Phospholipase C (cid:2) 2 Activation Redirects Vesicle Trafficking by Regulating F-actin *

Background: Ca 2 (cid:2) and PI(4,5)P 2 regulate F-actin and vesicle exocytosis in neuroendocrine cells. Results: Phospholipase C (cid:2) 2 knockdown inhibits Ca 2 (cid:2) -stimulated PI(4,5)P 2 hydrolysis, F-actin disassembly, and vesicle recruitment in PC12 cells. Conclusion: Phospholipase C (cid:2) 2, which localizes with actin, links Ca 2 (cid:2) rises to F-actin disassembly and vesicle trafficking. Significance: The results reveal a new role for phospholipase C (cid:2) 2 as a Ca 2 (cid:2) -dependent regulator of actin cytoskeletal dynamics and vesicle trafficking. PI(4,5)P 2 localizes to sites of dense core vesicle exocytosis in neuroendocrine cells and is required for Ca 2 (cid:3) -triggered vesicle exocytosis, but the impact of local PI(4,5)P 2 hydrolysis on exocytosis is poorly understood. Previously, we reported that Ca 2 (cid:3) dependent activation of phospholipase C (cid:2) 2 (PLC (cid:2) 2) catalyzes PI(4,5)P 2 hydrolysis, vesicle exocytosis regulating

Neuroendocrine cells possess a plasma membrane-resident pool of vesicles that undergo exocytosis in response to Ca 2ϩ rises. Cytoplasmic vesicles are also recruited to the plasma membrane for exocytosis during stimulation (33,34). We found that varying Ca 2ϩ influx in PC12 cells markedly affected whether resident or recruited vesicles undergo exocytosis. Stronger depolarization stimulated more Ca 2ϩ entry that uniquely promoted PI(4,5)P 2 hydrolysis and F-actin disassembly, which in turn enhanced exocytosis of cytoplasmic vesicles arriving during stimulation. PLC2 was the critical link between increased Ca 2ϩ and PI(4,5)P 2 hydrolysis, F-actin disassembly, and redirected vesicle exocytosis. These studies reveal a functional role for PLC2 as a Ca 2ϩ -dependent regulator of the actin cytoskeleton and the secretory pathway in neuroendocrine cells.
Antibodies and Reagents-Anti-mouse PLC2 polyclonal antibody was kindly provided by K. Fukami, anti-PLC␦1 (D-7) mouse monoclonal antibody was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX), and anti-GAPDH monoclonal antibody was purchased from Ambion (Austin, TX). Fluo-4 AM and Alexa Fluor 568 phalloidin were purchased from Molecular Probes, Inc. (Eugene, OR). Other materials and chemicals were obtained from commercial sources.
Cell Culture and Transfection-PC12 cells were cultured in Dulbecco's modified Eagle's medium (Sigma) supplemented with 5% horse serum and 5% calf serum at 37°C in an air plus 10% CO 2 atmosphere at constant humidity. Transfections for plasmid DNAs were performed by electroporation using an ECM 830 system (BTX, Holliston, MA) set at 230 V, 8 ms, and 1 pulse. PC12 cells (grown to ϳ80% confluence in a 10-cm dish) suspended in 0.5 ml of cytomix buffer (25 mM HEPES, 120 mM KCl, 10 mM KH 2 PO 4 , 0.15 mM CaCl 2 , 5 mM MgCl 2 , 2 mM EGTA, pH 7.6) were transfected with 10 -50 g of plasmid DNA(s) using a 4-mm gap size cuvette. Transfections for siRNAs were performed by electroporation using an ECM830 set at 90 V, 8 ms, and 1 pulse. PC12 cells were transfected with 1.33 M siRNA and 2.5 g of plasmid DNA using a 1-mm gap size cuvette.
Monitoring of DAG Generation on the Plasma Membrane-PC12 cells were transfected with 40 g of C1-mKate2 plasmid DNA or co-transfected with 25 g of C1-mKate2 and 25 g of EGFP, EGFP-PLC␦1, or EGFP-PLC2 plasmid DNAs and plated on poly-D-lysine-coated (Sigma) and type I collagencoated (BD Biosciences) 35-mm glass bottom dishes (MatTek Corp., Ashland, MA). After a 48-h incubation, the culture medium was replaced with basal buffer (15 mM HEPES, pH 7.4, 145 mM NaCl, 5.6 mM KCl, 2.2 mM CaCl 2 , 0.5 mM MgCl 2 , 5.6 mM glucose, 0.5 mM ascorbic acid, 0.1% BSA), and then cells were stimulated with 56 (moderate stimulation; MS) and 95 mM K ϩ (strong stimulation; SS) depolarization buffer (basal buffer adjusted to 95 mM NaCl and 56 mM KCl or 56 mM NaCl and 95 mM KCl). Cells were imaged on a Nikon total internal reflection fluorescence (TIRF) microscope evanescent wave imaging system used with a TE2000-U inverted microscope (Nikon) and an Apo TIRF ϫ100, numerical aperture 1.45 (Nikon) objective lens. EGFP and mKate2 fluorescence were excited with the 488-nm laser line and the 514-nm laser line, respectively. Images were acquired at 250-ms intervals with a CoolSNAP-ES digital monochrome CCD camera system (Photometrics, Tucson, AZ) controlled by Metamorph software (Universal Imaging Corp., Downingtown, PA). All data analysis was conducted with ImageJ software.
TIRF Analysis of BDNF-EGFP Secretion-PC12 cells were transfected with 30 g of BDNF-EGFP plasmid DNA and plated on poly-D-lysineand collagen-coated 35-mm glass bottom dishes. After a 48-h incubation, the culture medium was replaced with basal buffer, and cells were stimulated with MS or SS buffer. Cells were imaged on the Nikon TIRF microscope at 250-ms intervals with a CoolSNAP-ES digital monochrome CCD camera system (Photometrics) controlled by Metamorph software (Universal Imaging Corp.). The penetration depth (1/e) of the evanescent field was estimated to be 160 nm based on a calibration with fluorescent beads. At this penetration depth, resident vesicles (estimated d ϭ 100 nm) at the plasma membrane are evident, whereas vesicles enmeshed deeper in the 400-nm actin cortex (34), termed non-resident cytoplasmic vesicles, are dim or not evident. Exocytic events were manually counted and scored for whether exocytosis occurred from vesicles resident in the evanescent field for Ն0.5 s (resident) or Ͻ0.5 s (non-resident) prior to fusion. All data analysis used Metamorph software.
Ca 2ϩ Imaging-PC12 cells were plated on poly-D-lysine-and collagen-coated 35-mm glass bottom dishes. After a 24-h incubation, cells were washed, and the culture medium was replaced with basal buffer. Cells were loaded with fluo-4 by incubation with 2 M fluo-4, AM and 0.02% Pluronic F-127 (Molecular Probes) mixture at room temperature for 30 min in the dark. Cells were then washed with basal buffer and incubated at 37°C for 20 min to allow de-esterification of loaded dye in basal buffer. Cells were stimulated with MS or SS buffer, and images were acquired at 250-ms intervals on an epifluorescence microscope (Nikon). Cells were treated with 5 M ionomycin and 5 mM EGTA to obtain maximum (f max ) and minimum (f min ) fluorescence values, respectively. Average fluorescence intensity at each time point (f t ) was measured using Metamorph software. Relative fluorescence intensity of fluo-4-Ca 2ϩ (F) and concentration of intracellular Ca 2ϩ ([Ca 2ϩ ] i ) were determined as F ϭ Knockdown of PLC2 by shRNA and siRNA-PC12 cells were co-transfected with 30 g of pSM2-PLC2 shRNA vector; V2MM_89060 (shRNA 1) and V2MM_197066 (shRNA 2) targeting mouse PLC2 mRNA (accession number NM_ 001113360) sequence corresponding to nucleotides 2117-2135 (CCCTCTCGGACCTAGTGAA) and 2119 -2137 (CTCTCG-GACCTAGTGAAAT), respectively (Open Biosystems, Huntsville, AL); or pSM2 empty vector (Open Biosystems) and 10 g of C1-mKate2 plasmid DNAs and plated on poly-D-lysineand collagen-coated 35-mm glass bottom dishes for TIRF analysis and 6-well dishes for Western blotting. After a 72-h incubation, cells were lysed and subjected to blotting analysis with anti-PLC2 polyclonal antibody and anti-GAPDH monoclonal antibody. For rescue experiments, PC12 cells were triple-transfected with 30 g of pSM2-PLC2 shRNA or pSM2 empty vector, 10 g of C1-mKate2 plasmid DNA, and 5 g of EGFP-PLC2 3M plasmid DNA. Monitoring of DAG generation was performed as described above. PC12 cells were co-transfected with 35 g of pSM2-PLC2 shRNA or pSM2 empty vector and 15 g of BDNF-EGFP plasmid DNAs and plated on poly-D-lysineand collagen-coated 35-mm glass bottom dishes for TIRF analysis and a 6-well dish for blotting. After a 72-h incubation, blotting analysis and TIRF analysis were performed as described above. Knockdown of PLC2 by endoribonuclease-prepared siRNA (target sequence corresponds to nucleotides 1406 -1942 of mRNA isolated from PC12 cells) was performed as described previously (9).
Co-localization Analysis-Co-localization analysis of signals corresponding to PLC2 and F-actin was performed using a pixel by pixel analysis algorithm with Fiji-win32 software. The calculated percentage of random co-localization was subtracted from co-localization values.
Quantification of Cortical F-actin-EGFP-, EGFP-PLC␦1-, and EGFP-PLC2-overexpressing and PLC2 knockdown (EGFP co-transfection marker indicated that transfection efficiency was 82%) PC12 cells were plated on poly-D-lysineand collagen-coated 35-mm glass bottom dishes. After a 48-h (or 72-h for PLC2 knockdown) incubation, cells were treated with MS or SS buffers at room temperature for 0.5, 1, 2, and 3 min. After treatment, the cells were immediately fixed with 3.7% formaldehyde in PBS at room temperature for 8 min and permeabilized by incubation with 0.1% Triton X-100 in PBS containing 1% BSA in PBS at room temperature for 10 min. F-actin was visualized with Alexa Fluor 568-phalloidin at room temperature for 20 min followed by washing. Cells were imaged on a TIRF microscope with a CoolSNAP-ES digital monochrome CCD camera system controlled by Metamorph software, and data analysis utilized Metamorph software.

Results
Strong Stimulation Promotes Greater Ca 2ϩ Rises and PI(4,5)P 2 Hydrolysis-PI(4,5)P 2 is required for regulated vesicle exocytosis and is distributed in membrane domains present at sites of exocytosis (8,9,19). To determine the impact of PI(4,5)P 2 hydrolysis on regulated vesicle exocytosis, we utilized a range of stimulation conditions in the well characterized PC12 cell model for neuroendocrine secretion. TIRF microscopy was used to monitor the exocytosis of vesicles containing fluorescent cargo proteins (35,36) and to detect PI(4,5)P 2 hydrolysis and DAG generation (9,37). Depolarizing the cells by incubation in high KCl buffers promotes depolarization and Ca 2ϩ influx. PC12 cells have a low density of L-type Ca 2ϩ channels so that substantial depolarization in KCl buffers is required to elicit Ca 2ϩ increases (38,39). We found that 56 mM KCl (MS) was optimal for promoting maximal number of dense core vesicle fusion events of BDNF-EGFP within 3 min (Fig. 1A). Stronger depolarization at 95 mM KCl (SS) did not further increase the number of fusion events (Figs. 1A and 3C). Analysis of the time courses of averaged cumulative fusion events that fit well to an exponential function indicated that the time course of SS-evoked fusion events tended to be slower than MS-evoked fusion events (time constant values for MS and SS were 19.7 Ϯ 2.3 s and 29.5 Ϯ 4.9 s, respectively, p ϭ 0.09). MS conditions promoted a peak rise in [Ca 2ϩ ] to ϳ400 nM, whereas stronger depolarization with SS conditions evoked a peak [Ca 2ϩ ] rise to ϳ800 nM (Fig. 1B), similar to a previous report (39). The results indicate that Ca 2ϩ -dependent vesicle exocytosis is saturated by the Ca 2ϩ concentration rises elicited by MS conditions. In subsequent studies, we utilized MS and SS conditions to promote optimal (ϳ400 nM) or greater than optimal (ϳ800 nM) Ca 2ϩ rises, respectively.
Under the MS and SS buffer stimulation conditions, lipid signaling events differed markedly. A fluorescent PKC␦-C1-mKate2 probe was used to detect DAG generation in the plasma membrane by TIRF microscopy (37). Under MS conditions, where vesicle exocytosis was maximally stimulated (Fig.  1A), plasma membrane DAG levels did not differ from those in unstimulated cells (Fig. 1, C and D). By contrast, SS conditions resulted in a rapid (ϳ15 s), transient increase in DAG levels, as inferred from the translocation of the C1-mKate2 protein ( Fig.  1, C and D). The translocated C1-mKate2 was evident as bright puncta and diffuse fluorescence (Fig. 1C), which suggested that high concentration domains of DAG may be generated from high concentration domains of PI(4,5)P 2 (9) followed by diffusion. Using a PLC␦4-PH-mKate2 domain probe with TIRF (9), we detected a corresponding partial loss of PI(4,5)P 2 from the plasma membrane under SS but not under MS buffer conditions (Fig. 1E). The results indicate that PI(4,5)P 2 hydrolysis with DAG generation was only promoted at Ca 2ϩ concentrations higher (SS conditions) than those needed to maximally stimulate vesicle exocytosis (MS conditions).

PLC2 Mediates PI(4,5)P 2 Hydrolysis at Elevated Ca 2ϩ
Levels-The greater Ca 2ϩ elevation under SS conditions probably stimulated DAG generation from the Ca 2ϩ -dependent activation of a PI(4,5)P 2 -hydrolyzing PLC. Because PLC␦1 and PLC2 are expressed in PC12 cells (data not shown) (40) and are strongly activated by Ca 2ϩ (21), we determined which if either was responsible for DAG generation in response to Ca 2ϩ elevations under SS conditions. We first determined the effect of PLC␦1 and PLC2 overexpression on DAG generation. A PKC␦-C1-mKate2 probe that monitored DAG was translocated to the plasma membrane in EGFP-expressing control cells under SS conditions but not under MS conditions (Fig. 2, A (top) and B), whereas expression of a EGFP-PLC2 protein enhanced DAG generation even under MS conditions (Fig. 2, A (middle) and B). This contrasted with cells expressing EGFP-PLC␦1 (Fig. 2, A (bottom panels) and B), where there was no DAG generation beyond that of control cells. These findings indicate that PLC2 rather than PLC␦1 responds to Ca 2ϩ influx by generating DAG in PC12 cells, which was consistent with in vitro studies indicating the greater Ca 2ϩ sensitivity for PLC2 activation compared with PLC␦1 (41).
To determine whether endogenous PLC2 was responsible for DAG generation in control cells under SS conditions, we utilized shRNA plasmids and siRNA that were effective in reducing PLC2 by more than 90 and 80%, respectively (Fig. 2,  C and D). Depletion of PLC2 did not affect the expression level of PLC␦1 (Fig. 2E). In cells depleted of PLC2 by either shRNA plasmid or siRNA, DAG generation in response to SS buffer was completely abolished (Fig. 2, F-H). Both shRNA plasmids, but not a control plasmid, had similar effects (data not shown). To confirm that the loss of DAG generation in PLC2-depleted cells was due to the lack of PLC2, we conducted rescue experiments with an shRNA-resistant EGFP-PLC2 construct (EGFP-PLC2 3M) that contained three nucleotide substitutions in the shRNA target. Expression of EGFP-PLC2 3M restored DAG generation (Fig. 2, F and G). These findings indicate that PLC2 is the major PLC in PC12 cells that is activated at the plasma membrane by the higher . E, plasma membrane PI(4,5)P 2 levels in PC12 cells incubated in MS and SS buffers. Cells expressing PLC␦4-PH-mKate2 were incubated in MS or SS buffers for the times indicated and imaged by TIRF microscopy at 4 Hz. Representative images (top) indicate a transient decrease in PI(4,5)P 2 levels in cells incubated with SS but not MS buffers. The relative fluorescence of cell footprints was quantitated at 10-s intervals (mean Ϯ S.E., n ϭ 8 cells; *, p Ͻ 0.005; **, p Ͻ 0.05). The PI(4,5)P 2 decrease in SS buffers was partial. NOVEMBER  Strong Stimulation Shifts Exocytosis from Docked to Newly Arrived Vesicles-PI(4,5)P 2 is required for dense core vesicle exocytosis (3,18,42). However, the SS conditions that promoted PI(4,5)P 2 hydrolysis by PLC2 did not reduce the total number of exocytic events in 5 min (Fig. 3C). This is accounted for by the fact that PI(4,5)P 2 hydrolysis is partial under SS stimulation conditions (Fig. 1E) and that the DAG generated (Fig.  1D) further activates ubMunc13-2 (9). To determine more fully the impact of PI(4,5)P 2 hydrolysis on Ca 2ϩ -triggered vesicle exocytosis, we examined individual exocytic events by TIRF microscopy. As previously characterized for PC12 cells (34,36,43), two types of evoked vesicle exocytic events were observed: from resident vesicles present at the plasma membrane for Ն0.5 s before fusion or from non-resident cytoplasmic vesicles that arrived Ͻ0.5 s before fusion in stimulated cells (Fig. 3A). The vesicle exocytosis assay utilized in TIRF studies employing BDNF-EGFP cargo has been extensively characterized (35). Resident and non-resident vesicles that fuse are readily distin-guished from non-fusing, vesicles as shown by the traces of fluorescent changes in Fig. 3B. Fusing vesicles exhibit hallmark features of an initial brightening upon fusion pore formation (due to vesicle pH change) followed by slow dimming as the fusion pore closes and the vesicles re-acidify in cavicapture exocytosis (Fig. 3B, left). In accord with this, a similar brightening was obtained by neutralizing vesicle pH by treatment with 50 mM NH 4 Cl (Fig. 3B, left, arrow). The initial fluorescence of resident vesicles prior to fusion is greater than that for non-resident vesicles, which is at background (Fig. 3, A and B (left)). By contrast, non-fusing vesicles that approach the plasma membrane and dock or approach the plasma membrane and leave lack a fusion spike (Fig. 3B, right). Such vesicles are rare in stimulated cells and are readily distinguished from fusing vesicles. Under MS stimulation conditions, ϳ70% of exocytic events occurred from resident vesicles and ϳ30% from nonresident vesicles (Fig. 3D), similar to previous studies (36,43). By contrast, under SS stimulation conditions, the number of exocytic events from resident vesicles decreased, and those from non-resident vesicles increased (Fig. 3D), resulting in ϳ40% of exocytic events from resident vesicles and ϳ60% from newly arrived non-resident vesicles. We analyzed the cumulative time courses for each type of fusion event evoked under MS and SS conditions. In both MS and SS conditions, the time course of resident versus non-resident vesicle fusion events did not differ significantly ( value for MS-resident versus non-resident: 16 (Fig. 3E). However, the time course of resident vesicle fusion events under SS conditions ( ϭ 26.93 Ϯ 3.5 s) was significantly (p Ͻ 0.05, n ϭ 10) slower than that under MS condition ( ϭ 16.1 Ϯ 1.5 s). Increased Ca 2ϩ influx under SS conditions favored the fusion of newly arriving vesicles and reduced the number of and slowed down resident vesicle fusion events. Most fusion events occurred within 1 min after stimulation under both MS and SS conditions (Fig. 3, F and G).

PLC2 Activation Switches Exocytosis from Docked to Newly
Arrived Vesicles-Because PLC2 activation was restricted to SS stimulation conditions, we determined whether PLC2 was essential for switching the pathway for vesicle exocytosis from resident to non-resident vesicles. PLC2 knockdown did not affect the density of resident vesicles evident in the evanescent field (Fig. 4A). In both control and PLC2 knockdown cells, the number of total exocytic events was similar under MS and SS conditions (Fig. 4B, bar 1 versus bar 2 and bar 3 versus bar 4), although there was a trend for PLC2 knockdown to increase the total number of exocytic events under SS conditions (Fig.  4B, bar 1 versus bar 3 and bar 2 versus bar 4; not significant), as expected for increased PI(4,5)P 2 levels in the cells (18,19,44). The pathway for vesicle exocytosis shifted from resident to non-resident vesicles under SS stimulation conditions in control cells (Fig. 4C). However, in PLC2 knockdown cells, the shift from resident to non-resident vesicle exocytosis under SS conditions failed to occur (Fig. 4C). Similar results were obtained in PLC2 siRNA knockdown cells (Fig. 4D). These results establish that PLC2 activation is responsible for the shift in the exocytic pathway from resident to non-resident vesicles promoted by greater elevations in Ca 2ϩ . F-actin Disassembly Is Necessary for the Shift in the Exocytic Pathway-Cortical F-actin acts as a barrier or cage to restrict vesicle access to the plasma membrane for fusion (25,26). Because PI(4,5)P 2 regulates the actin cytoskeleton during vesicle exocytosis (45,46), we determined whether PLC2-catalyzed PI(4,5)P 2 hydrolysis caused disassembly of F-actin, which would increase the access of non-resident vesicles to the plasma membrane. The assembled state of cortical F-actin was assessed by fluorescent phalloidin staining of cells viewed by TIRF microscopy. The assembled state of cortical F-actin did not significantly change after 0.5, 1, 2, or 3 min after application of MS buffer. By contrast, in SS buffer, cortical F-actin strongly decreased by 0.5 min (ϳ42%) and 1 min (ϳ62%) (Fig. 5, A and B) but reassembled by 2-3 min (Fig. 5B). The F-actin dynamics correlated with the dynamics of PI(4,5)P 2 hydrolysis/DAG generation (Fig. 1, C-E). Moreover, non-resident vesicle fusion events were completed within this period (Fig. 3, E-G). The data suggest that PI(4,5)P 2 hydrolysis-dependent cortical F-actin disassembly might enable non-resident vesicle fusion events under SS condition.
Pharmacological treatments were used to determine whether F-actin disassembly was necessary or sufficient to shift the exocytic pathway from resident to non-resident vesicles. Treatment with jasplakinolide, an F-actin-stabilizing drug, inhibited fusion events under both MS and SS conditions (Fig.  5C, bar 1 versus bar 3 and bar 2 versus bar 4). In control cells (ϪJasp), the number of exocytic events from resident vesicles was reduced, and that from non-resident vesicles was increased under SS conditions (Fig. 5D, bars 1-4). Treatment with jasplakinolide (ϩJasp) mainly suppressed exocytic events from non-resident vesicles, especially under SS conditions (Fig. 5D, bars [5][6][7][8]. These data suggested that stabilization of F-actin by jasplakinolide preferentially affected the exocytosis of non-resident vesicles, thereby preventing an increase under SS conditions. Conversely, we promoted F-actin disassembly by treatment with halichondramide (Hali), an actin filament-severing and capping drug (47). Under MS conditions, halichondramide treatment increased the number of non-resident vesicle exocytic events without affecting the number of resident events (Fig. 5E). Halichondramide treatment mimicked the effect of SS conditions in enhancing non-resident exocytic events under MS condition. These data indicated that F-actin disassembly was sufficient to enhance the exocytosis of non-resident vesicles. Overall, the opposite effects of jasplakinolide and halichondramide treatment were consistent with a role for F-actin disassembly in enabling the fusion of non-resident vesicles.
PLC2 Regulates F-actin Disassembly-The previous results indicated that PLC2 activation shifts exocytosis from resident to non-resident vesicles and that F-actin disassembly was in part responsible for the shift. To determine the relationship of PLC2 to cortical F-actin, co-localization studies were conducted. TIRF microscopy revealed that EGFP-PLC2 was distributed in punctate and in filamentous structures, with the latter co-localizing with F-actin (Fig. 6, A and B). Because PLC2 was reported to localize to the plasma membrane by binding to PI(4,5)P 2 via its PH domain (41,48), the observed filamentous distribution of EGFP-PLC2 (Fig. 6A) could result from binding to PI(4,5)P 2 -rich membrane domains that co-localize along F-actin filaments. Alternatively, the filamentous distribution might correspond to direct binding of EGFP-PLC2 to F-actin or to actin-binding proteins. To distinguish these alternatives, we determined the localization of EGFP-PLC2 following treatment with latrunculin A to disassemble F-actin. Latrunculin A treatment effectively disassembled F-actin (Fig. 6C) and eliminated the filamentous distribution of EGFP-PLC2 (Fig. 6D) detected by TIRF microscopy. Latrunculin treatment produced small structures and puncta of EGFP-PLC2 with some nearly residual actin-containing structures (Fig. 6D, arrows). By contrast, the PH domain of PLC2 (EGFP-PLC2-PH), which localized to the plasma membrane similarly to full-length EGFP-PLC2 by epifluorescence (Fig. 6E), localized rather differently into broadly distributed small puncta rather than filaments by TIRF microscopy, and this distribution was not altered by latrunculin A treatment (Fig. 6F). The results suggest that the filamentous distribution of EGFP-PLC2 near the plasma membrane is mediated by a direct interaction with F-actin or actin-binding proteins utilizing a domain of PLC2 other than its PH domain.
To further link the activation of PLC2 to the state of F-actin assembly, we determined the effect of PLC overexpression on F-actin disassembly. In control EGFP-expressing cells, cortical F-actin visualized by fluorescent phalloidin was disassembled when cells were incubated under SS but not MS conditions (Fig. 7, A and D). Cells expressing EGFP-PLC␦1 exhibited changes very similar to control cells (Fig. 7,  B and D). By contrast, overexpression of EGFP-PLC2 resulted in the disassembly of cortical F-actin even under MS conditions (Fig. 7, C and D), which was similar to the results for DAG generation (Fig. 2, A and B). Last, to confirm that PLC2 activation affects F-actin disassembly, we determined the impact of PLC2 knockdown. In control cells, cortical F-actin was disassembled under SS but not MS conditions (Fig. 7, E and F). By contrast, neither MS nor SS conditions induced cortical F-actin disassembly in PLC2 knockdown cells (Fig. 7, E and F). The results indicate that PLC2 activation promotes F-actin disassembly.

Discussion
Studies in many cell types have described the important role of PI(4,5)P 2 in enabling F-actin assembly (24). In addition, multiple roles for F-actin in vesicle trafficking and exocytosis have been characterized for neuroendocrine cells (49). It has been shown that increases in cytoplasmic Ca 2ϩ trigger F-actin disassembly (25, 28, 49 -52), which increases the access of cytoplasmic recruitment vesicles to the plasma membrane for fusion (33,34). The current study reveals that PLC2 is a critical link between Ca 2ϩ rises and the disassembly of the F-actin cytoskeleton for regulating vesicle trafficking to the plasma membrane for fusion.
PLC2 is mainly expressed in neural and endocrine secretory cells, but a functional cellular role for the enzyme has not previously been characterized (41,53,54). The strong Ca 2ϩ dependence of the activation of PLC2 suggested that it was a Ca 2ϩdependent effector for unidentified neural/endocrine processes (41). G␤␥ subunits also activate PLC2, which could indicate receptor-regulated roles for this enzyme as well (55). However, PLC2 knock-out mice did not exhibit obvious phenotypes that would suggest a functional role (56). By contrast, the loss of PLC2 in PC12 cells attenuated the evoked fusion of vesicles recruited to the plasma membrane under enhanced Ca 2ϩ influx conditions. PLC2 was activated at cytoplasmic Ca 2ϩ levels (ϳ800 nM) greater than those required to elicit maximal vesicle exocytosis (ϳ400 nM) in PC12 cells, which corresponds closely to the observed in vitro Ca 2ϩ -dependent acti-  NOVEMBER 27, 2015 • VOLUME 290 • NUMBER 48

JOURNAL OF BIOLOGICAL CHEMISTRY 29017
vation of PLC2 but not PLC␦1 (41). The hydrolysis of PI(4,5)P 2 with DAG generation, F-actin disassembly, and the increased exocytosis of non-resident vesicles was also only evident at the higher Ca 2ϩ concentrations. Extrapolation of these results into the nervous system suggests that loss of PLC2 could slow neuropeptide secretion under conditions of high demand (e.g. sustained or high frequency stimulation), where higher Ca 2ϩ levels are attained. A phenotype in the PLC2 knock-out mouse (56) may only be evident under such conditions of stress. The current study indicates a functional role for PLC2 as a Ca 2ϩ -dependent effector that regulates vesicle trafficking through its hydrolysis of PI(4,5)P 2 and consequent remodeling of the actin cytoskeleton. The unanticipated localization of PLC2 to F-actin supports such a role. Compared with MS conditions, SS conditions uniquely activated PLC2, PI(4,5)P 2 hydrolysis, and F-actin disassembly and promoted a shift in the exocytic pathway toward new arriving vesicles without markedly altering the total number of vesicle exocytic events. PLC2 activation both inhibited the exocytosis of resident vesicles and facilitated the exocytosis of vesicles trafficking to the plasma membrane during stimulation, as shown by PLC2 knockdown. PLC2 activation was also responsible for the F-actin disassembly promoted under SS conditions. However, unlike the effects of F-actin disassembly promoted by PLC2 activation, halichondramide treatment mainly affected the fusion of non-resident vesicles without affecting the fusion of resident vesicles. The partial inhibition of resident vesicle fusion from PI(4,5)P 2 hydrolysis might result from the inhibition of the local F-actin assembly, mediated by actin-associated proteins such as N-WASP and Arp2/3, that enhances vesicle exocytosis (57,58). Because halichondramide causes F-actin disassembly without PI(4,5)P 2 hydrolysis, it is possible that PI(4,5)P 2 -dependent F-actin assembly still occurs to support resident vesicle fusion.
The facilitation of the exocytosis of newly recruited vesicles by PI(4,5)P 2 hydrolysis is readily understood as a consequence  . C, effect of latrunculin A (Lat A) on cortical F-actin. Cells were incubated with DMSO or with 1 M latrunculin A at 37°C for 5 min and fixed, permeabilized, and stained with Alexa Fluor 568 phalloidin for imaging by TIRF microscopy. D, effect of latrunculin A treatment on localization of EGFP-PLC2. EGFP-PLC2-expressing cells were incubated with DMSO or with 1 M latrunculin A at 37°C for 5 min and imaged by TIRF microscopy. In latrunculin A-treated cells, EGFP-PLC2 distributed to puncta and small structures that were near residual F-actin-containing structures stained with Alexa Fluor 568 phalloidin (arrows). E, the localization of EGFP-PLC2 and EGFP-PLC2-PH by epifluorescence microscopy. F, effect of latrunculin A on localization of EGFP-PLC2-PH domain. EGFP-PLC2-PHexpressing cells were incubated with DMSO or with 1 M latrunculin A at 37°C for 5 min and imaged by TIRF microscopy.
of the disassembly of a cortical F-actin meshwork that hinders vesicle access to the plasma membrane. This is consistent with the inhibition and stimulation of non-resident vesicle exocytosis by jasplakinolide and halichondramide, respectively. PI(4,5)P 2 regulates numerous actin-binding proteins, such as scinderin (also known as adseverin), gelsolin, profilin, villin, and cofilin (24). Cofilin and scinderin are sequestered by PI(4,5)P 2 and released upon PI(4,5)P 2 hydrolysis to sever F-actin filaments (31,45). Scinderin in chromaffin cells has been implicated in the Ca 2ϩ -induced disassembly of F-actin (27,52). The Ca 2ϩ -dependent activation of PLC2 and PI(4,5)P 2 hydrolysis would release bound scinderin and related proteins to promote their actin-severing activity. Although PKC activation was also reported to promote cortical F-actin disassembly in chromaffin cells (59), we found that overexpression of a phosphoinositide 5-phosphatase that hydrolyzes PI(4,5)P 2 without DAG generation closely mimicked the impact of PLC2 overexpression on vesicle exocytosis (data not shown), which suggests that it is the loss of PI(4,5)P 2 , rather than an increase in DAG that is principally responsible for the cytoskeletal remodeling and changes in vesicle trafficking in PC12 cells. It was also recently reported that PI(4,5)P 2 -dependent F-actin remodeling was required for vesicle translocation to the plasma membrane in chromaffin cells (60). PI(4,5)P 2 is required for multiple steps in vesicle exocytosis, which suggests additional roles for PLC2 as a Ca 2ϩ -dependent modulator. The priming factors CAPS and ubMunc13-2 are required for the regulated fusion of both resident and newly arrived vesicles (36). Studies in PC12 cells showed that the partial hydrolysis of PI(4,5)P 2 by PLC2 at elevated Ca 2ϩ levels was sufficient to reduce the PI(4,5)P 2 -dependent activity of CAPS while maintaining the PI(4,5)P 2 -dependent membrane recruitment of Munc13 (9). Membrane-recruited Munc13 was further activated by DAG to compensate for the loss of CAPS activity (9). DAG production at elevated Ca 2ϩ has been inferred to promote augmentation of synaptic neurotransmitter release mediated by Munc13-2 (20), but the PLC responsible for DAG generation had not been identified. Our results suggest that PLC2 may mediate synaptic augmentation promoted by elevated Ca 2ϩ . The extensive role of PI(4,5)P 2 in regulating plasma membrane-associated processes indicates that PLC2 may have other functions in response to sustained Ca 2ϩ elevations that are linked to the actin cytoskeleton, such as endocytosis, cytokinesis, and neurite outgrowth. The current work charac-  terizes one of the roles of PLC2 as a Ca 2ϩ -dependent regulator of the actin cytoskeleton and secretory pathway in neuroendocrine cells.
Author Contributions-M. Y. and T. F. J. M. conceived the study and wrote the manuscript. M. Y. and D. M. K.-G. performed the experiments, analyzed the data, and prepared the figures.