Activity-dependent retrograde laminin A signaling regulates synapse growth at Drosophila neuromuscular junctions

Retrograde signals induced by synaptic activities are derived from postsynaptic cells to potentiate presynaptic properties, such as cytoskeletal dynamics, gene expression, and synaptic growth. However, it is not known whether activity-dependent retrograde signals can also depotentiate synaptic properties. Here we report that laminin A (LanA) functions as a retrograde signal to suppress synapse growth at Drosophila neuromuscular junctions (NMJs). The presynaptic integrin pathway consists of the integrin subunit βν and focal adhesion kinase 56 (Fak56), both of which are required to suppress crawling activity-dependent NMJ growth. LanA protein is localized in the synaptic cleft and only muscle-derived LanA is functional in modulating NMJ growth. The LanA level at NMJs is inversely correlated with NMJ size and regulated by larval crawling activity, synapse excitability, postsynaptic response, and anterograde Wnt/Wingless signaling, all of which modulate NMJ growth through LanA and βν. Our data indicate that synaptic activities down-regulate levels of the retrograde signal LanA to promote NMJ growth.     

A distinct pool of phosphatidylinositol 4,5-bisphosphate in caveolae revealed by a nanoscale labeling technique

Multiple functionally independent pools of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂] have been postulated to occur in the cell membrane, but the existing techniques lack sufficient resolution to unequivocally confirm their presence. To analyze the distribution of PI(4,5)P₂ at the nanoscale, we developed an electron microscopic technique that probes the freeze-fractured membrane preparation by the pleckstrin homology domain of phospholipase C-δ1. This method does not require chemical fixation or expression of artificial probes, it is applicable to any cell in vivo and in vitro, and it can define the PI(4,5)P₂ distribution quantitatively. By using this method, we found that PI(4,5)P₂ is highly concentrated at the rim of caveolae both in cultured fibroblasts and mouse smooth muscle cells in vivo. PI(4,5)P₂ was also enriched in the coated pit, but only a low level of clustering was observed in the flat undifferentiated membrane. When cells were treated with angiotensin II, the PI(4,5)P₂ level in the undifferentiated membrane decreased to 37.9% within 10 sec and then returned to the initial level. Notably, the PI(4,5)P₂ level in caveolae showed a slower but more drastic change and decreased to 20.6% at 40 sec, whereas the PI(4,5)P₂ level in the coated pit was relatively constant and decreased only to 70.2% at 10 sec. These results show the presence of distinct PI(4,5)P₂ pools in the cell membrane and suggest a unique role for caveolae in phosphoinositide signaling.     

Trio engagement via plasma membrane phospholipids and the myristoyl moiety governs HIV-1 matrix binding to bilayers

Localization of the HIV type-1 (HIV-1) Gag protein on the plasma membrane (PM) for virus assembly is mediated by specific interactions between the N-terminal myristoylated matrix (MA) domain and phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P₂]. The PM bilayer is highly asymmetric, and this asymmetry is considered crucial in cell function. In a typical mammalian cell, the inner leaflet of the PM is enriched in phosphatidylserine (PS) and phosphatidylethanolamine (PE) and contains minor populations of phosphatidylcholine (PC) and PI(4,5)P₂. There is strong evidence that efficient binding of HIV-1 Gag to membranes is sensitive not only to lipid composition and net negative charge, but also to the hydrophobic character of the acyl chains. Here, we show that PS, PE, and PC interact directly with MA via a region that is distinct from the PI(4,5)P₂ binding site. Our NMR data also show that the myristoyl group is readily exposed when MA is bound to micelles or bicelles. Strikingly, our structural data reveal a unique binding mode by which the 2′-acyl chain of PS, PE, and PC lipids is buried in a hydrophobic pocket whereas the 1′-acyl chain is exposed. Sphingomyelin, a major lipid localized exclusively on the outer layer of the PM, does not bind to MA. Our findings led us to propose a trio engagement model by which HIV-1 Gag is anchored to the PM via the 1′-acyl chains of PI(4,5)P₂ and PS/PE/PC and the myristoyl group, which collectively bracket a basic patch projecting toward the polar leaflet of the membrane.     

Opposing mechanisms involving RNA and lipids regulate HIV-1 Gag membrane binding through the highly basic region of the matrix domain

Membrane binding of Gag, a crucial step in HIV-1 assembly, is facilitated by bipartite signals within the matrix (MA) domain: N-terminal myristoyl moiety and the highly basic region (HBR). We and others have shown that Gag interacts with a plasma-membrane-specific acidic phospholipid, phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P₂], via the HBR, and that this interaction is important for efficient membrane binding and plasma membrane targeting of Gag. Generally, in protein–PI(4,5)P₂ interactions, basic residues promote the interaction as docking sites for the acidic headgroup of the lipid. In this study, toward better understanding of the Gag–PI(4,5)P₂ interaction, we sought to determine the roles played by all of the basic residues in the HBR. We identified three basic residues promoting PI(4,5)P₂-dependent Gag-membrane binding. Unexpectedly, two other HBR residues, Lys25 and Lys26, suppress membrane binding in the absence of PI(4,5)P₂ and prevent promiscuous intracellular localization of Gag. This inhibition of nonspecific membrane binding is likely through suppression of myristate-dependent hydrophobic interaction because mutating Lys25 and Lys26 enhances binding of Gag with neutral-charged liposomes. These residues were reported to bind RNA. Importantly, we found that RNA also negatively regulates Gag membrane binding. In the absence but not presence of PI(4,5)P₂, RNA bound to MA HBR abolishes Gag-liposome binding. Altogether, these data indicate that the HBR is unique among basic phosphoinositide-binding domains, because it integrates three regulatory components, PI(4,5)P₂, myristate, and RNA, to ensure plasma membrane specificity for particle assembly.     

Golgi and plasma membrane pools of PI(4)P contribute to plasma membrane PI(4,5)P2 and maintenance of KCNQ2/3 ion channel current

Plasma membrane (PM) phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂] regulates the activity of many ion channels and other membrane-associated proteins. To determine precursor sources of the PM PI(4,5)P₂ pool in tsA-201 cells, we monitored KCNQ2/3 channel currents and translocation of PH_PLCδ1 domains as real-time indicators of PM PI(4,5)P₂, and translocation of PH_OSH2×2, and PH_OSH1 domains as indicators of PM and Golgi phosphatidylinositol 4-phosphate [PI(4)P], respectively. We selectively depleted PI(4)P pools at the PM, Golgi, or both using the rapamycin-recruitable lipid 4-phosphatases. Depleting PI(4)P at the PM with a recruitable 4-phosphatase (Sac1) results in a decrease of PI(4,5)P₂ measured by electrical or optical indicators. Depleting PI(4)P at the Golgi with the 4-phosphatase or disrupting membrane-transporting motors induces a decline in PM PI(4,5)P₂. Depleting PI(4)P simultaneously at both the Golgi and the PM induces a larger decrease of PI(4,5)P₂. The decline of PI(4,5)P₂ following 4-phosphatase recruitment takes 1–2 min. Recruiting the endoplasmic reticulum (ER) toward the Golgi membranes mimics the effects of depleting PI(4)P at the Golgi, apparently due to the trans actions of endogenous ER Sac1. Thus, maintenance of the PM pool of PI(4,5)P₂ appears to depend on precursor pools of PI(4)P both in the PM and in the Golgi. The decrease in PM PI(4,5)P₂ when Sac1 is recruited to the Golgi suggests that the Golgi contribution is ongoing and that PI(4,5)P₂ production may be coupled to important cell biological processes such as membrane trafficking or lipid transfer activity.This paper concerns the dynamics of cellular pools of phosphoinositides, a family of phospholipids located on the cytoplasmic leaflet of cellular membranes, that maintain cell structure, cell motility, membrane identity, and membrane trafficking; they also play key roles in signal transduction (1). Phosphatidylinositol (PI) can be phosphorylated at three positions to generate seven additional species. The subcellular localization of each phosphoinositide is tightly governed by the concurrent presence of lipid kinases and lipid phosphatases (2, 3), giving each membrane within the cell a unique and dynamic phosphoinositide signature (4). Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂] is localized to the inner leaflet of the plasma membrane (PM) and is the major substrate of phospholipase C (PLC). As a consequence, PI(4,5)P₂ levels are dynamically regulated by G_q-coupled receptors activating PLC. The activity of lipid kinases and phosphatases also can be modulated by signaling; for example, a PI 4-kinase, when associated with neuronal calcium sensor-1, is accelerated in response to elevated calcium that occurs with PI(4,5)P₂ cleavage (5). In addition, transient apposition between organelles can alter phosphoinositide levels by presenting membrane-bound phosphatases in trans. For example, the endoplasmic reticulum (ER) can make contacts with the Golgi, allowing 4-phosphatases of the ER to dephosphorylate Golgi phosphatidylinositol 4-phosphate [PI(4)P] (6–8).PI(4,5)P₂ is a dynamically regulated positive cofactor required for the activity of many plasma membrane ion channels (9). Current in KCNQ2/3 channels (the molecular correlate of neuronal M-current) can be turned off in a few seconds by depletion of PI(4,5)P₂ following activation of PLC through G_q-coupled M₁ muscarinic receptors (M₁Rs) (10–12). Given the importance of PI(4,5)P₂, we wanted to understand better how it is sourced from its precursor PI(4)P within the cell. How do subcellular compartments influence PI(4,5)P₂ abundance at the plasma membrane? PI(4,5)P₂ is derived from PI in two steps: PI 4-kinases make PI(4)P, and PI(4)P 5-kinases make PI(4,5)P₂. Thus, PI(4)P is the immediate precursor of PI(4,5)P₂. Mammalian cells express at least four distinct isoforms of PI 4-kinase that phosphorylate PI on the 4 position to generate PI(4)P and are commonly referred to as PI4K types II (α and β) and III (α and β) (1, 13). PI 4-kinase type IIIα generates PI(4)P at both the Golgi and the PM (14–16). Originally thought to be localized to an ER/Golgi compartment (17, 18), recent experiments show that it is targeted to the plasma membrane by a palmitoylated peripheral membrane protein (16). PI 4-kinase IIIβ is said to be localized to the Golgi and nucleus and contributes to the biosynthesis of Golgi PI(4)P through its association with Arf1 and neuronal calcium sensor 1 (19–21). Inhibition of PI 4-kinase IIIα and -β with micromolar concentrations of wortmannin prevents the replenishment of PM PI(4,5)P₂ following PLC activation (10, 22, 23). Type IIα and IIβ PI4Ks are membrane-bound proteins due to the palmitoylation of a conserved stretch of cysteines in their catalytic domains (24). Immunocytochemical analysis has revealed that they are mostly associated with trans-Golgi, endoplasmic reticulum, and endosomal membranes (25–27). These type IIα and IIβ enzymes are blocked by adenosine and calcium, but are resistant to wortmannin. Therefore, they are not thought to contribute to the recovery of PM PI(4,5)P₂ following G_q-receptor activation (10, 24). Our understanding of the contribution of the PI 4-kinase isoforms is undergoing refinement by accumulating information concerning the unique localization, trafficking, and activity of each PI 4-kinase.Although PI 4-kinase isoforms are present in the membranes of several organelles, the most abundant pools of PI(4)P appear to be those of the PM, Golgi, and secretory vesicles (13–15, 28). A need for Golgi PI(4)P in the maintenance of PM PI(4,5)P₂ was indirectly revealed when plasma membrane PI(4,5)P₂ recovery was slowed following recruitment of a 4-phosphatase to the trans-Golgi network (28). Depletion of PM PI(4)P has been shown to result in small changes to PM PI(4,5)P₂ (15, 22), and knockout of the PM-bound PI 4-kinase IIIα resulted in loss of PI(4)P and a relocation of PI(4,5)P₂ biosensors to intracellular membranes (16). Nevertheless, others have proposed that PM PI(4)P is redundant for the synthesis of PM PI(4,5)P₂ (29–31) and may not serve as its immediate precursor because treatment with the type III PI 4-kinase inhibitor wortmannin or recruiting a 4-phosphatase to the PM had little effect on the PM localization of the PI(4,5)P₂ reporter, the pleckstrin homology (PH) domain from PLCδ1 (PH_PLCδ1).Here, we revisit the relative contributions of PI(4)P pools to PM PI(4,5)P₂. We find that the majority of PM PI(4,5)P₂ needed for maintenance of KCNQ currents comes from two precursor pools of PI(4)P in the cell, one in the PM and the other in the Golgi. The PM pool makes the larger contribution, but the contribution from both locations is significant and ongoing.     

NMR Structure of the Myristylated Feline Immunodeficiency Virus Matrix Protein

Membrane targeting by the Gag proteins of the human immunodeficiency viruses (HIV types-1 and -2) is mediated by Gag’s N-terminally myristylated matrix (MA) domain and is dependent on cellular phosphatidylinositol-4,5-bisphosphate [PI(4,5)P₂]. To determine if other lentiviruses employ a similar membrane targeting mechanism, we initiated studies of the feline immunodeficiency virus (FIV), a widespread feline pathogen with potential utility for development of human therapeutics. Bacterial co-translational myristylation was facilitated by mutation of two amino acids near the amino-terminus of the protein (Q5A/G6S; myrMA^Q5A/G6S). These substitutions did not affect virus assembly or release from transfected cells. NMR studies revealed that the myristyl group is buried within a hydrophobic pocket in a manner that is structurally similar to that observed for the myristylated HIV-1 protein. Comparisons with a recent crystal structure of the unmyristylated FIV protein [myr(-)MA] indicate that only small changes in helix orientation are required to accommodate the sequestered myr group. Depletion of PI(4,5)P₂ from the plasma membrane of FIV-infected CRFK cells inhibited production of FIV particles, indicating that, like HIV, FIV hijacks the PI(4,5)P₂ cellular signaling system to direct intracellular Gag trafficking during virus assembly.     

Activity-dependent PI(3,5)P2 synthesis controls AMPA receptor trafficking during synaptic depression

Dynamic regulation of phosphoinositide lipids (PIPs) is crucial for diverse cellular functions, and, in neurons, PIPs regulate membrane trafficking events that control synapse function. Neurons are particularly sensitive to the levels of the low abundant PIP, phosphatidylinositol 3,5-bisphosphate [PI(3,5)P₂], because mutations in PI(3,5)P₂-related genes are implicated in multiple neurological disorders, including epilepsy, severe neuropathy, and neurodegeneration. Despite the importance of PI(3,5)P₂ for neural function, surprisingly little is known about this signaling lipid in neurons, or any cell type. Notably, the mammalian homolog of yeast vacuole segregation mutant (Vac14), a scaffold for the PI(3,5)P₂ synthesis complex, is concentrated at excitatory synapses, suggesting a potential role for PI(3,5)P₂ in controlling synapse function and/or plasticity. PI(3,5)P₂ is generated from phosphatidylinositol 3-phosphate (PI3P) by the lipid kinase PI3P 5-kinase (PIKfyve). Here, we present methods to measure and control PI(3,5)P₂ synthesis in hippocampal neurons and show that changes in neural activity dynamically regulate the levels of multiple PIPs, with PI(3,5)P₂ being among the most dynamic. The levels of PI(3,5)P₂ in neurons increased during two distinct forms of synaptic depression, and inhibition of PIKfyve activity prevented or reversed induction of synaptic weakening. Moreover, altering neuronal PI(3,5)P₂ levels was sufficient to regulate synaptic strength bidirectionally, with enhanced synaptic function accompanying loss of PI(3,5)P₂ and reduced synaptic strength following increased PI(3,5)P₂ levels. Finally, inhibiting PI(3,5)P₂ synthesis alters endocytosis and recycling of AMPA-type glutamate receptors (AMPARs), implicating PI(3,5)P₂ dynamics in AMPAR trafficking. Together, these data identify PI(3,5)P₂-dependent signaling as a regulatory pathway that is critical for activity-dependent changes in synapse strength.Phosphorylated phosphoinositide lipids (PIPs) regulate diverse cellular processes (reviewed in refs. 1, 2). These seven interconvertible PIP species are synthesized and turned over by highly regulated lipid kinases and phosphatases. PIPs likely assemble complex protein machines on membrane subdomains through binding of specific downstream protein effectors, which provides tight spatial and temporal control of cellular processes. Such precision is likely critical for complex cellular functions, including regulation of synaptic strength in the CNS.Pleiotropic defects are associated with impairments in phosphatidylinositol 3,5-bisphosphate [PI(3,5)P₂] synthesis (reviewed in ref. 3). Mutations in FIG4, the gene that encodes a positive regulator of PI(3,5)P₂ (4–10), are linked to several neurological disorders, including Charcot–Marie–Tooth type 4J (CMT4J) (4, 11), ALS, and primary lateral sclerosis (12), familial epilepsy with polymicrogyria (13) and Yunis–Varón syndrome (14). Little is known about how perturbations in PI(3,5)P₂ synthesis cause disease.Fig4 is a member of a protein complex that includes the phosphatidylinositol 3-phosphate (PI3P) 5-kinase (PIKfyve; Fab1 in yeast) (10, 15–18) and the scaffolding protein Vac14 (8, 9, 19–22) (Fig. S1). PIKfyve provides the sole source of PI(3,5)P₂ (10, 15, 17, 23–28). The pools of PI3P that are converted to PI(3,5)P₂ may derive from the class III PI 3-kinase VPS34 (29) and/or the class II PI 3-kinase C2α (30). In vivo, depletion of PIKfyve affects both PI(3,5)P₂ and PI5P pools (10, 21, 24, 28). Identification of PI(3,5)P₂ and PI5P protein effectors will likely reveal specific roles for each lipid.The ability to control PI(3,5)P₂ levels dynamically in mammalian cells is likely crucial for cellular function. In yeast, hyperosmotic stress transiently increases and decreases PI(3,5)P₂ levels (6, 31). Similarly, in multicellular organisms, diverse external cues, such as hormones, growth factors, or neurotransmitters, may lead to dynamic regulation of PI(3,5)P₂ levels. Indeed, analysis of the CMT4J disease mutation Fig4-I>T in yeast showed an impairment in stimulus-induced PI(3,5)P₂ synthesis without an effect on basal PI(3,5)P₂ levels (4). In cultured cortical neurons, knockdown of PIKfvye impairs the internalization of an AMPA-type glutamate receptor (AMPAR) subunit, HA-tagged GluA2 (32), and loss of Vac14 and/or Fig4 is associated with strengthened synapses (33). Together, these findings suggest that Vac14 and Fig4 regulate synapse strength via positive regulation of PIKfyve.Here, using multiple approaches, we show that PIKfyve kinase activity negatively regulates postsynaptic strength and plays specialized roles during two distinct forms of synaptic weakening. Chronic down-regulation of PIKfyve activity using shRNA increases postsynaptic strength, whereas brief chemical inhibition of PIKfyve blocks NMDA receptor (NMDAR)-dependent long-term depression (LTD) and reverses homeostatic synaptic weakening (downscaling). Notably, we developed methods to measure the activity-dependent changes in each PIP species in cultured hippocampal neurons and identified that two low abundant PIPs, PI(3,4,5)P₃ and PI(3,5)P₂, are highly dynamic during LTD. Moreover, PI(3,5)P₂ levels increase during homeostatic downscaling, and increasing PI(3,5)P₂ via a dominant-active PIKfyve mutant (PIKfyve^KYA) is sufficient to weaken postsynaptic strength. We further show that these effects on synapses derive, in part, from PI(3,5)P₂-dependent trafficking of AMPARs. Together, these findings demonstrate that PIKfyve lipid kinase activity plays a critical role in regulation of synapse strength.     

Neuronal activity-dependent membrane traffic at the neuromuscular junction

During development and also in adulthood, synaptic connections are modulated by neuronal activity. To follow such modifications in vivo, new genetic tools are designed. The nontoxic C-terminal fragment of tetanus toxin (TTC) fused to a reporter gene such as LacZ retains the retrograde and transsynaptic transport abilities of the holotoxin itself. In this work, the hybrid protein is injected intramuscularly to analyze in vivo the mechanisms of intracellular and transneuronal traffics at the neuromuscular junction (NMJ). Traffic on both sides of the synapse are strongly dependent on presynaptic neural cell activity. In muscle, a directional membrane traffic concentrates beta-galactosidase-TTC hybrid protein into the NMJ postsynaptic side. In neurons, the probe is sorted across the cell to dendrites and subsequently to an interconnected neuron. Such fusion protein, sensitive to presynaptic neuronal activity, would be extremely useful to analyze morphological changes and plasticity at the NMJ.     

Genetically encoded fluorescent probe to visualize intracellular phosphatidylinositol 3,5-bisphosphate localization and dynamics

Phosphatidylinositol 3,5-bisphosphate [PI(3,5)P₂] is a low-abundance phosphoinositide presumed to be localized to endosomes and lysosomes, where it recruits cytoplasmic peripheral proteins and regulates endolysosome-localized membrane channel activity. Cells lacking PI(3,5)P₂ exhibit lysosomal trafficking defects, and human mutations in the PI(3,5)P₂-metabolizing enzymes cause lysosome-related diseases. The spatial and temporal dynamics of PI(3,5)P₂, however, remain unclear due to the lack of a reliable detection method. Of the seven known phosphoinositides, only PI(3,5)P₂ binds, in the low nanomolar range, to a cytoplasmic phosphoinositide-interacting domain (ML1N) to activate late endosome and lysosome (LEL)-localized transient receptor potential Mucolipin 1 (TRPML1) channels. Here, we report the generation and characterization of a PI(3,5)P₂-specific probe, generated by the fusion of fluorescence tags to the tandem repeats of ML1N. The probe was mainly localized to the membranes of Lamp1-positive compartments, and the localization pattern was dynamically altered by either mutations in the probe, or by genetically or pharmacologically manipulating the cellular levels of PI(3,5)P₂. Through the use of time-lapse live-cell imaging, we found that the localization of the PI(3,5)P₂ probe was regulated by serum withdrawal/addition, undergoing rapid changes immediately before membrane fusion of two LELs. Our development of a PI(3,5)P₂-specific probe may facilitate studies of both intracellular signal transduction and membrane trafficking in the endosomes and lysosomes.Phosphorylated phosphoinositide lipids are produced on the cytosolic side of cellular lipid bilayer membranes (1, 2). There are seven different known phosphoinositides lipids, which localize to distinct membrane subdomains to regulate organelle-specific membrane signaling pathways and membrane-trafficking events (1, 2). One such phosphoinositide lipid is phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂], which is predominantly localized to the plasma membrane (PM). PI(4,5)P₂ serves as a permissive cofactor that is required for the activity of PM channels/transporters, as a precursor for the generation of second messengers, and as a PM recruiter for cytosolic peripheral proteins (3, 4). Likewise, PI(3,4)P₂ and PI(3,4,5)P₃ are transiently produced at the PM to regulate various signaling effectors, such as v-akt-murine thymoma viral oncogene homolog 1 (Akt) (1, 5). Conversely, PI(3)P is primarily found on early endosomes, phagosomes, and autophagosomes to regulate the maturation of these compartments (5, 6).Unlike the aforementioned phosphoinositides, the subcellular localization and functions of PI(3,5)P₂ are poorly understood. PI(3,5)P₂ is proposed to be mainly localized to late endosomes and lysosomes (LELs) (7), and also to early endosomes (8), based on the location of its synthesizing enzyme complex, which in mammalian cells consists of the phosphoinositide kinase, FYVE finger-containing (PIKfyve) the phosphatase FIG4 homolog, SAC1 lipid phosphatase domain-containing (Fig4), and the scaffolding protein VAC 14 homolog (Vac14) (9–12). Genetic disruption of any of these components in mice results in a decrease in the intracellular PI(3,5)P₂ level, lysosomal trafficking defects at the cellular level, and embryonic/neonatal lethality at the animal level (9–12). Consistent with the LEL localization of PI(3,5)P₂, we recently reported that PI(3,5)P₂ is an endogenous agonist for mucolipin TRP (TRPML) and two-pore TPC channels, which are LEL-localized membrane channel proteins (13, 14). Despite its genetic importance, the localization and dynamics of PI(3,5)P₂ remain to be established at the single-cell level, largely due to the lack of a direct method to visualize this low-abundance phosphoinositide.The recent development of specific fluorescent probes has greatly enhanced our understanding of phosphoinositide signaling (2, 15). These probes include those for PI(3)P, PI(4)P, PI(3,4)P₂, PI(4,5)P₂, and PI(3,4,5,)P₃, which are generated by fusing fluorescent tags with the phosphoinositide-interacting domains of their effector proteins. For example, GFP or mCherry proteins are fused directly with the FAPP1-PH domain [for PI(4)P], the phospholipase C (PLC)_δ-PH domain [for PI(4,5)P₂], the AKT-PH domain [for both PI(3,4)P₂ and PI(3,4,5)P₃], and the hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs)-FYVE and early endosome antigen 1 (EEA1)-FYVE domains [for PI(3)P] (15, 16). However, an effective, high-affinity PI(3,5)P₂ probe is still lacking, possibly due to two barriers. First, most peripheral phosphoinositide-binding proteins exhibit low affinity binding and often require the simultaneous binding of additional factors to increase the affinity and specificity (2, 17). PI(3,5)P₂ is a poorly characterized phosphoinositide with very few known effectors, most of which exhibit relatively low affinities (18, 19). Second, intracellular PI(3,5)P₂ levels are reportedly low [around 1/10th of that of PI(3)P and less than 1/100th of that of PI(4,5)P₂] (2, 10, 12).Thus, it has proven difficult to develop an effective PI(3,5)P₂ probe for in situ use by using the phosphoinositide-binding domains of peripheral proteins (18). We recently reported that TRPML1, a LEL-localized membrane channel protein, is potently activated by PI(3,5)P₂ in the low nanomolar range. We further showed that PI(3,5)P₂ binds to the cytosolic N-terminal polybasic domain of TRPML1 (ML1N). This specific activation/binding is abolished by mutations of basic residues within the polybasic domain (20). In this study, we report the development of a genetically encoded PI(3,5)P₂ fluorescent probe based on ML1N.     

Activity-dependent coordination of presynaptic release probability and postsynaptic GluR2 abundance at single synapses

The strength of an excitatory synapse depends on both the presynaptic release probability (p_r) and the abundance of functional postsynaptic AMPA receptors. How these parameters are related or balanced at a single synapse remains unknown. By taking advantage of live fluorescence imaging in cultured hippocampal neurons where individual synapses are readily resolved, we estimate p_r by labeling presynaptic vesicles with a styryl dye, FM1-43, while concurrently measuring postsynaptic AMPA receptor abundance at the same synapse by immunolabeling surface GluR2. We find no appreciable correlation between p_r and the level of surface synaptic GluR2 under basal condition, and blocking basal neural activity has no effect on the observed lack of correlation. However, elevating network activity drives their correlation, which accompanies a decrease in mean GluR2 level. These findings provide the direct evidence that the coordination of pre- and postsynaptic parameters of synaptic strength is not intrinsically fixed but that the balance is tuned by synaptic use at individual synapses.     

β-arrestin–dependent PI(4,5)P2 synthesis boosts GPCR endocytosis

β-arrestins regulate many cellular functions including intracellular signaling and desensitization of G protein–coupled receptors (GPCRs). Previous studies show that β-arrestin signaling and receptor endocytosis are modulated by the plasma membrane phosphoinositide lipid phosphatidylinositol-(4, 5)-bisphosphate (PI(4,5)P₂). We found that β-arrestin also helped promote synthesis of PI(4,5)P₂ and up-regulated GPCR endocytosis. We studied these questions with the G_q-coupled protease-activated receptor 2 (PAR2), which activates phospholipase C, desensitizes quickly, and undergoes extensive endocytosis. Phosphoinositides were monitored and controlled in live cells using lipid-specific fluorescent probes and genetic tools. Applying PAR2 agonist initiated depletion of PI(4,5)P₂, which then recovered during rapid receptor desensitization, giving way to endocytosis. This endocytosis could be reduced by various manipulations that depleted phosphoinositides again right after phosphoinositide recovery: PI(4)P, a precusor of PI(4,5)P_2, could be depleted at either the Golgi or the plasma membrane (PM) using a recruitable lipid 4-phosphatase enzyme and PI(4,5)P₂ could be depleted at the PM using a recruitable 5-phosphatase. Endocytosis required the phosphoinositides. Knock-down of β-arrestin revealed that endogenous β-arrestin normally doubles the rate of PIP5-kinase (PIP5K) after PAR2 desensitization, boosting PI(4,5)P₂-dependent formation of clathrin-coated pits (CCPs) at the PM. Desensitized PAR2 receptors were swiftly immobilized when they encountered CCPs, showing a dwell time of ∼90 s, 100 times longer than for unactivated receptors. PAR2/β-arrestin complexes eventually accumulated around the edges or across the surface of CCPs promoting transient binding of PIP5K-Iγ. Taken together, β-arrestins can coordinate potentiation of PIP5K activity at CCPs to induce local PI(4,5)P₂ generation that promotes recruitment of PI(4,5)P₂-dependent endocytic machinery.

Membrane phosphatidylinositide lipids (PPIs) are dynamic regulators of diverse cell functions, and their dysregulation underlies numerous human diseases (1). This paper concerns the key involvement of plasma membrane (PM) phosphatidylinositol-(4, 5)-bisphosphate (PI(4,5)P₂) in refining receptor–G protein and receptor–β-arrestin coupling (2, 3) and preparing for the endocytosis of receptors (4). Endocytosis requires clustering of adapter proteins on the PM, nucleation of clathrin-coated membrane pits, capture of receptors with β-arrestin (5–7), and pinching off of pits as intracellular vesicles by dynamin GTPase (4, 8–10). In clathrin-mediated endocytosis, PI(4,5)P₂ is typically needed for the assembly of the adaptor protein complexes, clathrin-coated pits (CCPs), and dynamin complexes (4, 11–14). Hence, receptor internalization should be compromised if PI(4,5)P₂ pools are depleted. This raises the question of how receptors that signal by depleting PI(4,5)P₂ can still be internalized. In this study, we found roles of receptor stimulation and β-arrestin in promoting resynthesis of PI(4,5)P₂, thus enabling endocytosis at the PM.Synthesis of PPIs starts with phosphatidylinositol and families of lipid kinases that generate the mono-, bis-, and tris-phosphorylated inositol ring. PM phosphatidylinositol 4-phosphate (PI(4)P) and PI(4,5)P₂ are produced by several mechanisms potentially involving other membrane compartments. They can be synthesized by lipid 4-kinases acting on PM phosphatidylinositol and by lipid 5-kinases acting on PM PI(4)P; they can be delivered in exchange for other lipids by phosphatidylinositol exchange proteins; and they can be delivered through fusion with other membranes (15–23). Such studies show that the PPI pools in different membranes are interdependent (21). For example, depleting PI(4)P locally in the trans-Golgi using a recruitable PI(4)P 4-phosphatase tool reduces the generation of PI(4,5)P₂ at the PM (24). Conversely, depleting PI(4,5)P₂ at the PM by activating muscarinic or angiotensin II receptors also strongly decreases total cellular PI(4)P (25–27). New evidence is emerging that the PPI composition controls membrane trafficking between organelles. For instance, trafficking of mannose 6-phosphate receptors from the Golgi to the PM can be slowed by reduction of PPI synthesis (28) presumably because PPIs are important for fusion of receptor-containing vesicles with the PM.Here, we study contributions of PPI pools to the endocytosis of the G_q-coupled protease-activated receptor 2 (PAR2). This receptor is involved in inflammatory responses (29), sensation of inflammatory pain (30), and cancer metastasis (31). It has been a target of drug development (32) facilitated by recent crystal structures (33). Stimulation of this receptor activates phospholipase C (PLC) to cleave and deplete PI(4,5)P₂ with accompanying production of diacylglycerol, inositol trisphosphate, and calcium signals (34, 35). Activation of the PAR-receptor family has unique properties. The receptor is activated by cleavage of the N terminus by serine proteases such as thrombin, tryptase, or trypsin (34, 36), which generates a tethered N-terminal ligand. The activation stimulates G_q but is followed quickly by desensitization that terminates G_q signaling (34, 35, 37). Our previous experimental results and mathematical modeling suggest that rapid phosphorylation of PAR2 precedes desensitization and that β-arrrestin clamps the phosphorylated and ligand-bound state of the receptor, protecting it from dephosphorylation by serine/threonine phosphatases (38). Then, the receptor is internalized slowly via a clathrin- and dynamin-dependent pathway (8). This rapidly desensitizing receptor is well suited to address mechanisms involved in PPI lipid–dependent G_qPCR endocytosis.Using genetic and optical tools to manipulate and measure PI(4)P and PI(4,5)P₂ levels acutely at the Golgi or the PM, we now demonstrate that PAR2 internalization can be controlled by PM PI(4,5)P₂ that is replenished using both PM and Golgi pools of PI(4)P. A β-arrestin–dependent activation of PIP5-kinase (PIP5K) at the PM turned out to be critical in the formation of PI(4,5)P₂- and PI(4)P-requiring CCPs and potentially other endocytic machinery for receptor internalization.     

Pheromone-induced anisotropy in yeast plasma membrane phosphatidylinositol-4,5-bisphosphate distribution is required for MAPK signaling

During response of budding yeast to peptide mating pheromone, the cell becomes markedly polarized and MAPK scaffold protein Ste5 localizes to the resulting projection (shmoo tip). We demonstrated before that this recruitment is essential for sustained MAPK signaling and requires interaction of a pleckstrin homology (PH) domain in Ste5 with phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P₂] in the plasma membrane. Using fluorescently tagged high-affinity probes specific for PtdIns(4,5)P₂, we have now found that this phosphoinositide is highly concentrated at the shmoo tip in cells responding to pheromone. Maintenance of this strikingly anisotropic distribution of PtdIns(4,5)P₂, stable tethering of Ste5 at the shmoo tip, downstream MAPK activation, and expression of a mating pathway-specific reporter gene all require continuous function of the plasma membrane-associated PtdIns 4-kinase Stt4 and the plasma membrane-associated PtdIns4P 5-kinase Mss4 (but not the Golgi-associated PtdIns 4-kinase Pik1). Our observations demonstrate that PtdIns(4,5)P₂ is the primary determinant for restricting localization of Ste5 within the plasma membrane and provide direct evidence that an extracellular stimulus-evoked self-reinforcing mechanism generates a spatially enriched pool of PtdIns(4,5)P₂ necessary for the membrane anchoring and function of a signaling complex.     

Allosteric modulation of alternatively spliced Ca2+-activated Cl− channels TMEM16A by PI(4,5)P2 and CaMKII

Transmembrane 16A (TMEM16A, anoctamin1), 1 of 10 TMEM16 family proteins, is a Cl⁻ channel activated by intracellular Ca²⁺ and membrane voltage. This channel is also regulated by the membrane phospholipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂]. We find that two splice variants of TMEM16A show different sensitivity to endogenous PI(4,5)P₂ degradation, where TMEM16A(ac) displays higher channel activity and more current inhibition by PI(4,5)P₂ depletion than TMEM16A(a). These two channel isoforms differ in the alternative splicing of the c-segment (exon 13). The current amplitude and PI(4,5)P₂ sensitivity of both TMEM16A(ac) and (a) are significantly strengthened by decreased free cytosolic ATP and by conditions that decrease phosphorylation by Ca²⁺/calmodulin-dependent protein kinase II (CaMKII). Noise analysis suggests that the augmentation of currents is due to a rise of single-channel current (i), but not of channel number (N) or open probability (P_O). Mutagenesis points to arginine 486 in the first intracellular loop as a putative binding site for PI(4,5)P₂, and to serine 673 in the third intracellular loop as a site for regulatory channel phosphorylation that modulates the action of PI(4,5)P₂. In silico simulation suggests how phosphorylation of S673 allosterically and differently changes the structure of the distant PI(4,5)P₂-binding site between channel splice variants with and without the c-segment exon. In sum, our study reveals the following: differential regulation of alternatively spliced TMEM16A(ac) and (a) by plasma membrane PI(4,5)P₂, modification of these effects by channel phosphorylation, identification of the molecular sites, and mechanistic explanation by in silico simulation.

TMEM16A (anoctamin1) plays a wide range of physiological roles in diverse cell types, including contraction of smooth muscle and gastrointestinal motility, secretion of Cl⁻ in epithelial cells, detection of noxious heat in nociceptive neurons, modulation of neuronal excitability, and regulation of cell volume (1). TMEM16A channels, from a family of 10 anoctamin proteins (TMEM16A–K), continuously monitor the concentration of intracellular Ca²⁺ and function as Ca²⁺-activated Cl⁻ channels (2–4). Several splice variants of TMEM16A generated by combinatorial inclusion or exclusion of four exon segments, a, b, c, and d (5–7), display unique electrophysiological properties in tissues. Segments a and b lie in the N terminus, and segments c and d lie in the first intracellular loop of TMEM16A. Among the four segments, it is known that b and c help regulate the cytosolic Ca²⁺ sensitivity and voltage dependence of channel gating. For example, inclusion of the b-segment results in decreased channel sensitivity to intracellular Ca²⁺ rise, whereas skipping of the c-segment reduces channel activity and also impairs Ca²⁺ sensitivity (5, 8, 9). In addition to inclusion or skipping of each segment, calmodulin (10–13), phosphorylation (14–16), protons (17–19), and lipids (20–27) also impact on the gating of TMEM16A channels.Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂] is a key signaling phospholipid in the inner leaflet of the plasma membrane. It acts as a cofactor that regulates many types of ion channels and receptors (28–30), and thus depletion of membrane PI(4,5)P₂ by the activation of either phospholipase C (PLC) or phosphoinositide 5-phosphatases leads to decreases or increases in gating activity of ion channels. Of the TMEM16 family, TMEM16A, TMEM16B, and TMEM16F are ion channels best known to be modulated by PI(4,5)P₂ (21–27, 31). Several studies showed that PI(4,5)P₂ is required for sustained TMEM16A channel activity and stabilizes the Ca²⁺-bound open state of the channels (23, 24, 32). Further work located a PI(4,5)P₂ regulatory region and demonstrated how PI(4,5)P₂ interacts with TMEM16A to regulate channel gating by performing computational simulation. Le et al. (25) proposed that channel activation and desensitization are mediated by two distinct structural modules; one is a PI(4,5)P₂-binding module formed by putative PI(4,5)P₂-binding residues of TMs 3–5 located near the cytoplasmic membrane interface and another is a Ca²⁺-binding module of TMs 6–8 involved in the primary opening of the channel pore by Ca²⁺. Yu et al. (26) identified three key binding sites involved in TMEM16A–PI(4,5)P₂ interaction. When PI(4,5)P₂ interacts with these binding residues, which form networks with each other, it affects TMEM16A channel gating as a result of the conformational change of TM6.In our study, using exogenous lipid phosphatase tools and mutagenesis, we found that PI(4,5)P₂ differentially regulates channel activity depending on the TMEM16A splice variant. In addition, we found that the presence or absence of intracellular ATP is a key determinant of the PI(4,5)P₂ sensitivity of TMEM16A. Through structural analysis partly based on a recent cryogenic electron microscopy (cryo-EM) structure of TMEM16A, we also confirmed that phosphorylation of serine 673 by CaMKII allosterically regulates the structure of a PI(4,5)P₂ interaction site in the RDR domain of TMEM16A(ac) near to transmembrane segment 3 (TM3). Together, our data reveal a molecular mechanism of TMEM16A channel regulation by PI(4,5)P₂, demonstrating that PI(4,5)P₂-dependent TMEM16A channel activation can be allosterically modulated by phosphorylation and alternative splicing.     

Inhibition of Phosphatidylinositol 4-phosphate 5-kinase by Cyclic AMP in Human Platelets

The effects of cyclic AMP (cAMP) on phosphatidylinositol 4,5-bisphosphate (PI 4,5-P₂) synthesis were examined in human platelets. In ³²P-prelabeled intact platelets, although the level of [³²P]phosphatidylinositol 4-phosphate (PI 4-P) was increased by forskolin and prostaglandin-I₂ (PGI₂), the formation of [³²P]PI 4,5-P₂ time-and concentration-dependently decreased, suggesting inhibition of phosphatidylinositol 4-phosphate 5-kinase (PI 4-P 5-kinase). In saponin-permeabilized platelets, formation of PI 4-P and PI 4,5-P₂ can be measured by utilizing [γ-³²P] ATP. In this system, PGI₂ and cAMP inhibited the generation of [³²P)PI 4,5-P₂. The PI 4-P 5-kinase activity was mostly located in the platelet membrane fraction and was inhibited by cAMP; H-8 and H-89, inhibitors of cAMP-dependent protein kinase (PKA), abolished this inhibitory effect, suggesting that cAMP exerted its action on PI 4-P 5-kinase via PKA. Adenosine, which is reported to directly inhibit phosphatidylinositol 4-kinase (PI 4-kinase) in some types of cells, had no effect on platelet membrane PI 4-P 5-kinase activity. In dbc AMP-pretreated membranes, PI 4-P 5-kinase activity was lower than that of control membranes. The involvement of PKA with the inhibitory action of cAMP in PI 4-P 5-kinase activity was further confirmed using the catalytic subunit of PKA. The synthesis of [³²P] PI 4,5-P₂ in permeabilized platelets and the specific activity of partially purified PI 4-P 5-kinase were decreased by incubation with the PKA catalytic subunit. The present results indicate that the cAMP-PKA system inhibits PI 4-P 5-kinase activity, leading to decreased formation of PI 4,5-P₂ in human platelets.     

Multiple signaling pathways are essential for synapse formation induced by synaptic adhesion molecules

Little is known about the cellular signals that organize synapse formation. To explore what signaling pathways may be involved, we employed heterologous synapse formation assays in which a synaptic adhesion molecule expressed in a nonneuronal cell induces pre- or postsynaptic specializations in cocultured neurons. We found that interfering pharmacologically with microtubules or actin filaments impaired heterologous synapse formation, whereas blocking protein synthesis had no effect. Unexpectedly, pharmacological inhibition of c-jun N-terminal kinases (JNKs), protein kinase-A (PKA), or AKT kinases also suppressed heterologous synapse formation, while inhibition of other tested signaling pathways—such as MAP kinases or protein kinase C—did not alter heterologous synapse formation. JNK and PKA inhibitors suppressed formation of both pre- and postsynaptic specializations, whereas AKT inhibitors impaired formation of post- but not presynaptic specializations. To independently test whether heterologous synapse formation depends on AKT signaling, we targeted PTEN, an enzyme that hydrolyzes phosphatidylinositol 3-phosphate and thereby prevents AKT kinase activation, to postsynaptic sites by fusing PTEN to Homer1. Targeting PTEN to postsynaptic specializations impaired heterologous postsynaptic synapse formation induced by presynaptic adhesion molecules, such as neurexins and additionally decreased excitatory synapse function in cultured neurons. Taken together, our results suggest that heterologous synapse formation is driven via a multifaceted and multistage kinase network, with diverse signals organizing pre- and postsynaptic specializations.

Synapse formation is the universal process that underlies construction of all of the brain’s circuits, but little is known about its mechanisms. Unknown signaling pathways presumably organize synapses, but what pathways are involved remains unclear. Synapse formation likely requires interactions between pre- and postsynaptic neurons via adhesion molecules that transmit bidirectional signals to pre- and postsynaptic neurons and organize pre- and postsynaptic specializations (reviewed in refs. 1–3). Synapses exhibit canonical features that include a presynaptic side that releases neurotransmitters rapidly and transiently and a postsynaptic side that recognizes these neurotransmitters. Interestingly, only the presynaptic side of a synapse harbors canonical features that are shared by all synapses, such as synaptic vesicles and active zones with the same components in excitatory and inhibitory synapses. In contrast, the postsynaptic sides differ dramatically between excitatory and inhibitory synapses. Even excitatory and inhibitory neurotransmitter receptors exhibit no homology, and few if any molecular components are shared among excitatory and inhibitory postsynaptic specializations.At present, it is unknown what intracellular signaling pathways are involved in the assembly of pre- and postsynaptic specializations, whether different types of signaling pathways exist for pre- vs. postsynaptic specializations, and how excitatory vs. inhibitory synapses are organized. In the present study, we chose the heterologous synapse formation assay as an approach in order to begin to address these fundamental questions (4). In the heterologous synapse formation assay, nonneuronal cells, such as HEK293T cells, express a synaptic adhesion molecule that then induces pre- or postsynaptic specializations when these nonneuronal cells are cocultured with neurons (5–9). For example, if a postsynaptic adhesion molecule, such as neuroligin-1 (Nlgn1) or latrophilin-3, is expressed in HEK293T cells, and the HEK293T cells are cocultured with neurons, these neurons form presynaptic specializations on the HEK293T cells (5, 10). If, conversely, a presynaptic adhesion molecule, such as a neurexin or teneurin, is expressed in HEK293T cells, postsynaptic specializations are induced in cocultured neurons (8, 9, 11).Many adhesion molecules have been shown to induce heterologous synapse formation, including neurexins, neuroligins, latrophilins, teneurins, SynCAMs, neuronal pentraxin receptors, SALMs, LAR-type PTPRs, and others (5, 6, 8–15), suggesting that there are common “synapse signaling” pathways and that the heterologous synapse formation assay nonspecifically transduces different adhesion molecules signals into a response that organizes pre- or postsynaptic specializations. Even engagement of neuronal AMPA-type glutamate receptors by the neuronal pentraxin receptor, when expressed in HEK293T cells, causes organization of postsynaptic specializations in the heterologous synapse formation assay, testifying to the broad nature of the signals that mediate heterologous synapse formation (12). Strikingly, any given adhesion molecule triggers only either pre- or postsynaptic specializations, but not both, indicating signaling specificity. Most adhesion molecules—with the exception of teneurin splice variants (11)—induce both excitatory and inhibitory synaptic specializations at the same time. Heterologous synapses resemble real synapses and are functional (6, 7). Overall, these observations suggest that specific signaling pathways regulate synapse formation and that the heterologous synapse formation assay provides a plausible and practical paradigm to dissect such signaling pathways, even though it represents an artificial system that lacks much of the specificity of physiological synapse formation.In the present study, we have employed pharmacological inhibitors and molecular interventions to probe the nature of the signals mediating heterologous synapse formation. Our data reveal that multiple parallel protein kinase signaling pathways are required for heterologous synapse formation. We identified a role for both JNK and PKA signaling in the formation of pre- and postsynaptic specializations and found that the PI3 kinase pathway is specifically required for the formation of post- but not presynaptic specializations. Thus, our data provide initial insight into the signaling mechanisms underlying heterologous synapse formation that may be relevant for synapse formation in general.     

Calcium oscillations-coupled conversion of actin travelling waves to standing oscillations

Dynamic spatial patterns of signaling factors or macromolecular assemblies in the form of oscillations or traveling waves have emerged as important themes in cell physiology. Feedback mechanisms underlying these processes and their modulation by signaling events and reciprocal cross-talks remain poorly understood. Here we show that antigen stimulation of mast cells triggers cyclic changes in the concentration of actin regulatory proteins and actin in the cell cortex that can be manifested in either spatial pattern. Recruitment of FBP17 and active Cdc42 at the plasma membrane, leading to actin polymerization, are involved in both events, whereas calcium oscillations, which correlate with global fluctuations of plasma membrane PI(4,5)P₂, are tightly linked to standing oscillations and counteract wave propagation. These findings demonstrate the occurrence of a calcium-independent oscillator that controls the collective dynamics of factors linking the actin cytoskeleton to the plasma membrane. Coupling between this oscillator and the one underlying global plasma membrane PI(4,5)P2 and calcium oscillations spatially regulates actin dynamics, revealing an unexpected pattern-rendering mechanism underlying plastic changes occurring in the cortical region of the cell.     

Spontaneous transmitter release is critical for the induction of long-term and intermediate-term facilitation in Aplysia

Long-term plasticity can differ from short-term in recruiting the growth of new synaptic connections, a process that requires the participation of both the presynaptic and postsynaptic components of the synapse. How does information about synaptic plasticity spread from its site of origin to recruit the other component? The answer to this question is not known in most systems. We have investigated the possible role of spontaneous transmitter release as such a transsynaptic signal. Until recently, relatively little has been known about the functions of spontaneous release. In this paper, we report that spontaneous release is critical for the induction of a learning-related form of synaptic plasticity, long-term facilitation in Aplysia. In addition, we have found that this signaling is engaged quite early, during an intermediate-term stage that is the first stage to involve postsynaptic as well as presynaptic molecular mechanisms. In a companion paper, we show that spontaneous release from the presynaptic neuron acts as an orthograde signal to recruit the postsynaptic mechanisms of intermediate-term facilitation and initiates a cascade that can culminate in synaptic growth with additional stimulation during long-term facilitation. Spontaneous release could make a similar contribution to learning-related synaptic plasticity in mammals.     

Extensive morphological divergence and rapid evolution of the larval neuromuscular junction in Drosophila

Although the complexity and circuitry of nervous systems undergo evolutionary change, we lack understanding of the general principles and specific mechanisms through which it occurs. The Drosophila larval neuromuscular junction (NMJ), which has been widely used for studies of synaptic development and function, is also an excellent system for studies of synaptic evolution because the genus spans >40 Myr of evolution and the same identified synapse can be examined across the entire phylogeny. We have now characterized morphology of the NMJ on muscle 4 (NMJ4) in >20 species of Drosophila. Although there is little variation within a species, NMJ morphology and complexity vary extensively between species. We find no significant correlation between NMJ phenotypes and phylogeny for the species examined, suggesting that drift alone cannot explain the phenotypic variation and that selection likely plays an important role. However, the nature of the selective pressure is still unclear because basic parameters of synaptic function remain uniform. Whatever the mechanism, NMJ morphology is evolving rapidly in comparison with other morphological features because NMJ phenotypes differ even between several sibling species pairs. The discovery of this unexpectedly extensive divergence in NMJ morphology among Drosophila species provides unique opportunities to investigate mechanisms that regulate synaptic growth; the interrelationships between synaptic morphology, neural function, and behavior; and the evolution of nervous systems and behavior in natural populations.