosphatidylinositol 4,5-bisphosphate regulation of SNARE function in membrane fusion mediated by CAPS

Ca²⁺-triggered vesicle exocytosis in neuroendocrine cells requires priming reactions that follow vesicle tethering/docking and precede triggered fusion. Priming requires PI(4,5)P₂ and priming factors, and likely involves SNARE protein complex assembly. In studies with proteoliposomes, the priming factor CAPS interacts with PI(4,5)P₂, binds the SNARE protein syntaxin-1, promotes trans SNARE complex formation, and stimulates PI(4,5)P₂- and SNARE-dependent liposome fusion. We propose that CAPS functions in priming vesicle exocytosis by coupling membrane binding to SNARE complex assembly.     

Phosphatidylinositol 4,5-bisphosphate phosphodiesterase in higher plants.

A phospholipase C which hydrolyses phosphatidylinositol 4,5-bisphosphate to release inositol trisphosphate was detected in a sedimentable fraction from celery and from some other higher plants. The particulate enzyme also hydrolyses phosphatidylinositol, whereas the soluble phosphatidylinositol phosphodiesterase described previously [Irvine, Letcher & Dawson (1980) Biochem. J. 192, 279-283] acts only on phosphatidylinositol, and we were unable to detect activity of this soluble activity on phosphatidylinositol 4,5-bisphosphate. Activity of the particulate enzyme is markedly enhanced in the presence of deoxycholate, but not of other detergents; the particulate enzyme can also be solubilized by extraction with deoxycholate.     

Phosphatidylinositol 4,5-bisphosphate functions as a second messenger that regulates cytoskeleton-plasma membrane adhesion

Binding interactions between the plasma membrane and the cytoskeleton define cell functions such as cell shape, formation of cell processes, cell movement, and endocytosis. Here we use optical tweezers tether force measurements and show that plasma membrane phosphatidylinositol 4,5-bisphosphate (PIP2) acts as a second messenger that regulates the adhesion energy between the cytoskeleton and the plasma membrane. Receptor stimuli that hydrolyze PIP2 lowered adhesion energy, a process that could be mimicked by expressing PH domains that sequester PIP2 or by targeting a 5'-PIP2-phosphatase to the plasma membrane to selectively lower plasma membrane PIP2 concentration. Our study suggests that plasma membrane PIP2 controls dynamic membrane functions and cell shape by locally increasing and decreasing the adhesion between the actin-based cortical cytoskeleton and the plasma membrane.     

Phosphatidylinositol 4,5-bisphosphate: Light-mediated breakdown in the vertebrate retina

Isolated frog ($\text{Rana}\text{Pipiens}$) retinas were labeled in the dark with either [³²P]PO₄-orthophosphate or $\text{myo}$-[2-³H]inositol for 2.5–4 hrs. After washing the retinas with cold buffer, they were exposed to brief flashes of light (5 secs or 15 secs) and their rod outer segments isolated. Upon separation of labeled phospholipids, a specific decrease in label in phosphatidylinositol 4,5-bisphosphate was observed, whereas there was no significant effect on the labeling of phosphatidylinositol 4-phosphate, phosphatidylinositol, or phosphatidic acid. These results are indicative of a light-activated phosphatidylinositol 4,5-bisphosphate-specific phospholipase C in frog rod outer segments.     

Phosphatidylinositol 4,5-bisphosphate alters synaptotagmin 1 membrane docking and drives opposing bilayers closer together

Synaptotagmin 1 (syt1) is a synaptic vesicle-anchored membrane protein that acts as the calcium sensor for the synchronous component of neuronal exocytosis. Using site-directed spin labeling, the position and membrane interactions of a fragment of syt1 containing its two C2 domains (syt1C2AB) were assessed in bilayers containing phosphatidylcholine (PC), phosphatidylserine (PS), and phosphatidylinositol 4,5-bisphosphate (PIP(2)). Addition of 1 mol % PIP(2) to a lipid mixture of PC and PS results in a deeper membrane penetration of the C2A domain and alters the orientation of the C2B domain so that the polybasic face of C2B comes into the proximity of the bilayer interface. The C2B domain is found to contact the membrane interface in two regions, the Ca(2+)-binding loops and a region opposite the Ca(2+)-binding loops. This suggests that syt1C2AB is configured to bridge two bilayers and is consistent with a model generated previously for syt1C2AB bound to membranes of PC and PS. Point-to-plane depth restraints, obtained by progressive power saturation, and interdomain distance restraints, obtained by double electron-electron resonance, were obtained in the presence of PIP(2) and used in a simulated annealing routine to dock syt1C2AB to two membrane interfaces. The results yield an average structure different from what is found in the absence of PIP(2) and indicate that bilayer-bilayer spacing is decreased in the presence of PIP(2). The results indicate that PIP(2), which is necessary for bilayer fusion, alters C2 domain orientation, enhances syt1-membrane electrostatic interactions, and acts to drive vesicle and cytoplasmic membrane surfaces closer together.     

Syndecan-4 promotes the retention of phosphatidylinositol 4,5-bisphosphate in the plasma membrane

Although phosphatidylinositol 4,5-bisphosphate (PIP₂) regulates syndecan-4 function, the potential influence of syndecan-4 on PIP₂ remains unknown. GFP containing PIP₂-binding-PH domain of phospholipase Cδ (GFP-PHδ) was used to monitor PIP₂. Syndecan-4 overexpression in COS-7 cells enhanced membrane translocation of GFP-PHδ, while the opposite was observed when syndecan-4 was knocked-down. PIP₂ levels were higher in total phospholipids extracted from rat embryo fibroblasts expressing syndecan-4. Syndecan-4-induced membrane targeting of GFP-PHδ was further enhanced by phosphoinositide-3-kinase inhibitor, but not by phospholipase C (PLC) inhibitor. Besides, both ionomycin and epidermal growth factor caused dissociation of GFP-PHδ from plasma membrane, an effect that was significantly delayed by syndecan-4 over-expression. Collectively, these data suggest that syndecan-4 promotes plasma membrane retention of PIP₂ by negatively regulating PLC-dependent PIP₂ degradation.     

Phosphatidylinositol 4,5-bisphosphate phosphatase regulates the rearrangement of actin filaments.

Phosphatidylinositol 4,5-bisphosphate (PIP2) reorganizes actin filaments by modulating the functions of a variety of actin-regulatory proteins. Until now, it was thought that bound PIP2 is hydrolyzed only by tyrosine-phosphorylated phospholipase Cgamma (PLCgamma) after the activation of tyrosine kinases. Here, we show a new mechanism for the hydrolysis of bound PIP2 and the regulation of actin filaments by PIP2 phosphatase (synaptojanin). We isolated a 150-kDa protein (p150) from brains that binds the SH3 domains of Ash/Grb2. The sequence of this protein was found to be homologous to that of synaptojanin. The expression of p150 in COS 7 cells produces a decrease in the number of actin stress fibers in the center of the cells and causes the cells to become multinuclear. On the other hand, the expression of a PIP2 phosphatase-negative mutant does not disrupt actin stress fibers or produce the multinuclear phenotype. We have also shown that p150 forms the complexes with Ash/Grb2 and epidermal growth factor (EGF) receptors only when the cells are treated with EGF and that it reorganizes actin filaments in an EGF-dependent manner. Moreover, the PIP2 phosphatase activity of native p150 purified from bovine brains is not inhibited by profilin, cofilin, or alpha-actinin, although PLCdelta1 activity is markedly inhibited by these proteins. Furthermore, p150 suppresses actin gelation, which is induced by smooth muscle alpha-actinin. All these data suggest that p150 (synaptojanin) hydrolyzes PIP2 bound to actin regulatory proteins, resulting in the rearrangement of actin filaments downstream of tyrosine kinase and Ash/Grb2.     

Phosphatidylinositol 4,5-bisphosphate competitively inhibits phorbol ester binding to protein kinase C

Calcium phospholipid dependent protein kinase C (PKC) is activated by diacylglycerol (DG) and by phorbol esters and is recognized to be the phorbol ester receptor of cells; DG displaces phorbol ester competitively from PKC. A phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP2), can also activate PKC in the presence of phosphatidylserine (PS) and Ca2+ with a KPIP2 of 0.04 mol %. Preliminary experiments have suggested a common binding site for PIP2 and DG on PKC. Here, we investigate the effect of PIP2 on phorbol ester binding to PKC in a mixed micellar assay. In the presence of 20 mol % PS, PIP2 inhibited specific binding of [3H]phorbol 12,13-dibutyrate (PDBu) in a dose-dependent fashion up to 85% at 1 mol %. Inhibition of binding was more pronounced with PIP2 than with DG. Scatchard analysis indicated that the decrease in binding of PDBu in the presence of PIP2 is the result of an altered affinity for the phorbol ester rather than of a change in maximal binding. The plot of apparent dissociation constants (Kd') against PIP2 concentration was linear over a range of 0.01-1 mol % with a Ki of 0.043 mol % and confirmed the competitive nature of inhibition between PDBu and PIP2. Competition between PIP2 and phorbol ester could be demonstrated in a liposomal assay system also. These results indicate that PIP2, DG, and phorbol ester all compete for the same activator-receiving region on the regulatory moiety of protein kinase C, and they lend support to the suggestion that PIP2 is a primary activator of the enzyme.     

Phosphatidylinositol 4,5-bisphosphate regulates two steps of homotypic vacuole fusion

Yeast vacuoles undergo cycles of fragmentation and fusion as part of their transmission to the daughter cell and in response to changes of nutrients and the environment. Vacuole fusion can be reconstituted in a cell free system. We now show that the vacuoles synthesize phosphoinositides during in vitro fusion. Of these phosphoinositides, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) are important for fusion. Monoclonal antibodies to PI(4,5)P(2), neomycin (a phosphoinositide ligand), and phosphatidylinositol-specific phospholipase C interfere with the reaction. Readdition of PI(4, 5)P(2) restores fusion in each case. Phosphatidylinositol 3-phosphate and PI(3,5)P(2) synthesis are not required. PI(4,5)P(2) is necessary for priming, i.e., for the Sec18p (NSF)-driven release of Sec17p (alpha-SNAP), which activates the vacuoles for subsequent tethering and docking. Therefore, it represents the kinetically earliest requirement identified for vacuole fusion so far. Furthermore, PI(4,5)P(2) is required at a step that can only occur after docking but before the BAPTA sensitive step in the latest stage of the reaction. We hence propose that PI(4,5)P(2) controls two steps of vacuole fusion.     

Phosphatidylinositol 4,5-bisphosphate formation in rabbit skeletal and heart muscle membranes

Incubation of rabbit skeletal muscle microsomes or isolated triads with gamma 32P-ATP/Mg2+ in the absence and in the presence of added phosphatidylinositol resulted in the formation of phosphatidylinositol 4-phosphate catalyzed by phosphatidylinositol kinase. When phosphatidylinositol 4-phosphate was added as exogenous substrate, phosphatidylinositol 4,5-bisphosphate was also formed demonstrating the presence of a membrane bound phosphatidylinositol 4-phosphate kinase. Triads were broken mechanically in a French press and separated on a continuous sucrose gradient. Incubation of these fractions with gamma 32P-ATP/Mg2+ resulted in a rapid labeling of phospholipid in a membrane fraction banding between transverse tubules and the terminal cisternae. Partial triad breakage and triad reformation experiments indicated that this phosphatidylinositol kinase was associated with T-tubules. When exogenous phosphatidylinositol 4-phosphate was employed as substrate phosphatidylinositol 4,5-bisphosphate and phosphatidic acid were formed, indicating the presence of all the enzymes of the polyphosphoinositide signaling system in this special membrane fraction. In contrast, heart muscle microsomes or plasma membranes can catalyze this reaction sequence from endogenous formed phosphatidylinositol 4-phosphate.     

Phosphatidylinositol 4,5-bisphosphate stimulates phosphorylation of the adaptor protein Shc by c-Src

The adaptor protein Shc was prepared as glutathione S-transferase fusion proteins (GST–Shc) and used as in vitro substrate for c-Src. Since phosphotyrosine-binding domain of Shc has been shown to bind phosphatidyl-inositol 4,5-bisphosphate (PtdIns(4,5)P2) [Zhou et al. (1995) Nature 378, 584–592], effect of PtdIns(4,5)P2 on the phosphorylation of GST–Shc by c-Src was examined. PtdIns(4,5)P2 stimulated the phosphorylation of GST–Shc without any effect on the c-Src activity as judged by both its autophosphorylation and phosphorylation of exogenous substrate, Cdc2 peptide. On the other hand, phosphatidylserine, phosphatidic acid, phosphatidylinositol, and phosphatidylinositol 4-phosphate but not phosphatidylcholine stimulated the c-Src activity itself. K_m for GST–Shc in the presence of 1 μM PtdIns(4,5)P2 was calculated to be 90 nM. The PtdIns(4,5)P2-dependent phosphorylation of GST–Shc was inhibited by a GST–fusion protein containing the phosphotyrosine-binding domain of Shc. These results suggest that PtdIns(4,5)P2 can act as a regulator of phosphorylation of Shc by c-Src through its binding to Shc.     

Phosphatidylinositol 4,5-bisphosphate may represent the site of release of plasma membrane-bound calcium upon stimulation of human platelets

Thrombin stimulation of human blood platelets caused an extensive (up to 45%) and rapid (5-10 s) decline in endogenous phosphatidylinositol 4,5-bisphosphate (PI-P2). Thrombin initiated an equally rapid loss of membrane-bound Ca, as indicated by the decrease in fluorescence of chlortetracycline (CTC)-loaded platelets. PI-P2 breakdown also correlated with decreased CTC fluorescence upon use of other platelet stimuli: Arachidonate caused moderate and slow decreases in both PI-P2 and CTC fluorescence, while ionophore only induced minimal changes. Thrombin-induced decreases in PI-P2 content could account for release of sufficient membrane-bound Ca to raise cytoplasmic free [Ca2+] to 1-2 microM, supporting the hypothesis that PI-P2 represents the Ca-binding site involved in the stimulus-dependent increase in cytoplasmic Ca2+ evoked by receptor-ligand interactions.     

Phosphatidylinositol 4,5-bisphosphate promotes both [3H]-noradrenaline and [14C]-glutamate exocytosis from nerve endings

Inhibition of phosphatidylinositol 4-phosphate (PI4P) and phosphatidylinositol 4,5-bisphosphate (PI4,5P(2)) synthesis by phenylarsine oxide (PAO) inhibits both [3H]-noradrenaline ([3H]-NA) and [14C]-glutamate ([14C]-glu) exocytosis from streptolysin-O (SLO)-perforated synaptosomes. When PI4,5P(2) is blocked by an antibody or chelated by neomycin, neurotransmitter exocytosis again is inhibited. Also, when phosphoinositide (PI) synthesis is indirectly decreased by shunting phosphatidic acid (PA) synthesis into phosphatidylbutanol production, both [14C]-glutamate and [3H]-noradrenaline exocytosis are inhibited. All of these results indicate that PI4,5P(2) is necessary for exocytosis of both synaptic vesicles (SVs) and dense core vesicles (DCVs).