Decision letter: A positive feedback loop between Flower and PI(4,5)P2 at periactive zones controls bulk endocytosis in Drosophila (2020)

Article Figures and data Abstract Introduction Results Discussion Materials and methods Appendix 1 Data availability References Decision letter Author response Article and author information Metrics Abstract Synaptic vesicle (SV) endocytosis is coupled to exocytosis to maintain SV pool size and thus neurotransmitter release. Intense stimulation induces activity-dependent bulk endocytosis (ADBE) to recapture large quantities of SV constituents in large endosomes from which SVs reform. How these consecutive processes are spatiotemporally coordinated remains unknown. Here, we show that Flower Ca2+ channel-dependent phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) compartmentalization governs control of these processes in Drosophila. Strong stimuli trigger PI(4,5)P2 microdomain formation at periactive zones. Upon exocytosis, Flower translocates from SVs to periactive zones, where it increases PI(4,5)P2 levels via Ca2+ influxes. Remarkably, PI(4,5)P2 directly enhances Flower channel activity, thereby establishing a positive feedback loop for PI(4,5)P2 microdomain compartmentalization. PI(4,5)P2 microdomains drive ADBE and SV reformation from bulk endosomes. PI(4,5)P2 further retrieves Flower to bulk endosomes, terminating endocytosis. We propose that the interplay between Flower and PI(4,5)P2 is the crucial spatiotemporal cue that couples exocytosis to ADBE and subsequent SV reformation. Introduction Proper synaptic vesicle (SV) exocytosis dictates the robustness of brain activity. Coupling SV exocytosis with proper endocytosis is crucial for maintaining a balance of SV proteins at the release site, plasma membrane equilibrium, SV identity, and SV pool size (Chanaday et al., 2019; Haucke et al., 2011; Lou, 2018; Wu et al., 2014a). Currently, four modes of SV endocytosis are proposed. These differ in terms of stimulation intensity for their induction, formation, and molecular components (Haucke et al., 2011; Kononenko and Haucke, 2015; Wu et al., 2014a). Under mild neuronal stimulation, the SV partially fuses with the plasma membrane and reforms at the active zone, the so called ‘kiss and run’ mode. During clathrin-mediated endocytosis (CME), the SV fully collapses into the plasma membrane, followed by reformation of a single SV. Ultrafast endocytosis was also shown to recycle SVs at a sub-second timescale by forming a ~ 80 nm-sized bulk endosome predominantly at the edge of the active zone. SVs subsequently regenerate from this bulk endosome (Granseth et al., 2006; Watanabe et al., 2013a; Watanabe et al., 2013b; Zhu et al., 2009). High frequency stimulation and thus exocytosis could easily surpass the capacity of the three above-described endocytic mechanisms. It has therefore been proposed that activity-dependent bulk endocytosis (ADBE) has the necessary recapture capacity upon intense stimulation (Clayton et al., 2008; Soykan et al., 2017; Wu and Wu, 2007). This retrieval mode is elicited at the periactive zone to recapture large quantities of SV constituents via bulk endosome (~100–500 nm) formation, from which SVs subsequently reform. Hence, specific routes of SV recycling may fit the specific demands of a wide range of neuronal activities at the synapse. However, how stimulation intensity dictates the choice between these different endocytic modes is not well understood. ADBE has been documented in many different types of neurons in invertebrates and vertebrates (Clayton et al., 2008; Heerssen et al., 2008; Heuser and Reese, 1973; Holt et al., 2003; Kasprowicz et al., 2008; Kittelmann et al., 2013; Miller and Heuser, 1984; Richards et al., 2000; Soykan et al., 2017; Stevens et al., 2012; Vijayakrishnan et al., 2009; Wenzel et al., 2012; Wu and Wu, 2007; Yao et al., 2017). Although the precise mechanism regulating ADBE is not known, multiple lines of evidence suggest that actin polymerization may serve as the membrane invagination force responsible for generating bulk endosomes (Gormal et al., 2015; Holt et al., 2003; Kokotos and Low, 2015; Nguyen et al., 2012; Richards et al., 2004; Soykan et al., 2017; Wu et al., 2016). Furthermore, phosphatidylinositol metabolism has also been implicated in controlling this recycling mode (Gaffield et al., 2011; Holt et al., 2003; Richards et al., 2004; Vijayakrishnan et al., 2009). Importantly, loss of Synaptojanin, the major phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) catalytic enzyme in neurons (Tsujishita et al., 2001), leads to reduced SV endocytosis elicited by intense stimulation, presumably by affecting ADBE (Mani et al., 2007). PI(4,5)P2 is known to promote actin polymerization by activating a number of actin modulators (Janmey et al., 2018). PI(4,5)P2 is clustered to form microdomains upon the demand for diverse cellular functions (Aoyagi et al., 2005; Chen et al., 2015; Honigmann et al., 2013; Kabeche et al., 2015; Mu et al., 2018; Picas et al., 2014; Riggi et al., 2018; van den Bogaart et al., 2011). It has also been reported that formation of PI(4,5)P2 microdomains precedes actin polymerization during a process reminiscent of ADBE in neurosecretory cells (Gormal et al., 2015). Thus, subcellular compartmentalization of PI(4,5)P2 may provide the spatial information dictating where bulk membranes will invaginate. However, the mechanism initiating the formation of the PI(4,5)P2 microdomains is unknown. Coordinated SV protein and membrane retrieval plays an important role in maintaining the identity of newly formed SVs during SV recycling (Kaempf and Maritzen, 2017; McMahon and Boucrot, 2011; Saheki and De Camilli, 2012; Traub and Bonifacino, 2013). It has been well documented that proper sorting of SV proteins to the nascent SV is achieved in CME by the cooperative action of PI(4,5)P2 and adaptor protein complexes (Saheki and De Camilli, 2012; Traub and Bonifacino, 2013). Recent studies have also revealed a distinct sorting mechanism that retrieves selective SV cargoes to the bulk endosome via ADBE (Kokotos et al., 2018; Nicholson-Fish et al., 2015). Several lines of evidence further suggest that clathrin and adaptor protein complexes are required for reforming SVs from the bulk endosome (Cheung and Cousin, 2012; Glyvuk et al., 2010; Kokotos et al., 2018; Kononenko et al., 2014; Park et al., 2016). A dynaminI/dynaminIII/clathrin-independent mechanism has also been reported as being involved in this process (Wu et al., 2014c). Thus, during SV regeneration via ADBE, multiple protein sorting steps may be required to ensure that the SVs harbor the proper compositions of lipids and proteins, thereby endowing specific release probabilities in relation to other modes of endocytosis (Cheung et al., 2010; Hoopmann et al., 2010; Nicholson-Fish et al., 2015; Silm et al., 2019). The mechanism by which protein sorting and membrane retrieval are coordinated in this process remains to be explored. Here, we show that, upon intense stimulation, PI(4,5)P2 is compartmentalized into microdomains at periactive zones in the synaptic boutons of Drosophila larval neuromuscular junctions (NMJs). Blockade of PI(4,5)P2 microdomain formation diminishes ADBE and SV reformation from the bulk endosome. Increased intracellular Ca2+ and SV exocytosis are prerequisites for initiating ADBE (Morton et al., 2015; Wu and Wu, 2007). We have previously shown that Flower (Fwe), a SV-associated Ca2+ channel, regulates both CME and ADBE, and that its channel activity is strongly activated upon intense stimulation to elicit ADBE (Yao et al., 2017). We show that Fwe initiates a positive feedback loop upon PI(4,5)P2 increase to ensure the formation of PI(4,5)P2 microdomains and thus trigger ADBE and subsequent SV reformation. Intriguingly, PI(4,5)P2 also participates in retrieval of Fwe to the bulk endosome, thereby stopping membrane recycling. Hence, spatiotemporal interplays between Flower and PI(4,5)P2 coordinate the retrieval of SV cargos and membranes, coupling exocytosis to ADBE and subsequent SV reformation. Results Intense neuronal activity induces formation of PI(4,5)P2 microdomains at the presynaptic periactive zone of Drosophila synapses To investigate the dynamics of PI(4,5)P2 in the presynaptic compartment, we expressed a GFP fusion protein of the pleckstrin homology (PH) domain of PLCδ1 (PLCδ1-PH-EGFP) in synaptic boutons of Drosophila larval NMJs using nSyb-GAL4, a pan-neuronal driver. PLCδ1-PH-EGFP binds to PI(4,5)P2 with high affinity and is widely used to label subcellular compartments in which PI(4,5)P2 is enriched (Chen et al., 2014; Khuong et al., 2010). We delivered 20 Hz stimuli for three minutes to synaptic boutons in a 2 mM extracellular Ca2+ solution and performed live imaging in the third minute. Consecutive snapshot images were taken before stimulation, during the third minute of stimulation, and after stimulation. As shown in Figure 1a–b, we observed a very subtle increase in PLCδ1-PH-EGFP fluorescence in individual boutons (white arrows), similar to findings of a previous study (Verstreken et al., 2009). However, when we raised the stimulus intensity to 40 Hz, we recorded a robust increase in fluorescence relative to a GFP fusion protein of the plasma membrane-integrated mCD8 domain (UAS-mCD8-GFP). Fluorescence signals rapidly returned to basal levels within tens of seconds when the stimuli were removed. We have previously documented that treatment with 40 Hz electric pulses or 90 mM high KCl solution can cause comparable stimulation intensities in Drosophila NMJ boutons (Yao et al., 2017). High K+ treatment also increased the fluorescence signal of PLCδ1-PH-EGFP. No increase in the presynaptic protein level of PLCδ1-PH-EGFP was found under this condition (Figure 1—figure supplement 1a–b), arguing that this stimulation does not induce protein synthesis. These results suggest that, in response to intense stimulation, PLCδ1-PH-EGFP is redistributed and concentrated to PI(4,5)P2-enriched subdomains of the plasma membrane, thereby enhancing the overall fluorescence. Figure 1 with 3 supplements see all Download asset Open asset PI(4,5)P2 forms microdomains at periactive zones under conditions of intense stimulation. (a–b) Increased fluorescence of PLCδ1-PH-EGFP but not mCD8-GFP in NMJ boutons upon intense stimulation. (a) (Top) Live images of the boutons (arrows) expressing UAS-PLCδ1-PH-EGFP or UAS-mCD8-GFP. The larvae were reared at 25°C. Electrical (20 or 40 Hz) or chemical (90 mM K+) stimulation was conducted in a 2 mM-Ca2+ solution for 3 min (electrical) or 5 min (chemical) and then rested in 0 mM Ca2+ and 5 mM K+. Snapshot images taken before stimulation, at the third (electrical) and fifth (chemical) min of stimulation, and after stimulation. (Bottom) Traces of probe fluorescence for single boutons. The number of boutons imaged (N). (b) Quantification data for EGFP fluorescence change. The resting fluorescence level (F0). Fluorescence change evoked by stimulation (ΔF). (c–f) PLCδ1-PH-EGFP enrichment at periactive zones is dependent on Ca2+ upon intense stimulation. Single-plane confocal (c) or SIM (e) images of the boutons expressing PLCδ1-PH-EGFP. The larvae were reared at 25°C. The boutons subjected to high K+/2 mM Ca2+ (10 min stimulation of 90 mM K+/2 mM Ca2+), high K+/0 mM Ca2+ (10 min stimulation of 90 mM K+/0 mM Ca2+), or high K+/0.5 mM Ca2+ (1 min stimulation of 90 mM K+/0.5 mM Ca2+) treatments were fixed immediately and immunostained for PLCδ1-PH-EGFP (green) and Bruchpilot (Brp) [an active zone scaffold protein; magenta (c), red (e)]. The PLCδ1-PH-EGFP-enriched puncta (Arrows). (d) Quantification data for PLCδ1-PH-EGFP staining intensities, normalized to the value of the resting condition. (f) Average area of PLCδ1-PH-EGFP clusters in individual boutons, as measured by SIM. Individual data values are shown in graphs. p values: ns, not significant; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Mean ± SEM. Scale bar: 1 µm (e), 2 µm (a, c). Statistics: one-way ANOVA with Tukey’s post hoc test (b, d). Figure 1—source data 1 Source data for Figure 1. https://cdn.elifesciences.org/articles/60125/elife-60125-fig1-data1-v2.xlsx Download elife-60125-fig1-data1-v2.xlsx To characterize the subcellular distribution of the stimulus-dependent PI(4,5)P2 induction, we conducted a chemical fixation protocol whereby the NMJ boutons were fixed at rest or immediately after high K+ stimulation and then immunostained for PLCδ1-PH-EGFP using an α-GFP antibody to enhance the signal for high-resolution microscopic imaging. Confocal images revealed that, for neurons at rest, native PLCδ1-PH-EGFP fluorescence was weakly detected on the presynaptic plasma membrane labeled by α-Hrp staining, with some additional fluorescent signal being dispersed in the cytosol (Figure 1—figure supplement 1c). We then elevated PI(4,5)P2 on the plasma membrane by removing a copy of synaptojanin (synj), which encodes the major neuronal PI(4,5)P2 phosphatase (Tsujishita et al., 2001; Verstreken et al., 2003). Consistent with previous studies (Chen et al., 2014; Verstreken et al., 2009), this reduction in Synj levels enhanced PLCδ1-PH-EGFP fluorescence (Figure 1—figure supplement 1c–d). In this context, we did not observe a significant change in protein expression of PLCδ1-PH-EGFP in the presynaptic compartment (Figure 1—figure supplement 1e–f). By using α-GFP immunostaining, these PLCδ1-PH-EGFP signals could be faithfully amplified (Figure 1—figure supplement 1c–d). Therefore, this immunostaining approach was subsequently used to monitor presynaptic PI(4,5)P2 levels. Next, we stimulated the boutons expressing PLCδ1-PH-EGFP with 90 mM K+ and 2 mM Ca2+ for 10 min. Similar to our live-imaging results, PLCδ1-PH-EGFP signals were significantly increased on the presynaptic plasma membrane (Figure 1—figure supplement 1g–h). In particular, we observed high-level induction of PLCδ1-PH-EGFP puncta (Figure 1c–d). To assess the possibility that chemical fixation may have altered membrane properties and consequently plasma membrane PLCδ1-PH-EGFP clustering upon intense stimulation, we further examined the distributions of mCD8-GFP and PLCδ1-PHS39R-EGFP, a PI(4,5)P2-binding mutant (Khuong et al., 2010; Verstreken et al., 2009). There was no obvious change in the plasma membrane pattern of mCD8-GFP in fixed boutons stimulated with high K+ (Figure 1—figure supplement 2a–b). Unlike PLCδ1-PH-EGFP, PLCδ1-PHS39R-EGFP was found mainly in the cytosol at rest. After intense stimulation, immunostaining signals of PLCδ1-PHS39R-EGFP did not accumulate on the plasma membrane (Figure 1—figure supplement 2c–d) and were not increased within the boutons (Figure 1—figure supplement 2e). Thus, clustering of PLCδ1-PH-EGFP on the plasma membrane upon stimulation results from an increase in local PI(4,5)P2 concentration, although a potential effect of chemical fixation, if any, on PLCδ1-PH-EGFP clustering cannot be excluded. To assess potential dominant-negative effects of PI(4,5)P2 binding by PLCδ1-PH-EGFP on the stimulation-induced accumulation of PI(4,5)P2, we further examined the recruitment of the AP-2 complex by PI(4,5)P2. Similar to PLCδ1-PH-EGFP, levels of the α subunit of the AP-2 complex (AP-2α) were also increased on the plasma membrane upon high K+ stimulation (Figure 1—figure supplement 3a–b). Together, these data indicate that intense stimulation can promote the formation of PI(4,5)P2 microdomains. We noted that the induced PI(4,5)P2 microdomains were primarily sited at periactive zones marked by activity-dependent localization of Eps15 (Figure 1c; Figure 1—figure supplement 3c; Koh et al., 2007; Winther et al., 2013). Periactive zones are known hot-spots for ADBE (Chanaday et al., 2019; Kononenko and Haucke, 2015; Wu et al., 2014a). By using structured illumination microscopy (SIM), we estimated that the average size of these PI(4,5)P2 microdomains formed after stimulation is ~300 nm in diameter (Figure 1e–f) (0.07655 ± 0.004 μm2, mean ± S.E.M., n = 37 boutons). Next, to determine the Ca2+ dependence of the PI(4,5)P2 microdomains, we stimulated the boutons in a solution of 90 mM K+ and 0 mM Ca2+, which resulted in failure to induce PI(4,5)P2 microdomain formation (Figure 1d). We obtained a similar result using 1 min stimulation of 90 mM K+ and 0.5 mM Ca2+ (Figure 1d), which was previously shown to primarily elicit CME but not ADBE (Yao et al., 2017). Hence, these results suggest that intense stimulation can elicit Ca2+-driven compartmentalization of PI(4,5)P2 at the periactive zone in synaptic boutons of Drosophila NMJs. PI(4,5)P2 microdomains are involved in ADBE initiation and SV reformation from bulk endosomes Next, we investigated the function of PI(4,5)P2 microdomains in ADBE. High K+ treatment is widely used to trigger ADBE in a broad range of synapses (Akbergenova and Bykhovskaia, 2009; Clayton et al., 2008; Jin et al., 2019; Stevens et al., 2012; Vijayakrishnan et al., 2009; Wu and Wu, 2007; Wu et al., 2014c). To measure ADBE, we induced ADBE with 90 mM K+ and 2 mM Ca2+ for 10 min and then conducted transmission electron microscopy (TEM). ADBE was evoked in wild-type control boutons under these conditions and generated the formation of bulk endosomes (red asterisks, defined as >80 nm in diameter) (Figure 2c–d). To suppress the function of PI(4,5)P2, we expressed PLCδ1-PH-EGFP, anticipating that binding of the PLCδ1-PH domain to PI(4,5)P2 would restrict availability of PI(4,5)P2 to its effectors and metabolic enzymes (Figure 2a; Khuong et al., 2013). By using the GAL4/UAS system, we were able to adjust expression levels of the PLCδ1-PH domain via temperature manipulation (Brand and Perrimon, 1993; D'Avino and Thummel, 1999; Wilder, 2000). When we neuronally expressed PLCδ1-PH-EGFP using nSyb-GAL4 and grew larvae at 25°C, we found that mild expression of PLCδ1-PH-EGFP had a mild inhibitory effect on ADBE induction relative to wild-type control boutons (Figure 2c–d). In contrast, when larvae were grown at 29°C to effect a two-fold increase in PLCδ1-PH-EGFP expression (Figure 2—figure supplement 1a–b), ADBE was almost completely abolished (Figure 2c–d). However, when larvae expressing PLCδ1-PHS39R-EGFP were grown at 29°C, ADBE was not suppressed by the mutant protein (Figure 2c–d). Figure 2 with 2 supplements see all Download asset Open asset PI(4,5)P2 microdomains drive ADBE and SV reformation from bulk endosomes. Reducing PI(4,5)P2 availability suppresses ADBE and subsequent SV reformation. (a) A schematic for PI(4,5)P2 suppression by PLCδ1-PH-EGFP or Synj expression. (b) Expression of Synj reduced presynaptic plasma membrane PI(4,5)P2 upon high K+ treatment. The boutons co-expressing UAS-PLCδ1-PH-EGFP with UAS-RFP (control) or UAS-synj using nSyb-GAL4 were reared at 25°C and subjected to resting condition (10 min incubation of 5 mM K+/0 mM Ca2+) or high K+ stimulation (10 min stimulation of 90 mM K+/2 mM Ca2+), followed by α-GFP immunostaining. Single-plane confocal images of the boutons are shown in Figure 2—figure supplement 1c. Quantification data for PLCδ1-PH-EGFP staining intensity are shown, normalized to the value of the resting condition of controls. (c) TEM images of the boutons of controls (nSyb-GAL4/+ at 29°C), mild PLCδ1-PH-EGFP expression (nSyb-GAL4/UAS-PLCδ1-PH-EGFP at 25°C), high PLCδ1-PH-EGFP expression (nSyb-GAL4/UAS-PLCδ1-PH-EGFP at 29°C), high PLCδ1-PHS39R-EGFP expression (nSyb-GAL4/UAS-PLCδ1-PHS39R-EGFP at 29°C), mild Synj expression (nSyb-GAL4/UAS-synj at 25°C), or high Synj expression (nSyb-GAL4/UAS-synj at 29°C). At rest (10 min incubation of 5 mM K+/0 mM Ca2+). High K+ (10 min stimulation of 90 mM K+/2 mM Ca2+). 10 min recovery (10 min stimulation of 90 mM K+/2 mM Ca2+, followed by 10 min incubation of 5 mM K+/0 mM Ca2+). 20 min recovery (10 min stimulation of 90 mM K+/2 mM Ca2+, followed by 20 min incubation of 5 mM K+/0 mM Ca2+). Bulk endosomes (>80 nm in diameter, red asterisks). Mitochondria (mt). Quantification data for total number of bulk endosomes per bouton area (d). Individual data values are shown in graphs. p values: ns, not significant; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Mean ± SEM. Scale bar: 500 nm. Statistics: one-way ANOVA with Tukey’s post hoc test. Figure 2—source data 1 Source data for Figure 2. https://cdn.elifesciences.org/articles/60125/elife-60125-fig2-data1-v2.xlsx Download elife-60125-fig2-data1-v2.xlsx Synj comprises a central 5-phosphatase domain that specifically dephosphorylates the 5' position of PI(4,5)P2 to produce PI(4)P (McPherson et al., 1996; Woscholski et al., 1997). In addition, an N-terminal Sac1 domain converts several phosphatidylinositides—including PI(3,5)P2, PI(3)P, and PI(4)P—to PI (Figure 2a; Guo et al., 1999). Indeed, overexpression of Synj reduced the formation of the PI(4,5)P2 microdomains induced by high K+ (Figure 2b; Figure 2—figure supplement 1c). Importantly, overexpression of Synj resulted in dosage-dependent inhibition of ADBE (Figure 2c–d), similar to the effect of increasing levels of the PLCδ1-PH domain. Together, these data suggest that the PI(4,5)P2 microdomains initiate ADBE to control SV membrane retrieval upon intense stimulation. SVs regenerate from bulk endosomes within minutes of their formation (Cheung and Cousin, 2012; Glyvuk et al., 2010; Kononenko et al., 2014; Stevens et al., 2012; Wu et al., 2014c). Using an approach employed previously (Stevens et al., 2012), we treated the boutons with high K+ followed by an incubation in 5 mM K+ and 0 mM Ca2+ solution for 10 or 20 min, allowing the SVs to reform from the bulk endosomes. In controls (nSyb-GAL4), the number and area of the induced bulk endosomes reverted to almost basal levels within 10 min (Figure 2c–d; Figure 2—figure supplement 2), demonstrating proper SV reformation. Interestingly, upon expression of low levels of PLCδ1-PH-EGFP, the induced bulk endosomes remained after 10 min and 20 min recovery periods, yet ADBE was only partially impaired (Figure 2c–d; Figure 2—figure supplement 2). In contrast, SV reformation in the boutons expressing high levels of PLCδ1-PHS39R-EGFP (Figure 2c–d; Figure 2—figure supplement 2). Furthermore, we observed a loss of SV reformation upon mild overexpression of Synj (Figure 2c–d; Figure 2—figure supplement 2). Hence, the formation of PI(4,5)P2 microdomains is for both ADBE and SV reformation from the bulk endosome, with the being elicited by high levels of PI(4,5)P2. that of PI(4,5)P2 microdomain formation did not cause SV accumulation on the bulk endosome during recovery periods, that PI(4,5)P2 microdomains may an role in SV reformation from bulk endosomes. PI(4,5)P2 microdomains are via a positive feedback loop of and PI(4,5)P2 We have previously shown that the SV-associated Ca2+ channel Flower presynaptic Ca2+ levels in response to stimuli to trigger ADBE (Yao et al., 2009; Yao et al., 2017). the Ca2+ dependence of PI(4,5)P2 microdomains (Figure 1d), we that exocytosis evoked by intense stimulation Fwe clustering at periactive zones, where it may provide the Ca2+ to induce PI(4,5)P2 microdomain To test this we conducted a et al., to investigate if is a between Fwe and PI(4,5)P2 in response to stimulation. In our (Figure was expressed in a mutant to Fwe protein with a and expression of UAS-PLCδ1-PH-EGFP reported the localization of PI(4,5)P2. the and EGFP protein were used to between and PLCδ1-PH-EGFP. The signal was low in resting boutons, high K+ treatment significantly increased signal intensity (Figure 3a–b). Therefore, these results suggest that Fwe and PI(4,5)P2 are when intense stimulation PI(4,5)P2 microdomain Figure 3 Download asset Open asset Fwe and PI(4,5)P2 form a positive feedback loop to PI(4,5)P2 microdomains. (a–b) Fwe and PLCδ1-PH-EGFP upon intense stimulation. (a) A schematic for confocal images of mutant boutons co-expressing and PLCδ1-PH-EGFP in at After resting condition or high K+ stimulation, the boutons were subjected to α-GFP and for PLCδ1-PH-EGFP (green) and signals (b) Quantification data for signal intensities, normalized to the value of the resting condition. (c–f) of Fwe or its Ca2+ channel activity PI(4,5)P2 microdomain and (c) Single-plane confocal images of the boutons neuronally expressing UAS-PLCδ1-PH-EGFP in (control) or at 25°C. The boutons were subjected to resting condition or high K+ stimulation, followed by α-GFP immunostaining. Quantification data for PLCδ1-PH-EGFP staining intensity are shown, normalized to the value of the resting condition of controls (d). (e) Single-plane confocal images of the boutons neuronally expressing using in wild-type Fwe boutons in at 25°C), or boutons in at When the low expression of this was used to produce levels of PLCδ1-PH-EGFP in boutons. Quantification data for PLCδ1-PH-EGFP staining intensity are shown, normalized to the value of the resting condition of boutons PI(4,5)P2 microdomain Single-plane confocal images of the boutons co-expressing UAS-PLCδ1-PH-EGFP with UAS-RFP or using The larvae were reared at 25°C. After resting condition or high K+ stimulation, the boutons were with Quantification data for PLCδ1-PH-EGFP staining intensities, normalized to the value of the resting condition of controls. Blockade of the PI(4,5)P2 microdomains Fwe Ca2+ Snapshot Ca2+ images of the boutons (arrows) expressing using The larvae of control mutant Synj overexpression expression or mutant expressing in were reared at 25°C. was taken in the fifth minute for minute after high K+ mM Ca2+) stimulation. Quantification data for evoked Ca2+ normalized to the value of controls. Ca2+ levels are shown as the increase in fluorescence under high K+ stimulation. Individual data values are shown in graphs. p values: ns, not significant; *p<0.05; **p<0.01; ****p<0.0001. Mean ± SEM. Scale bar: 2 Statistics: ANOVA with Tukey’s post hoc test Figure data 1 Source data for Figure Download Next, we investigated the effect of loss of Fwe on PI(4,5)P2 microdomain levels of PI(4,5)P2 were not in the mutant relative to control (Figure However, intense stimulation to elicit PI(4,5)P2 microdomain formation in the mutant (Figure a crucial role for Fwe in this To determine if the Ca2+ channel activity of Fwe is responsible for this activity, we conducted on our previous in which mutant boutons expression of the wild-type Fwe or the mutant that has reduced Ca2+ (Yao et al., 2017). We found that the wild-type Fwe PI(4,5)P2 microdomain formation, the mutant that (Figure Thus, Fwe the formation of PI(4,5)P2 microdomains in a Ca2+ channel-dependent and are to be the Ca2+ for ADBE and Cousin, 2007; Jin et al., 2019; and et al., 2010; Wu et al., 2009; Wu et al., The complex dephosphorylates PI(4,5)P2 metabolic Synj and and 2001; et al., 2005; et al., 2004; van den and 2009). Drosophila three of the catalytic subunit of and regulates of Drosophila NMJ boutons et al., Therefore, we in neurons by using et al., and and 2007). Similar to the effect of loss of levels via expression of suppressed the formation of PI(4,5)P2 microdomains to the control (Figure that the Ca2+ conducted by Fwe to induce PI(4,5)P2 microdomains. previous Ca2+ imaging data that intense stimulation Fwe to increase presynaptic Ca2+ (Yao et al., 2017). We measured intracellular Ca2+ levels with the Ca2+ (Chen et al., 20