Transmembrane tethering of synaptotagmin to synaptic vesicles controls multiple modes of neurotransmitter release

Synaptotagmin 1 (Syt1) is a synaptic vesicle integral membrane protein that regulates neurotransmitter release by activating fast synchronous fusion and suppressing slower asynchronous release. The cytoplasmic C2 domains of Syt1 interact with SNAREs and plasma membrane phospholipids in a Ca²⁺-dependent manner and can substitute for full-length Syt1 in in vitro membrane fusion assays. To determine whether synaptic vesicle tethering of Syt1 is required for normal fusion in vivo, we performed a structure-function study with tethering mutants at the Drosophila larval neuromuscular junction. Transgenic animals expressing only the cytoplasmic C2 domains or full-length Syt1 tethered to the plasma membrane failed to restore synchronous synaptic vesicle fusion, and also failed to clamp spontaneous vesicle release. In addition, transgenic animals with shorter, but not those with longer, linker regions separating the C2 domains from the transmembrane segment abolished Syt1’s ability to activate synchronous vesicle fusion. Similar defects were observed when C2 domain alignment was altered to C2B-C2A from the normal C2A-C2B orientation, leaving the tether itself intact. Although cytoplasmic and plasma membrane-tethered Syt1 variants could not restore synchronous release in syt1 null mutants, they were very effective in promoting fusion through the slower asynchronous pathway. As such, the subcellular localization of Syt1 within synaptic terminals is important for the temporal dynamics that underlie synchronous and asynchronous neurotransmitter release.Neurotransmitter release requires temporal and spatial coupling of action potential-triggered Ca²⁺ influx to synaptic vesicle fusion (1). The core fusion machine contains SNARE proteins found on the synaptic vesicle (v-SNAREs) and plasma membrane (t-SNAREs) that assemble into a four-helix bundle to bring the two bilayers into close apposition (2, 3). Besides SNAREs, Ca²⁺-binding proteins act to trigger release through fast synchronous and slow asynchronous pathways. Synaptotagmin 1 (Syt1) is a synaptic vesicle protein that binds Ca²⁺ and triggers synchronous vesicle fusion (4–9). Syt1 contains an intravesicular N-terminal tail, a single transmembrane segment, and a ∼60- residue linker that connects to two cytoplasmic Ca²⁺-binding C2 domains (10–13).Numerous Syt1 studies have focused on its cytoplasmic C2 domains, which interact with phospholipids and the SNARE complex in a Ca²⁺-dependent manner and are proposed to be the essential domains that trigger fusion (12, 14–21). In contrast, the significance of other structural elements of Syt1 remains poorly understood. Syt1 is predicted to facilitate synaptic vesicle fusion through a trans interaction with plasma membrane lipids (22–27). Tethering of Syt1 to synaptic vesicles through its transmembrane domain has been postulated to position the protein to properly target lipids and SNAREs, or to be required to generate force for pulling the membranes together. Although anchoring through the transmembrane tether is unlikely to generate the intramembrane proximity required for the final steps in fusion owing to the distance involved, binding of individual C2 domains simultaneously to both membranes might, because such binding can aggregate lipid bilayers in vitro (27–29).Despite these models, however, the role of vesicular tethering of Syt1 in vivo remains unclear. Injection of a cytoplasmic domain of rat Syt1 into crayfish motor axons facilitates exocytosis (30), implying that the cytoplasmic region alone may act as a fusion trigger. In contrast, in vitro studies indicate that the linker domain that connects the transmembrane region to the C2 domains may regulate docking, fusion pore opening, Syt1 multimerization, and intramolecular C2 domain interactions (31–34). The requirement of C2 domain order (C2A, then C2B) has been suggested to be dispensable for synaptic vesicle endocytosis in vitro (35), but the functional consequences of altered C2 domain order on Syt1’s role in triggering exocytosis in vivo remain unclear.Here we assayed the requirements of these Syt1 regions for neurotransmitter release in vivo. We generated transgenic animals expressing modified Syt1 proteins in the synaptotagmin 1 null mutant background and examined their function at the Drosophila larval neuromuscular junction (NMJ), a well-established model glutamatergic synapse. Our results indicate that synaptic vesicle tethering, optimal linker length, and specific C2 domain alignment are important for Syt1 to regulate vesicle fusion. In addition, synaptic vesicle-tethered and cytoplasmic Syt1 proteins differentially regulate synchronous vs. asynchronous release kinetics, indicating that synaptic vesicle localization of Syt1 is critical for regulating neurotransmitter release.     

In vivo synaptic recovery following optogenetic hyperstimulation

Local recycling of synaptic vesicles (SVs) allows neurons to sustain transmitter release. Extreme activity (e.g., during seizure) may exhaust synaptic transmission and, in vitro, induces bulk endocytosis to recover SV membrane and proteins; how this occurs in animals is unknown. Following optogenetic hyperstimulation of Caenorhabditis elegans motoneurons, we analyzed synaptic recovery by time-resolved behavioral, electrophysiological, and ultrastructural assays. Recovery of docked SVs and of evoked-release amplitudes (indicating readily-releasable pool refilling) occurred within ∼8–20 s (τ = 9.2 s and τ = 11.9 s), whereas locomotion recovered only after ∼60 s (τ = 20 s). During ∼11-s stimulation, 50- to 200-nm noncoated vesicles (“100nm vesicles”) formed, which disappeared ∼8 s poststimulation, likely representing endocytic intermediates from which SVs may regenerate. In endophilin, synaptojanin, and dynamin mutants, affecting endocytosis and vesicle scission, resolving 100nm vesicles was delayed (>20 s). In dynamin mutants, 100nm vesicles were abundant and persistent, sometimes continuous with the plasma membrane; incomplete budding of smaller vesicles from 100nm vesicles further implicates dynamin in regenerating SVs from bulk-endocytosed vesicles. Synaptic recovery after exhaustive activity is slow, and different time scales of recovery at ultrastructural, physiological, and behavioral levels indicate multiple contributing processes. Similar processes may jointly account for slow recovery from acute seizures also in higher animals.Efficient chemical synaptic neurotransmission requires synaptic vesicle (SV) biogenesis, transmitter loading, membrane approximation and docking, priming, fusion, and release of transmitter (1–3). These processes are followed by retrieval of membrane and proteins from the plasma membrane (PM) via endocytosis (4, 5). Particularly, sustained SV release relies on a tight coupling of exocytosis and endocytosis (5–8). During high-frequency or long-term neuronal activity, SVs need to be efficiently recycled, because, otherwise, the readily releasable pool and the (mobilized) resting pool of SVs would be depleted and transmission would seize (9, 10). After fusion, SV membranes and proteins are recycled (11). Coupling SV exocytosis with local recycling largely eliminates the dependence of chemical transmission on somatic de novo SV synthesis and transport. Thus far, these processes have been studied in dissected preparations or cultured cells and tissues; how and at which time scales this occurs within a live, nondissected animal (e.g., during seizures) is currently unclear. For example, patients suffering from a seizure often remain unconscious for minutes to hours (12, 13). Although fatigue at different levels of circuits and brain systems is likely to contribute, also physiological changes in chemical synapses may play a role in this slow recovery.Depending on the SV fusion rate, endocytosis occurs via different pathways: (i) clathrin-mediated endocytosis (14), supposedly accounting for most recycled SVs; (ii) fast “kiss-and-run” recycling, where SVs do not fully fuse but open a transient pore for transmitter release (15, 16); and (iii) clathrin-independent bulk-phase endocytosis, going along with high neuronal activity (17–20). Following membrane invagination, or to close the fusion pore, the GTPase dynamin finalizes, or at least speeds up, the process of membrane severing (21–25). Before scission by dynamin, the phospholipid phosphatase synaptojanin, via the membrane-binding Bin–amphiphysin–Rvs (BAR) domain protein endophilin, binds to the phospholipid enriched PM at endocytic sites and modifies lipids to promote scission (6, 26–28). Synaptojanin is also required after scission, particularly to uncoat endocytosed vesicles: by dephosphorylating the lipid head groups, synaptojanin releases the interaction of clathrin adaptors with the endocytosed membranes (27, 29). By recruiting clathrin adaptors, synaptotagmin/SNT-1 is also involved in SV recycling (30, 31).Across systems, different modes of endocytosis appear to be in effect: for example, in dynamin knockout mice, spontaneous activity induced endocytosed synaptic membrane, which appeared trapped as invaginations, tubulated and capped by clathrin-coated pits (21). Upon excessive stimulation, in inhibitory neurons, bulk-endocytosed, endosome-like structures resulted, which were severed from the PM despite the absence of dynamin (25). In neuron terminals of Drosophila in which clathrin function was acutely inhibited, SV recycling was impaired, whereas bulk endocytosis was still observed (22), and inhibition of dynamin uncovered different modes of SV recycling (32). Furthermore, when dynamin dephosphorylation was stalled, clathrin-mediated SV endocytosis was functional, but bulk endocytosis was affected (23). Thus, during moderate activity, SV recycling may occur by clathrin-mediated endocytosis (CME), to allow SV proteins to be retrieved and release sites to be “cleared” from integral membrane proteins. Upon prolonged or high-frequency activation, bulk endocytosis may follow in a clathrin-independent and, depending on synapse type, dynamin-dependent or -independent fashion, whereas resolution of the invaginated membrane structures may again be clathrin-dependent. However, in retinal bipolar cells, clathrin was required for a slow (τ ≈ 10–20 s) but not a fast (τ ≈ 1–2 s) phase of endocytosis (33), the latter of which was shown to depend on endophilin (34). In Caenorhabditis elegans, clathrin inactivation, surprisingly, had no effect on normal chemical transmission, and yet SV size was altered (35). Thus, it is unclear whether CME is required for SV endocytosis in C. elegans or is needed at a later step (e.g., following a different endocytic pathway) to shape new SVs. Bulk endocytosis can rapidly remove membrane material from the PM after excessive fusion of many SVs in vitro and was observed in experimental paradigms involving long-term electrical or chemical stimulation and pharmacological treatment (36–42), and yet it has not been studied in an intact animal.How the diverse endocytic events differentially contribute to the dynamic refilling of different SV pools is only partially understood. Because virtually all processes at active zones (AZs) occur at scales below the diffraction limit of light microscopy, it is difficult to study their dynamic behavior during and after stimulation. Therefore, electron microscopy (EM) has been the method of choice to analyze SV pools and AZ morphology at high resolution. Although previous work could visualize triggered SV exocytosis and endocytosis, these dynamic processes are difficult to analyze at high temporal resolution using classical EM (36–39). The dependence on slow chemical-fixation techniques precluded the capture of precise time points during dynamic events and limited the preservation of synaptic structures. Both problems may be overcome using cryofixation by high-pressure freeze (HPF)-EM (43, 44). The requirement of endophilin, synaptojanin, clathrin, and other proteins for SV endocytosis has been studied to some extent in C. elegans, also by EM (27, 28, 35, 43, 45–47). However, this was not done in a temporally resolved fashion relative to a stimulus (i.e., only steady-state “snapshots” were analyzed), and also HPF-EM has not yet been used in this context. How C. elegans synapses regulate endocytosis during and following periods of extreme activity, possibly by different modes of endocytosis, and which proteins are required for this, is unknown.Using a combination of channelrhodopsin-2 (ChR2)-mediated photostimulation of neurons and electrophysiological analysis in dissected animals (48, 49), as well as photostimulation followed by HPF-EM in intact animals, we monitored dynamic processes at AZs in three dimensions at EM resolution, and in a time-dependent manner. We studied the kinetics of docked SV depletion and recovery, as well as the generation and decomposition of bulk-endocytosed vesicles, during and following prolonged photostimulation. Whereas behavioral recovery required 60 s, synapses became fully competent to release transmitter only after ∼20 s, in line with morphological recovery of most docked SVs, whereas spontaneous release occurred at normal rates right after the stimulus. In addition, we found formation and disintegration of large (50–200 nm) bulk-endocytosed vesicles, within 11 and 8 s, respectively, the disassembly of which was largely delayed in animals expressing mutant endophilin, synaptojanin, and dynamin proteins.     

Variable priming of a docked synaptic vesicle

The priming of a docked synaptic vesicle determines the probability of its membrane (VM) fusing with the presynaptic membrane (PM) when a nerve impulse arrives. To gain insight into the nature of priming, we searched by electron tomography for structural relationships correlated with fusion probability at active zones of axon terminals at frog neuromuscular junctions. For terminals fixed at rest, the contact area between the VM of docked vesicles and PM varied >10-fold with a normal distribution. There was no merging of the membranes. For terminals fixed during repetitive evoked synaptic transmission, the normal distribution of contact areas was shifted to the left, due in part to a decreased number of large contact areas, and there was a subpopulation of large contact areas where the membranes were hemifused, an intermediate preceding complete fusion. Thus, fusion probability of a docked vesicle is related to the extent of its VM–PM contact area. For terminals fixed 1 h after activity, the distribution of contact areas recovered to that at rest, indicating the extent of a VM–PM contact area is dynamic and in equilibrium. The extent of VM–PM contact areas in resting terminals correlated with eccentricity in vesicle shape caused by force toward the PM and with shortness of active zone material macromolecules linking vesicles to PM components, some thought to include Ca²⁺ channels. We propose that priming is a variable continuum of events imposing variable fusion probability on each vesicle and is regulated by force-generating shortening of active zone material macromolecules in dynamic equilibrium.Synaptic vesicles (SVs) move toward and dock on (are held in contact with) the presynaptic plasma membrane (PM) of a neuron’s axon terminal before fusing with the PM and releasing their neurotransmitter into the synaptic cleft to mediate synaptic transmission (1, 2). Docking is achieved by force-generating interactions of the vesicle membrane (VM) protein synaptobrevin with the PM proteins syntaxin and SNAP25 (3, 4). These interactions, which produce force by forming a coiled coil called the SNARE core complex, are regulated by auxiliary proteins (1, 5–7). Such force on the VM–PM contact site may also play a role in their fusion (8). At typical synapses, docking and fusion take place at structurally specialized regions along the PM called active zones (9, 10). Several lines of evidence suggest that the formation of the SNARE core complex occurs in the macromolecules composing the common active zone organelle, active zone material (AZM) (2, 11–14), which is positioned near Ca²⁺ channels concentrated in the PM at active zones (15–17). Influx of Ca²⁺ through the channels after the arrival of a nerve impulse triggers fusion of the VM of docked SVs with the PM.Priming is a step in synaptic transmission between the docking of an SV on, and fusion with, the PM and accounts for the observation that relatively few docked SVs fuse with the PM after the arrival of a nerve impulse (18). It has been suggested that priming transitions docked SVs from fusion-incompetent (i.e., having 0% fusion probability) to fusion-competent (i.e., having 100% fusion probability), in a binary way (reviewed in ref. 19). However, as described for frog neuromuscular junctions (20–23), the number of SVs that can fuse with the PM after arrival of an impulse varies greatly with differing concentrations of cytosolic Ca²⁺, indicating that priming is more complex than a simple binary transition. Biochemical and electrophysiological approaches have provided evidence that priming is mediated by interactions between the SNARE proteins and their regulators (7, 12–14, 24) and can involve differences in positioning of docked SVs relative to Ca²⁺ channels (25). Biochemistry has also led to the suggestion that primed SVs may become deprimed (26).We have previously shown by electron tomography on frog neuromuscular junctions (NMJs) fixed at rest that there are, for docked SVs, variations in the extent of the VM–PM contact area and in the length of the several AZM macromolecules linking the VM to the PM, the so-called ribs, pegs, and pins (2, 27). Here, we examined these, and other, structural variations in the same axon terminals fixed at rest, during repetitive evoked synaptic activity, or after recovery from such activity, with a view toward testing and extending our understanding of the processes that regulate priming. Our findings suggest a model in which all docked SVs are primed to varying degrees by a reversible continuum of AZM-mediated forces on them, and it is the degree of priming at any moment that determines the probability of a docked SV fusing with the PM upon arrival of a nerve impulse.     

Readily releasable vesicles recycle at the active zone of hippocampal synapses

During the synaptic vesicle cycle, synaptic vesicles fuse with the plasma membrane and recycle for repeated exo/endocytic events. By using activity-dependent N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino) styryl) pyridinium dibromide dye uptake combined with fast (<1 s) microwave-assisted fixation followed by photoconversion and ultrastructural 3D analysis, we tracked endocytic vesicles over time, “frame by frame.” The first retrieved synaptic vesicles appeared 4 s after stimulation, and these endocytic vesicles were located just above the active zone. Second, the retrieved vesicles did not show any sign of a protein coat, and coated pits were not detected. Between 10 and 30 s, large labeled vesicles appeared that had up to 5 times the size of an individual synaptic vesicle. Starting at around 20 s, these large labeled vesicles decreased in number in favor of labeled synaptic vesicles, and after 30 s, labeled vesicles redocked at the active zone. The data suggest that readily releasable vesicles are retrieved as noncoated vesicles at the active zone.The mechanisms that govern synaptic vesicle (SV) retrieval have been debated since the discovery of the SV cycle (1, 2). Currently the two original mechanisms via coated vesicles and via “kiss and run” are proposed for mammalian excitatory central synapses (3–6). The proposed clathrin-mediated mechanism that retrieves the membrane via coated vesicles has comparable slow kinetics (7) (15–40 s until the endocytic vesicle separates from the plasma membrane) and a retrieval site outside the active zone (8, 9). First, the SV fully collapses into the release site and diffuses outside the active zone either as an entity or by its parts. At regions outside the active zone, coated pits form that sort SV proteins, and eventually a coated endocytic vesicle pinches off the plasma membrane. It is generally believed that coated vesicles shed their coat and fuse with early endosomes from which SVs bud off that join the SV cluster (8). This endosomal budding step is also believed to be mediated via coated vesicles. In contrast, SV retrieval via kiss and run has faster kinetics (10, 11) (<1 s), and SVs are retrieved at the active zone. During kiss and run, the SV is thought to maintain its identity and SVs are available for redocking and rapid reuse (12–14). Because SVs maintain their identity, fusion steps with potential endosomal compartments after endocytosis are not believed to occur after kiss and run.There is overwhelming evidence that SV retrieval at mammalian central synapses depends on the major coat protein clathrin, but the visualization of coated vesicles shortly after physiological stimulation has only been shown for lower vertebrate synapses (8, 15, 16). Kiss and run, on the other hand, is not generally accepted as a retrieval mechanism at mammalian central synapses. Several fluorescent imaging techniques (e.g., pHluorin-based SV protein chimeras and nanoparticles) recently provided unique insights into kiss and run (6), but many open questions remain. The visualization of a labeled endocytic vesicle at or near the active zone right after a physiological stimulus would provide elegant additional proof for kiss and run. More so, if one could follow such a labeled vesicle until “redocking,” it would greatly facilitate the investigation of the various steps SVs pass through on their way through the SV cycle.Here, we introduce a technique that is based on activity-dependent labeling of SV retrieval with N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino) styryl) pyridinium dibromide (FM1-43) followed by photoconversion and electron microscopic 3D analysis. This technique is combined with fast microwave-assisted fixation, ensuring a high time resolution that allows tracking of endocytic vesicles “frame by frame”—that is, at distinct time points (0, 4, 10, 20, 30, and 40 s) after stimulation.     

Reprogramming of G protein-coupled receptor recycling and signaling by a kinase switch

The postendocytic recycling of signaling receptors is subject to multiple requirements. Why this is so, considering that many other proteins can recycle without apparent requirements, is a fundamental question. Here we show that cells can leverage these requirements to switch the recycling of the beta-2 adrenergic receptor (B2AR), a prototypic signaling receptor, between sequence-dependent and bulk recycling pathways, based on extracellular signals. This switch is determined by protein kinase A-mediated phosphorylation of B2AR on the cytoplasmic tail. The phosphorylation state of B2AR dictates its partitioning into spatially and functionally distinct endosomal microdomains mediating bulk and sequence-dependent recycling, and also regulates the rate of B2AR recycling and resensitization. Our results demonstrate that G protein-coupled receptor recycling is not always restricted to the sequence-dependent pathway, but may be reprogrammed as needed by physiological signals. Such flexible reprogramming might provide a versatile method for rapidly modulating cellular responses to extracellular signaling.How proteins are sorted in the endocytic pathway is a fundamental question in cell biology. This is especially relevant for signaling receptors, given that relatively small changes in rates of receptor sorting into the recycling pathway can cause significant changes in surface receptors, and hence in cellular sensitivity (1–3). Our knowledge of receptor signaling and trafficking comes mainly from studying examples such as the beta-2 adrenergic receptor (B2AR), a prototypical member of G protein-coupled receptor (GPCR) family, the largest family of signaling receptors (2–5). B2AR activation initiates surface receptor removal and transport to endosomes, causing cellular desensitization (6, 7). The rate and extent of resensitization is then determined by B2AR surface recycling (1–3, 8, 9).Interestingly, the recycling of signaling receptors is functionally distinct from the recycling of constitutively cycling proteins like the transferrin receptor (TfR) (1, 6, 10, 11). TfR recycles by “bulk” geometric sorting, largely independent of specific cytoplasmic sequences (12, 13). B2AR recycling, in contrast, requires a specific PSD95-Dlg1-zo-1 domain (PDZ)-ligand sequence on its C-terminal tail, which links the receptor to the actin cytoskeleton (14, 15). Recent work has identified physically and biochemically distinct microdomains on early endosomes that mediate B2AR recycling independent of TfR (14–16). Although the exact mechanisms of B2AR sorting into these domains remain under investigation, this sorting clearly requires specific sequence elements on B2AR (1, 10, 11, 17). Importantly, why signaling receptor sorting is subject to such specialized requirements, considering that cargo like TfR apparently can recycle without specific sequence requirements, is not clear (1, 12–16). One possibility is that these requirements allow signaling pathways to regulate and redirect receptor trafficking between different pathways as needed (17–19). Although this is an attractive idea, whether and how physiological signals regulate receptor sorting remain poorly understood (7, 19).Here we show that adrenergic signaling can switch B2AR recycling between the sequence-dependent and bulk recycling pathways. Adrenergic activation, via protein kinase A (PKA)-mediated B2AR phosphorylation on the cytoplasmic tail, restricts B2AR to spatially defined PDZ- and actin-dependent endosomal microdomains. Dephosphorylation of B2AR switches B2AR to the bulk (PDZ-independent) recycling pathway, causing faster recycling of B2AR and increased cellular sensitivity. Our results suggest that cells may leverage sequence requirements for rapid adaptive reprogramming of signaling receptor trafficking and cellular sensitivity.     

Deconstructing complexin function in activating and clamping Ca2+-triggered exocytosis by comparing knockout and knockdown phenotypes

Complexin, a presynaptic protein that avidly binds to assembled SNARE complexes, is widely acknowledged to activate Ca²⁺-triggered exocytosis. In addition, studies of invertebrate complexin mutants and of mouse neurons with a double knockdown (DKD) of complexin-1 and -2 suggested that complexin maintains the readily releasable pool (RRP) of vesicles and clamps spontaneous exocytosis. In contrast, studies of mouse neurons with a double knockout (DKO) of complexin-1 and -2, largely carried out in hippocampal autapses, did not detect changes in the RRP size or in spontaneous exocytosis. To clarify complexin function, we here directly compared in two different preparations, cultured cortical and olfactory bulb neurons, the phenotypes of complexin DKD and DKO neurons. We find that complexin-deficient DKD and DKO neurons invariably exhibit a ∼50% decrease in vesicle priming. Moreover, the DKD consistently increased spontaneous exocytosis, but the DKO did so in cortical but not olfactory bulb neurons. Furthermore, the complexin DKD but not the complexin DKO caused a compensatory increase in complexin-3 and -4 mRNA levels; overexpression of complexin-3 but not complexin-1 increased spontaneous exocytosis. Complexin-3 but not complexin-1 contains a C-terminal lipid anchor attaching it to the plasma membrane; addition of a similar lipid anchor to complexin-1 converted complexin-1 from a clamp into an activator of spontaneous exocytosis. Viewed together, our data suggest that complexin generally functions in priming and Ca²⁺ triggering of exocytosis, and additionally contributes to the control of spontaneous exocytosis dependent on the developmental history of a neuron and on the subcellular localization of the complexin.Complexin is a small, evolutionarily conserved protein that binds to SNARE complexes and has been variably associated with clamping spontaneous synaptic vesicle exocytosis (“mini release”), priming vesicles for exocytosis, and assisting synaptotagmin in Ca²⁺ triggering of exocytosis (1, 2). Four complexins are expressed in brain. The highly abundant complexin-1 (Cpx1) and -2 (Cpx2) are soluble proteins (3), whereas the low-abundance complexin-3 (Cpx3) and -4 (Cpx4) are attached to plasma membranes by a C-terminal lipid anchor (4). Complexins are conserved in invertebrates, which usually also express soluble and membrane-anchored forms (5, 6). In human patients, significant evidence links complexin expression to schizophrenia, although it is unclear whether the observed changes are cause or consequence of the disorder (7–10).All complexins are composed of four domains that are packed into a short ∼130-residue sequence. These domains are composed of unstructured N-terminal and C-terminal regions that flank adjacent accessory and central α-helices (11). In Cpx1 and Cpx2, the N-terminal region is required for Ca²⁺ triggering of release, the accessory α-helix is essential for clamping release, the central α-helix binds to assembling SNARE complexes, and the C-terminal region is essential for both priming and clamping vesicles (5, 6, 11–18). Loss-of-function experiments combined with rescues demonstrated that the three principal functions of complexins (in priming, clamping, and Ca²⁺ triggering of exocytosis) are independent of each other because some mutations selectively impair one or two of the three functions, but leave the other function(s) intact (14, 15, 17). However, all complexin functions require its central α-helix that binds to SNARE complexes, suggesting that complexin always acts in association with SNARE complexes (14).It is generally agreed that complexins activate Ca²⁺-triggered exocytosis, but considerable uncertainty exists about the relative importance of the other two functions of complexin, i.e., their role in priming and clamping exocytosis. Most of this uncertainty is based on differences in approach. For example, in vitro fusion assays initially only described a clamping function of complexin, thus emphasizing this role (19–22). Analysis of mouse complexin double-knockout (DKO) neurons lacking Cpx1 and Cpx2, conversely, initially demonstrated only a Ca²⁺-triggering function of complexin, thus suggesting that mammalian complexins primarily function in this role (4, 13). Later experiments with liposomes, however, also revealed an activating function for complexin (23–26). Conversely, subsequent experiments suggested that the energy barrier to fusion may be increased in complexin DKO neurons, but did not detect an alteration in the total size of the readily releasable pool (RRP) of vesicles (16). In contrast to the DKO studies, studies with neurons overexpressing dominant-negative Cpx1 (12) or with double-knockdown (DKD) neurons containing dramatically reduced Cpx1 and Cpx2 levels identified phenotypes in all three presumed functional domains of complexin—clamping, priming, and Ca²⁺ triggering of exocytosis (14, 15, 17). Furthermore, studies of genetic complexin mutants in Drosophila and Caenorhabditis elegans also revealed phenotypes in clamping, priming, and Ca²⁺ triggering (5, 6, 18, 27–30).Viewed together, the studies in invertebrates and DKD neurons broadly agreed in suggesting complexin functions in the clamping, priming, and Ca²⁺ activation of exocytosis, whereas the studies in DKO neurons indicated a more restricted complexin function in Ca²⁺ activation of exocytosis. Because a genetic DKO is likely technically more reliable than an shRNA-mediated DKD, the discrepancies between the DKO and DKD results in mouse neurons give rise to concern. As a further complicating factor, the DKO studies were largely carried out in autapses in which a hippocampal neuron is grown in isolation so that it forms synapses on itself, whereas the DKD experiments were performed on synapses formed between neurons cultured at high density.The confusion about the functions of complexin has hindered our understanding of the mechanism of exocytosis. Increasing numbers of studies indicate that complexin performs a fundamental general function in Ca²⁺-stimulated exocytosis that is not limited to the synapse. For example, complexin is involved in chromaffin granule exocytosis, which resembles the exocytosis of large dense-core vesicles (31), and is essential for Ca²⁺-induced exocytosis of IGF1-containing secretory vesicles in mitral neurons that depends on a different synaptotagmin isoform (Syt10) than synaptic and neuroendocrine exocytosis (Syt1, Syt2, Syt7, and Syt9) (32). A major cause for the uncertainty of complexin function at least in mammalian neurons is that the phenotypes of the DKO and DKD neurons have not been directly compared in the same electrophysiological preparation. Therefore, we have in the present study systematically examined the effects of the separate or combined DKO and DKD manipulations on synaptic transmission in two different types of cultured neurons, cortical neurons and olfactory bulb neurons.     

N terminus of ASPP2 binds to Ras and enhances Ras/Raf/MEK/ERK activation to promote oncogene-induced senescence

The ASPP2 (also known as 53BP2L) tumor suppressor is a proapoptotic member of a family of p53 binding proteins that functions in part by enhancing p53-dependent apoptosis via its C-terminal p53-binding domain. Mounting evidence also suggests that ASPP2 harbors important nonapoptotic p53-independent functions. Structural studies identify a small G protein Ras-association domain in the ASPP2 N terminus. Because Ras-induced senescence is a barrier to tumor formation in normal cells, we investigated whether ASPP2 could bind Ras and stimulate the protein kinase Raf/MEK/ERK signaling cascade. We now show that ASPP2 binds to Ras–GTP at the plasma membrane and stimulates Ras-induced signaling and pERK1/2 levels via promoting Ras–GTP loading, B-Raf/C-Raf dimerization, and C-Raf phosphorylation. These functions require the ASPP2 N terminus because BBP (also known as 53BP2S), an alternatively spliced ASPP2 isoform lacking the N terminus, was defective in binding Ras–GTP and stimulating Raf/MEK/ERK signaling. Decreased ASPP2 levels attenuated H-RasV12–induced senescence in normal human fibroblasts and neonatal human epidermal keratinocytes. Together, our results reveal a mechanism for ASPP2 tumor suppressor function via direct interaction with Ras–GTP to stimulate Ras-induced senescence in nontransformed human cells.ASPP2, also known as 53BP2L, is a tumor suppressor whose expression is altered in human cancers (1). Importantly, targeting of the ASPP2 allele in two different mouse models reveals that ASPP2 heterozygous mice are prone to spontaneous and γ-irradiation–induced tumors, which rigorously demonstrates the role of ASPP2 as a tumor suppressor (2, 3). ASPP2 binds p53 via the C-terminal ankyrin-repeat and SH3 domain (4–6), is damage-inducible, and can enhance damage-induced apoptosis in part through a p53-mediated pathway (1, 2, 7–10). However, it remains unclear what biologic pathways and mechanisms mediate ASPP2 tumor suppressor function (1). Indeed, accumulating evidence demonstrates that ASPP2 also mediates nonapoptotic p53-independent pathways (1, 3, 11–15).The induction of cellular senescence forms an important barrier to tumorigenesis in vivo (16–21). It is well known that oncogenic Ras signaling induces senescence in normal nontransformed cells to prevent tumor initiation and maintain complex growth arrest pathways (16, 18, 21–24). The level of oncogenic Ras activation influences its capacity to activate senescence; high levels of oncogenic H-RasV12 signaling leads to low grade tumors with senescence markers, which progress to invasive cancers upon senescence inactivation (25). Thus, tight control of Ras signaling is critical to ensure the proper biologic outcome in the correct cellular context (26–28).The ASPP2 C terminus is important for promoting p53-dependent apoptosis (7). The ASPP2 N terminus may also suppress cell growth (1, 7, 29–33). Alternative splicing can generate the ASPP2 N-terminal truncated protein BBP (also known as 53BP2S) that is less potent in suppressing cell growth (7, 34, 35). Although the ASPP2 C terminus mediates nuclear localization, full-length ASPP2 also localizes to the cytoplasm and plasma membrane to mediate extranuclear functions (7, 11, 12, 36). Structural studies of the ASPP2 N terminus reveal a β–Grasp ubiquitin-like fold as well as a potential Ras-binding (RB)/Ras-association (RA) domain (32). Moreover, ASPP2 can promote H-RasV12–induced senescence (13, 15). However, the molecular mechanism(s) of how ASPP2 directly promotes Ras signaling are complex and remain to be completely elucidated.Here, we explore the molecular mechanisms of how Ras-signaling is enhanced by ASPP2. We demonstrate that ASPP2: (i) binds Ras-GTP and stimulates Ras-induced ERK signaling via its N-terminal domain at the plasma membrane; (ii) enhances Ras-GTP loading and B-Raf/C-Raf dimerization and forms a ASPP2/Raf complex; (iii) stimulates Ras-induced C-Raf phosphorylation and activation; and (iv) potentiates H-RasV12–induced senescence in both primary human fibroblasts and neonatal human epidermal keratinocytes. These data provide mechanistic insight into ASPP2 function(s) and opens important avenues for investigation into its role as a tumor suppressor in human cancer.     

Mitigation of acute kidney injury by cell-cycle inhibitors that suppress both CDK4/6 and OCT2 functions

Acute kidney injury (AKI) is a potentially fatal syndrome characterized by a rapid decline in kidney function caused by ischemic or toxic injury to renal tubular cells. The widely used chemotherapy drug cisplatin accumulates preferentially in the renal tubular cells and is a frequent cause of drug-induced AKI. During the development of AKI the quiescent tubular cells reenter the cell cycle. Strategies that block cell-cycle progression ameliorate kidney injury, possibly by averting cell division in the presence of extensive DNA damage. However, the early signaling events that lead to cell-cycle activation during AKI are not known. In the current study, using mouse models of cisplatin nephrotoxicity, we show that the G1/S-regulating cyclin-dependent kinase 4/6 (CDK4/6) pathway is activated in parallel with renal cell-cycle entry but before the development of AKI. Targeted inhibition of CDK4/6 pathway by small-molecule inhibitors palbociclib (PD-0332991) and ribociclib (LEE011) resulted in inhibition of cell-cycle progression, amelioration of kidney injury, and improved overall survival. Of additional significance, these compounds were found to be potent inhibitors of organic cation transporter 2 (OCT2), which contributes to the cellular accumulation of cisplatin and subsequent kidney injury. The unique cell-cycle and OCT2-targeting activities of palbociclib and LEE011, combined with their potential for clinical translation, support their further exploration as therapeutic candidates for prevention of AKI.Cell division is a fundamental biological process that is tightly regulated by evolutionarily conserved signaling pathways (1, 2). The initial decision to start cell division, the fidelity of subsequent DNA replication, and the final formation of daughter cells is monitored and regulated by these essential pathways (2–6). The cyclin-dependent kinases (CDKs) are the central players that orchestrate this orderly progression through the cell cycle (1, 2, 6, 7). The enzymatic activity of CDKs is regulated by complex mechanisms that include posttranslational modifications and expression of activating and inhibitory proteins (1, 2, 6, 7). The spatial and temporal changes in the activity of these CDK complexes are thought to generate the distinct substrate specificities that lead to sequential and unidirectional progression of the cell cycle (1, 8, 9).Cell-cycle deregulation is a universal feature of human cancer and a long-sought-after target for anticancer therapy (1, 10–13). Frequent genetic or epigenetic changes in mitogenic pathways, CDKs, cyclins, or CDK inhibitors are observed in various human cancers (1, 4, 11). In particular, the G1/S-regulating CDK4/6–cyclin D–inhibitors of CDK4 (INK4)–retinoblastoma (Rb) protein pathway frequently is disrupted in cancer cells (11, 14). These observations provided an impetus to develop CDK inhibitors as anticancer drugs. However, the earlier class of CDK inhibitors had limited specificity, inadequate clinical activity, poor pharmacokinetic properties, and unacceptable toxicity profiles (10, 11, 14, 15). These disappointing initial efforts now have been followed by the development of the specific CDK4/6 inhibitors palbociclib (PD0332991), ribociclib (LEE011), and abemaciclib (LY2835219), which have demonstrated manageable toxicities, improved pharmacokinetic properties, and impressive antitumor activity, especially in certain forms of breast cancer (14, 16). Successful early clinical trials with these three CDK4/6 inhibitors have generated cautious enthusiasm that these drugs may emerge as a new class of anticancer agents (14, 17). Palbociclib recently was approved by Food and Drug Administration for the treatment of metastatic breast cancer and became the first CDK4/6 inhibitor approved for anticancer therapy (18).In addition to its potential as an anticancer strategy, CDK4/6 inhibition in normal tissues could be exploited therapeutically for wide-ranging clinical conditions. For example, radiation-induced myelosuppression, caused by cell death of proliferating hematopoietic stem/progenitor cells, can be rescued by palbociclib (19, 20). Furthermore, cytotoxic anticancer agents cause significant toxicities to normal proliferating cells, which possibly could be mitigated by the concomitant use of CDK4/6 inhibitors (20, 21). More broadly, cell-cycle inhibition could have beneficial effects in disorders in which maladaptive proliferation of normal cells contributes to the disease pathology, as observed in vascular proliferative diseases, hyperproliferative skin diseases, and autoimmune disorders (22, 23). In support of this possibility, palbociclib treatment recently was reported to ameliorate disease progression in animal models of rheumatoid arthritis through cell-cycle inhibition of synovial fibroblasts (24).Abnormal cellular proliferation also is a hallmark of various kidney diseases (25), and cell-cycle inhibition has been shown to ameliorate significantly the pathogenesis of polycystic kidney disease (26), nephritis (27), and acute kidney injury (AKI) (28). Remarkably, during AKI, the normally quiescent renal tubular cells reenter the cell cycle (29–34), and blocking cell-cycle progression can reduce renal injury (28). Here, we provide evidence that the CDK4/6 pathway is activated early during AKI and demonstrate significant protective effects of CDK4/6 inhibitors in animal models of cisplatin-induced AKI. In addition, we found that the CDK4/6 inhibitors palbociclib and LEE011 are potent inhibitors of organic cation transporter 2 (OCT2), a cisplatin uptake transporter highly expressed in renal tubular cells (35–37). Our findings provide a rationale for the clinical development of palbociclib and LEE011 for the prevention and treatment of AKI.     

Fibroblasts secrete Slit2 to inhibit fibrocyte differentiation and fibrosis

Monocytes leave the blood and enter tissues. In healing wounds and fibrotic lesions, some of the monocytes differentiate into fibroblast-like cells called fibrocytes. In healthy tissues, even though monocytes enter the tissue, for unknown reasons, very few monocytes differentiate into fibrocytes. In this report, we show that fibroblasts from healthy human tissues secrete the neuronal guidance protein Slit2 and that Slit2 inhibits human fibrocyte differentiation. In mice, injections of Slit2 inhibit bleomycin-induced lung fibrosis. In lung tissue from pulmonary fibrosis patients with relatively normal lung function, Slit2 has a widespread distribution whereas, in patients with advanced disease, there is less Slit2 in the fibrotic lesions. These data may explain why fibrocytes are rarely observed in healthy tissues, may suggest that the relative levels of Slit2 present in healthy tissue and at sites of fibrosis may have a significant effect on the decision of monocytes to differentiate into fibrocytes, and may indicate that modulating Slit2 signaling may be useful as a therapeutic for fibrosis.To help form granulation tissue during wound healing, monocytes leave the circulation, enter the tissue, and differentiate into fibroblast-like cells called fibrocytes (1–4). Fibrocytes are also found in lesions associated with fibrotic diseases such as pulmonary fibrosis, congestive heart failure, cirrhosis of the liver, and nephrogenic systemic fibrosis (3, 5–9). Fibrocytes express markers of both hematopoietic cells (CD34, CD45, FcγR, LSP-1, and MHC class II) and stromal cells (collagens, fibronectin, and matrix metalloproteases) (2, 3, 10–12). Fibrocytes also promote angiogenesis by secreting VEGF, bFGF, IL-8, and PDGF and promote fibroblast proliferation, migration, and collagen production by secreting TGF-β and CTGF (13, 14). Fibrocyte recruitment and differentiation is regulated by a variety of factors (3, 15). In vitro, monocytes can differentiate into fibrocytes without the addition of any exogenous factors (5, 11, 12, 16–22). A key question about fibrocyte differentiation and fibrosis is why, in healthy tissues where monocytes and macrophages are readily identified, fibrocytes are rarely observed (3, 8, 23–26).In tissues, fibroblasts are a major cell population and can modulate the immune system (27–30). In this report, we show that fibroblasts secrete the neuronal guidance protein Slit2 and that Slit2 inhibits fibrocyte differentiation. In addition, we show that injections of Slit2 reduce bleomycin-induced pulmonary fibrosis in mice. Finally, we show that, in the mouse pulmonary fibrosis model as well as human patients with pulmonary fibrosis, there seems to be a decrease in Slit2 levels in the lungs, suggesting that pulmonary fibrosis may be in part a Slit2 deficiency disease. These data suggest that the relative level of Slit2 present at sites of wound healing, inflammation, and fibrosis may have a profound effect on the ability of monocytes to differentiate into fibrocytes.     

From the Cover: Feature Article: mTOR complex 2-Akt signaling at mitochondria-associated endoplasmic reticulum membranes (MAM) regulates mitochondrial physiology

The target of rapamycin (TOR) is a highly conserved protein kinase and a central controller of growth. Mammalian TOR complex 2 (mTORC2) regulates AGC kinase family members and is implicated in various disorders, including cancer and diabetes. Here we report that mTORC2 is localized to the endoplasmic reticulum (ER) subcompartment termed mitochondria-associated ER membrane (MAM). mTORC2 localization to MAM was growth factor-stimulated, and mTORC2 at MAM interacted with the IP₃ receptor (IP3R)-Grp75–voltage-dependent anion-selective channel 1 ER-mitochondrial tethering complex. mTORC2 deficiency disrupted MAM, causing mitochondrial defects including increases in mitochondrial membrane potential, ATP production, and calcium uptake. mTORC2 controlled MAM integrity and mitochondrial function via Akt mediated phosphorylation of the MAM associated proteins IP3R, Hexokinase 2, and phosphofurin acidic cluster sorting protein 2. Thus, mTORC2 is at the core of a MAM signaling hub that controls growth and metabolism.Mitochondria-associated endoplasmic reticulum (ER) membrane (MAM) is a subcompartment of the ER that forms a quasisynaptic structure with mitochondria. The main function of this membrane is to facilitate the transfer of lipids and calcium between the two organelles. MAM thereby controls mitochondrial physiology and apoptosis (1, 2). MAM also mediates ER homeostasis and lipid biosynthesis by harboring chaperones and several key lipid synthesis enzymes (3–6). In mammalian MAM, the ER and mitochondria are physically tethered to each other by the IP₃ receptor (IP3R)-Grp75-VDAC1 (voltage-dependent anion-selective channel 1) trimeric complex (7) and by dimers of the mitofusin (Mfn) proteins Mfn1 and Mfn2 (8) (Fig. S1H). The σ-1 receptor also stabilizes MAM by interacting with IP3R and VDAC (9). MAM formation is regulated by multiple signaling inputs, including calcium and possibly growth factors (10–12). However, the mechanism(s) that controls MAM formation is largely unknown other than it involves recruitment of MAM components by the MAM resident proteins phosphofurin acidic cluster sorting protein 2 (PACS2) and Rab32 (13–15). Akt, an AGC family kinase that is also found at MAM (16), phosphorylates PACS2 (17), but it remains to be determined whether Akt is involved in mediating MAM integrity.Akt, often up-regulated in cancer, also phosphorylates hexokinase 2 (HK2) to promote association of HK2 with the MAM protein VDAC1 (18, 19). This association, possibly at MAM (20, 21), enables HK2, using ATP exiting mitochondria through VDAC1, to phosphorylate glucose and thereby stimulate glycolysis (22). Conversely, upon inhibition of Akt, HK2 dissociates from VDAC1, causing VDAC1 closure and increased mitochondrial membrane potential (19). This regulation of HK2 by Akt has been proposed to account for enhanced glycolysis in cancer cells, also known as the Warburg effect (23). Furthermore, Akt regulates calcium release from MAM by phosphorylating IP3R, thereby controlling apoptosis (24–26). Thus, MAM is increasingly recognized as a signaling hub controlling cell physiology (15), and is implicated in a wide spectrum of diseases, including cancer, neurodegenerative disorders, inflammation, and infection (27).The target of rapamycin (TOR) pathway is a cellular signaling cascade that, like mitochondria, is present in all eukaryotes (28, 29). TOR integrates and relays signals from both extra- and intracellular sources (e.g., growth factors, nutrients, and cellular energy levels), and thereby instructs the cell to grow. TOR is found in two structurally and functionally distinct protein complexes that in mammalian cells are termed mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) (30). mTORC2 comprises mTOR, rictor, mammalian lethal with SEC13 protein 8 (mLST8), stress-activated protein kinase (SAPK)-interacting protein (Sin1), and protor [also known as proline-rich protein 5 (PRR5)] (31), and phosphorylates AGC kinases, such as Akt, serum/glucocorticoid-regulated kinase 1 (SGK1), and PKC, all of which are linked to cancer and diabetes (32). Growth factors activate mTORC2 by promoting mTORC2-ribosome association in a PI3K-dependent manner (33, 34). mTORC2 is antiapoptotic, presumably via its role in phosphorylating and activating Akt (34–38).Various observations indicate that mTORC2 is linked to both the ER and mitochondria. Recent findings suggest that mTORC2 is at the ER, possibly through interaction with ER-bound ribosomes (34, 39). mTORC2 phosphorylates Akt at the ER (39, 40), and mTORC2 signaling is sensitive to ER stress (41, 42). In Chlamydomonas, TOR associates with membranes from the ER (43). With regard to mitochondria, mTOR has been observed in close proximity to the outer mitochondrial membrane (44), and mTOR and mLST8 interact with the mitochondrial outer-membrane protein VDAC1 (45) and the mitochondria-associated protein Grp75 (46), respectively. mTORC2 regulates the cellular distribution of mitochondria (47), and mTORC2-activated Akt is associated with mitochondria (18, 48, 49). Pink1, a regulator of mitochondrial function, has been implicated in mTORC2 activation (50). mTORC2-addicted cancer cells exhibit enhanced dependence on mitochondria, Rab32 and HK2 (51). Finally, Barquilla et al. reported that TORC2 in trypanosomes is localized to both ER and mitochondria (52). Thus, mTORC2 has been physically and functionally linked to both the ER and mitochondria.Here we investigate the localization of mTORC2. We show that ribosome-bound mTORC2 is at MAM. Localization to MAM is growth factor-dependent. MAM-associated mTORC2 activates Akt and thereby controls MAM integrity, mitochondrial metabolism, and cell survival. Thus, our findings describe a critical role for mTORC2 in a MAM signaling hub.     

Electrochemical tuning of vertically aligned MoS2 nanofilms and its application in improving hydrogen evolution reaction

The ability to intercalate guest species into the van der Waals gap of 2D layered materials affords the opportunity to engineer the electronic structures for a variety of applications. Here we demonstrate the continuous tuning of layer vertically aligned MoS₂ nanofilms through electrochemical intercalation of Li⁺ ions. By scanning the Li intercalation potential from high to low, we have gained control of multiple important material properties in a continuous manner, including tuning the oxidation state of Mo, the transition of semiconducting 2H to metallic 1T phase, and expanding the van der Waals gap until exfoliation. Using such nanofilms after different degree of Li intercalation, we show the significant improvement of the hydrogen evolution reaction activity. A strong correlation between such tunable material properties and hydrogen evolution reaction activity is established. This work provides an intriguing and effective approach on tuning electronic structures for optimizing the catalytic activity.Layer-structured 2D materials are an interesting family of materials with strong covalent bonding within molecular layers and weak van der Waals interaction between layers. Beyond intensively studied graphene-related materials (1–4), there has been recent strong interest in other layered materials whose vertical thickness can be thinned down to less than few nanometers and horizontal width can also be reduced to nanoscale (5–9). The strong interest is driven by their interesting physical and chemical properties (2, 10) and their potential applications in transistors, batteries, topological insulators, thermoelectrics, artificial photosynthesis, and catalysis (4, 11–25).One of the unique properties of 2D layered materials is their ability to intercalate guest species into their van der Waals gaps, opening up the opportunities to tune the properties of materials. For example, the spacing between the 2D layers could be increased by intercalation such as lithium (Li) intercalated graphite or molybdenum disulfide (MoS₂) and copper intercalated bismuth selenide (26–29). The electronic structures of the host lattice, such as the charge density, anisotropic transport, oxidation state, and phase transition, may also be changed by different species intercalation (26, 27).As one of the most interesting layered materials, MoS₂ has been extensively studied in a variety of areas such as electrocatalysis (20–22, 30–36). It is known that there is a strong correlation between the electronic structure and catalytic activity of the catalysts (20, 37–41). It is intriguing to continuously tune the morphology and electronic structure of MoS₂ and explore the effects on MoS₂ hydrogen evolution reaction (HER) activity. Very recent studies demonstrated that the monolayered MoS₂ and WS₂ nanosheets with 1T metallic phase synthesized by chemical exfoliation exhibited superior HER catalytic activity to those with 2H semiconducting phase (35, 42), with a possible explanation that the strained 1T phase facilitates the hydrogen binding process during HER (42). However, it only offers two end states of materials and does not offer a continuous tuning. A systematic investigation to correlate the gradually tuned electronic structure, including oxidation state shift and semiconducting–metallic phase transition, and the corresponding HER activity is important but unexplored. We believe that the Li electrochemical intercalation method offers a unique way to tune the catalysts for optimization.In this paper, we demonstrate that the layer spacing, oxidation state, and the ratio of 2H semiconducting to 1T metallic phase of MoS₂ HER catalysts were continuously tuned by Li intercalation to different voltages vs. Li⁺/Li in nanofilms with molecular layers perpendicular to the substrates. Correspondingly, the catalytic activity for HER was observed to be continuously tuned. The lower oxidation state of Mo and 1T metallic phase of MoS₂ turn out to have better HER catalytic activities. The performance of MoS₂ catalyst on both flat and 3D electrodes was dramatically improved when it was discharged to low potentials vs. Li⁺/Li.     

Activity-dependent BDNF release via endocytic pathways is regulated by synaptotagmin-6 and complexin

Brain-derived neurotrophic factor (BDNF) is known to modulate synapse development and plasticity, but the source of synaptic BDNF and molecular mechanisms regulating BDNF release remain unclear. Using exogenous BDNF tagged with quantum dots (BDNF-QDs), we found that endocytosed BDNF-QDs were preferentially localized to postsynaptic sites in the dendrite of cultured hippocampal neurons. Repetitive neuronal spiking induced the release of BDNF-QDs at these sites, and this process required activation of glutamate receptors. Down-regulating complexin 1/2 (Cpx1/2) expression eliminated activity-induced BDNF-QD secretion, although the overall activity-independent secretion was elevated. Among eight synaptotagmin (Syt) isoforms examined, down-regulation of only Syt6 impaired activity-induced BDNF-QD secretion. In contrast, activity-induced release of endogenously synthesized BDNF did not depend on Syt6. Thus, neuronal activity could trigger the release of endosomal BDNF from postsynaptic dendrites in a Cpx- and Syt6-dependent manner, and endosomes containing BDNF may serve as a source of BDNF for activity-dependent synaptic modulation.Brain-derived neurotrophic factor (BDNF), a member of neurotrophin family of secreted factors, is known to play important regulatory roles in neuronal survival and differentiation, synaptic development and plasticity, as well as many cognitive functions (1, 2). The findings that the synthesis and secretion of neurotrophins are regulated by neuronal activity prompted the suggestion that neurotrophins may regulate activity-dependent neural plasticity in the brain (3). Indeed, there is now substantial evidence indicating that activity-induced BDNF secretion at glutamatergic synapses is essential for long-term potentiation (LTP) (4), a cellular substrate for the learning and memory functions of neural circuits.The BDNF protein is first synthesized in the endoplasmic reticulum as a precursor protein, prepro-BDNF, which is then converted to pro-BDNF by removal of the signal peptide and further cleaved to generate the mature BDNF (5). Immunostaining and electron microscope studies using specific antibodies to the pro- and mature form of BDNF showed that pro-BDNF is colocalized with mature BDNF in secretory granules in presynaptic axon terminals (6), suggesting that the cleavage may occur in the secretory granule. However, under some experimental conditions, the processing of pro-BDNF into mature BDNF may occur extracellularly (7, 8). The secretory granule containing BDNF and pro-BDNF could undergo exocytosis upon neuronal excitation, as readily demonstrated in cell cultures using ELISA or fluorescent protein-tagged BDNF expressed in the neuron (9, 10). Besides secretory granules, neurotrophins within neuronal cytoplasm could also reside in endosomal compartments, resulting from endocytic uptake of extracellular neurotrophins secreted by the neuron itself or other nearby cells. Initially discovered as factors derived by target tissues, neurotrophins exert their actions via binding to neuronal surface receptors, including tropomyosin related kinase B (TrkB) and pan-neurotrophin receptor p75 (11). Neurotrophin binding to its receptor leads to cytoplasmic signaling as well as internalization of the neurotrophin-receptor complexes. These endocytosed neurotrophin-receptor complexes remain active in the form of “signaling endosomes” that could be transported over long distances within neuronal cytoplasm to exert its regulatory functions within the neuron (12–15). In this study, we have examined the possibility that these endosomes may undergo activity-dependent exocytosis at postsynaptic dendrites, thus providing an additional source of synaptic BDNF.To mark endosomes containing BDNF via the endocytic pathway, it is necessary to monitor BDNF trafficking in neurons. Although YFP-tagged BDNF has been used to study internalization of exogenous BDNF (16), such fluorescent protein-labeled BDNF was not suitable for real-time tracking of BDNF-containing endosomes at a high spatiotemporal resolution. In this study, we used BDNF linked to quantum dots (QDs), which are fluorescent nanoparticles with excellent photostability (17) and could be tracked in live cells with high signal-to-noise ratio and over unprecedented duration. This method has been used to examine endocytic recycling of synaptic vesicles (18) and axonal transport of endosomes containing neurotrophins (19, 20). In this study, we used time-lapse imaging of BDNF-QDs within cultured hippocampal neurons to monitor intracellular transport and localization of these endosomes. Furthermore, the sudden disappearance of cytoplasmic QD fluorescence in a solution containing fluorescence quencher was used to indicate the exocytosis of QD-containing endosomes. Previous studies have shown that extracellular false transmitters, soluble fluorescent markers, and membrane-bound fluorescent lipid dyes could be loaded into endosomes, which undergo exocytosis upon membrane depolarization (21–25). However, whether endosomes formed by receptor-mediated endocytosis is similarly regulated by activity remains unclear. Furthermore, the Ca²⁺-dependence and the kinetics of exocytosis of different endosomal vesicle populations may be differentially regulated by distinct vesicle-associated proteins.In the present study, we have explored the role of synaptotagmin (Syt) and complexin (Cpx) in regulating activity-induced exocytosis of BDNF-containing endosomes. As a universal cofactor in all Ca²⁺-triggered vesicular fusion reactions that have been examined (26), Cpx is known to serve both activating and clamping functions for vesicular exocytosis, by interacting with the Ca²⁺ sensor Syt and the assembled SNARE complexes at the plasma membrane (27). Various isoforms of Syt play distinct regulatory roles in various types of neurosecretion, presumably via their differential Ca²⁺ sensitivity. By manipulating the expression of various Syt and Cpx isoforms in cultured hippocampal neurons, we found that Syt6 and Cpx1/2 play essential regulatory roles in activity-dependent exocytosis of BDNF-containing endosomes. These results support the notion that BDNF-containing endosomes may serve as a source of extracellular BDNF for activity-dependent synaptic modulation and that Syt6 specifically regulates the exocytosis of BDNF-containing endosomes.     

TIPE2 protein serves as a negative regulator of phagocytosis and oxidative burst during infection

Phagocytosis and oxidative burst are two major effector arms of innate immunity. Although it is known that both are activated by Toll-like receptors (TLRs) and Rac GTPases, how their strengths are controlled in quiescent and TLR-activated cells is not clear. We report here that TIPE2 (TNFAIP8L2) serves as a negative regulator of innate immunity by linking TLRs to Rac. TLRs control the expression levels of TIPE2, which in turn dictates the strengths of phagocytosis and oxidative burst by binding to and blocking Rac GTPases. Consequently, TIPE2 knockout cells have enhanced phagocytic and bactericidal activities and TIPE2 knockout mice are resistant to bacterial infection. Thus, TIPE2 sets the strengths of phagocytosis and oxidative burst and may be targeted to effectively control infections.Phagocytosis and oxidative burst (or respiratory burst) are two fundamental effector mechanisms of innate immunity that work in concert to eliminate infectious microbes (1, 2). Phagocytosis allows the phagocytes of the immune system (monocytes and granulocytes) to engulf infectious microbes and to contain them in a special vacuole called a phagosome. Oxidative burst in turn injects into the vacuole reactive oxygen species (ROS) (e.g., superoxide radical and hydrogen peroxide) that kill the microbes. Deficiency in either of these innate immune mechanisms leads to immune deficiency and uncontrolled infections (3–6).Both phagocytosis and oxidative burst are controlled by the Rac proteins of the Ras small GTPase superfamily (1–4). There are three mammalian Rac GTPases, which are designated as Rac1, Rac2, and Rac3. Small GTPases are enzymes that hydrolyze GTP. They are active when bound to GTP and inactive when bound to GDP and serve as molecular “on-and-off” switches of signaling pathways that control a wide variety of cellular processes including growth, motility, vesicle trafficking, and death (7). Rac GTPases control phagocytosis by promoting actin polymerization through their effector proteins such as p21-activated kinases (PAKs), WASP family Verprolin homology domain-containing protein (WAVE), and IQ motif containing GTPase-activating protein-1 (IQGAP1) (1). Rac GTPases also mediate ROS production by binding and activating the NADPH oxidase complex through the p67(Phox) protein (1). Rac GTPase deficiency in mice and humans leads to an immune-deficient syndrome, which is characterized by defective phagocytosis and oxidative burst, recurrent infection, and granulomas (3–6).Although quiescent phagocytes are capable of phagocytosis and ROS production, their levels are low. Toll-like receptor (TLR) activation or microbial infection significantly up-regulates these innate immune processes (8–11). However, the mechanisms whereby microbes promote them are not well understood. TIPE2, or tumor necrosis factor-α–induced protein 8 (TNFAIP8)-like 2 (TNFAIP8L2), is a member of the TNFAIP8 family, which is preferentially expressed in hematopoietic cells (12–18). It is significantly down-regulated in patients with infectious or autoimmune disorders (15, 19). The mammalian TNFAIP8 family consists of four members: TNFAIP8, TIPE1, TIPE2, and TIPE3, whose functions are largely unknown (14, 20). We recently generated TIPE2-deficient mice and discovered that TIPE2 plays a crucial role in immune homeostasis (14). We report here that TIPE2 controls innate immunity by targeting the Rac GTPases.     

From the Cover: Membrane raft association is a determinant of plasma membrane localization

The lipid raft hypothesis proposes lateral domains driven by preferential interactions between sterols, sphingolipids, and specific proteins as a central mechanism for the regulation of membrane structure and function; however, experimental limitations in defining raft composition and properties have prevented unequivocal demonstration of their functional relevance. Here, we establish a quantitative, functional relationship between raft association and subcellular protein sorting. By systematic mutation of the transmembrane and juxtamembrane domains of a model transmembrane protein, linker for activation of T-cells (LAT), we generated a panel of variants possessing a range of raft affinities. These mutations revealed palmitoylation, transmembrane domain length, and transmembrane sequence to be critical determinants of membrane raft association. Moreover, plasma membrane (PM) localization was strictly dependent on raft partitioning across the entire panel of unrelated mutants, suggesting that raft association is necessary and sufficient for PM sorting of LAT. Abrogation of raft partitioning led to mistargeting to late endosomes/lysosomes because of a failure to recycle from early endosomes. These findings identify structural determinants of raft association and validate lipid-driven domain formation as a mechanism for endosomal protein sorting.Recent advances in superresolution microscopy (1), lipid analysis (2, 3), and plasma membrane (PM) isolation (4, 5) have confirmed the coexistence of lipid-driven, fluid domains in biological membranes. The relatively ordered domains, known as “membrane rafts,” have been proposed to be involved in protein sorting (6), viral/pathogen trafficking (3, 7), and PM signaling in a variety of contexts (8). However, despite the increasing evidence confirming the existence of dynamic, nanoscopic membrane rafts, the functional consequences of this phenomenon remain speculative because of the limitations of the previously used methods for defining raft association, i.e., the resistance of membrane components to solubilization by nonionic detergents (9).Lipid-mediated domains have been implicated as a mechanism for protein sorting in the latter stages of the secretory pathway (trans-Golgi network to the PM) (2, 6, 10–12), with analogous pathways mediating endosomal sorting/recycling (13, 14). Raft lipids (i.e., sterols and sphingolipids) are significantly enriched at the PM (15–17), and recent observations confirm that these lipids also are enriched in sorting vesicles destined for the PM (2, 11). For proteins, several specific cytosolic signals exist for adapter/coat-mediated sorting between cellular organelles (18); in parallel, protein–lipid interactions through hydrophobic transmembrane domains (TMDs) also have been shown to regulate trafficking. For example, a strong correlation exists between the TMD length of bitopic proteins and their organelle specificity (19, 20), with longer TMDs targeting proteins to the PM and shorter TMDs found in the endoplasmic reticulum (ER), Golgi apparatus, and endocytic organelles. These findings suggest cargo sorting in the secretory and endocytic pathways, with proteins containing longer TMDs, together with sphingolipids and cholesterol, being specifically trafficked to the PM, although the mechanism for this observation remains unresolved.One possibility for sorting of specific lipid classes along with proteins containing longer TMDs is lateral segregation and coalescence of ordered domains, followed by either domain-induced (21) or cytoskeleton-assisted (22) budding of raft-enriched transport vesicles. Proteins using this “raft pathway” would not require cytosolic sorting signals but rather would be recruited to transport vesicles by their raft affinity, i.e., their propensity to interact with specific lipids, ordered domains, or other raft-embedded proteins. Because ordered phases in lipid model systems consistently have been shown to be 0.6–1.5 nm thicker than disordered domains (23, 24), raft-associated transmembrane (TM) proteins would be predicted to have longer TMDs. TMD length-dependent protein sorting between coexisting lipid domains has been addressed experimentally only recently by measuring partitioning of an oligomeric toxin (perfringolysin O) with multiple (35–40) TM segments in synthetic, phase-separated liposomes (25). Whether these observations extend to single-pass TM proteins in biological membranes is unknown.To evaluate the role of lipid-driven raft domains as a mechanism for subcellular protein sorting, we quantitatively compared the raft association of 30 TM protein variants with their subcellular localization. To quantify raft partitioning of the constructs comprising single-pass TM proteins with varying TMD lengths and sequences, we used giant PM vesicles (GPMVs). GPMVs are cell-detached PM blebs whose protein (26) and lipid (27) diversity mirrors that of the native PM. These PM vesicles separate into coexisting liquid phases (4) with different order (28), which recruit membrane components in accordance with their predicted raft affinity, i.e., saturated lipids, glycosphingolipids (29), glycosylphosphatidyl inositol-anchored proteins (4), and palmitoylated proteins (30) partition to the ordered phase, denoted here as the “raft phase.” Most importantly, these vesicles provide a platform for repeatable, direct, and quantitative analysis of raft partitioning (30), allowing investigation of the structural determinants of raft association and its effect on protein function. We find that perturbation of raft partitioning by three independent means (decreasing TMD length, mutation of palmitoylation sites, and TMD sequence manipulation) perturbed subcellular localization, leading to missorting of PM proteins to late endosomes and lysosomes because of a failure to recycle nonraft proteins from early endosomes (EEs). These results confirm the presence of a raft-mediated recycling route in nonpolarized cells, begin to define the molecular parameters for protein association with raft domains, and suggest an explanation for the accumulation of proteins with longer TMDs at the PM.     

Potentiation of cytotoxic chemotherapy by growth hormone-releasing hormone agonists

The dismal prognosis of malignant brain tumors drives the development of new treatment modalities. In view of the multiple activities of growth hormone-releasing hormone (GHRH), we hypothesized that pretreatment with a GHRH agonist, JI-34, might increase the susceptibility of U-87 MG glioblastoma multiforme (GBM) cells to subsequent treatment with the cytotoxic drug, doxorubicin (DOX). This concept was corroborated by our findings, in vivo, showing that the combination of the GHRH agonist, JI-34, and DOX inhibited the growth of GBM tumors, transplanted into nude mice, more than DOX alone. In vitro, the pretreatment of GBM cells with JI-34 potentiated inhibitory effects of DOX on cell proliferation, diminished cell size and viability, and promoted apoptotic processes, as shown by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide proliferation assay, ApoLive-Glo multiplex assay, and cell volumetric assay. Proteomic studies further revealed that the pretreatment with GHRH agonist evoked differentiation decreasing the expression of the neuroectodermal stem cell antigen, nestin, and up-regulating the glial maturation marker, GFAP. The GHRH agonist also reduced the release of humoral regulators of glial growth, such as FGF basic and TGFβ. Proteomic and gene-expression (RT-PCR) studies confirmed the strong proapoptotic activity (increase in p53, decrease in v-myc and Bcl-2) and anti-invasive potential (decrease in integrin α3) of the combination of GHRH agonist and DOX. These findings indicate that the GHRH agonists can potentiate the anticancer activity of the traditional chemotherapeutic drug, DOX, by multiple mechanisms including the induction of differentiation of cancer cells.Glioblastoma multiforme (GBM) is one of the most aggressive human cancers, and the afflicted patients inevitably succumb. The dismal outcome of this malignancy demands great efforts to find improved methods of treatment (1). Many compounds have been synthesized in our laboratory in the past few years that have proven to be effective against diverse malignant tumors (2–14). These are peptide analogs of hypothalamic hormones: luteinizing hormone-releasing hormone (LHRH), growth hormone-releasing hormone (GHRH), somatostatin, and analogs of other neuropeptides such as bombesin and gastrin-releasing peptide. The receptors for these peptides have been found to be widely distributed in the human body, including in many types of cancers (2–14). The regulatory functions of these hypothalamic hormones and other neuropeptides are not confined to the hypothalamo–hypophyseal system or, even more broadly, to the central nervous system (CNS). In particular, GHRH can induce the differentiation of ovarian granulosa cells and other cells in the reproductive system and function as a growth factor in various normal tissues, benign tumors, and malignancies (2–4, 6, 11, 14–18). Previously, we also reported that antagonistic cytototoxic derivatives of some of these neuropeptides are able to inhibit the growth of several malignant cell lines (2–14).Our earlier studies showed that treatment with antagonists of LHRH or GHRH rarely effects complete regression of glioblastoma-derived tumors (5, 7, 10, 11). Previous studies also suggested that growth factors such as EGF or agonistic analogs of LHRH serving as carriers for cytotoxic analogs and functioning as growth factors may sensitize cancer cells to cytotoxic treatments (10, 19) through the activation of maturation processes. We therefore hypothesized that pretreatment with one of our GHRH agonists, such as JI-34 (20), which has shown effects on growth and differentiation in other cell lines (17, 18, 21, 22), might decrease the pluripotency and the adaptability of GBM cells and thereby increase their susceptibility to cytotoxic treatment.In vivo, tumor cells were implanted into athymic nude mice, tumor growth was recorded weekly, and final tumor mass was measured upon autopsy. In vitro, proliferation assays were used for the determination of neoplastic proliferation and cell growth. Changes in stem (nestin) and maturation (GFAP) antigen expression was evaluated with Western blot studies in vivo and with immunocytochemistry in vitro. The production of glial growth factors (FGF basic, TGFβ) was verified by ELISA. Further, using the Human Cancer Pathway Finder real-time quantitative PCR, numerous genes that play a role in the development of cancer were evaluated. We placed particular emphasis on the measurement of apoptosis, using the ApoLive-Glo Multiplex Assay kit and by detection of the expression of the proapoptotic p53 protein. This overall approach permitted the evaluation of the effect of GHRH agonist, JI-34, on the response to chemotherapy with doxorubicin.     

Self-assembly of alkynylplatinum(II) terpyridine amphiphiles into nanostructures via steric control and metal–metal interactions

A series of mono- and dinuclear alkynylplatinum(II) terpyridine complexes containing the hydrophilic oligo(para-phenylene ethynylene) with two 3,6,9-trioxadec-1-yloxy chains was designed and synthesized. The mononuclear alkynylplatinum(II) terpyridine complex was found to display a very strong tendency toward the formation of supramolecular structures. Interestingly, additional end-capping with another platinum(II) terpyridine moiety of various steric bulk at the terminal alkyne would lead to the formation of nanotubes or helical ribbons. These desirable nanostructures were found to be governed by the steric bulk on the platinum(II) terpyridine moieties, which modulates the directional metal−metal interactions and controls the formation of nanotubes or helical ribbons. Detailed analysis of temperature-dependent UV-visible absorption spectra of the nanostructured tubular aggregates also provided insights into the assembly mechanism and showed the role of metal−metal interactions in the cooperative supramolecular polymerization of the amphiphilic platinum(II) complexes.Square-planar d⁸ platinum(II) polypyridine complexes have long been known to exhibit intriguing spectroscopic and luminescence properties (1–54) as well as interesting solid-state polymorphism associated with metal−metal and π−π stacking interactions (1–14, 25). Earlier work by our group showed the first example, to our knowledge, of an alkynylplatinum(II) terpyridine system [Pt(tpy)(C ≡ CR)]⁺ that incorporates σ-donating and solubilizing alkynyl ligands together with the formation of Pt···Pt interactions to exhibit notable color changes and luminescence enhancements on solvent composition change (25) and polyelectrolyte addition (26). This approach has provided access to the alkynylplatinum(II) terpyridine and other related cyclometalated platinum(II) complexes, with functionalities that can self-assemble into metallogels (27–31), liquid crystals (32, 33), and other different molecular architectures, such as hairpin conformation (34), helices (35–38), nanostructures (39–45), and molecular tweezers (46, 47), as well as having a wide range of applications in molecular recognition (48–52), biomolecular labeling (48–52), and materials science (53, 54). Recently, metal-containing amphiphiles have also emerged as a building block for supramolecular architectures (42–44, 55–59). Their self-assembly has always been found to yield different molecular architectures with unprecedented complexity through the multiple noncovalent interactions on the introduction of external stimuli (42–44, 55–59).Helical architecture is one of the most exciting self-assembled morphologies because of the uniqueness for the functional and topological properties (60–69). Helical ribbons composed of amphiphiles, such as diacetylenic lipids, glutamates, and peptide-based amphiphiles, are often precursors for the growth of tubular structures on an increase in the width or the merging of the edges of ribbons (64, 65). Recently, the optimization of nanotube formation vs. helical nanostructures has aroused considerable interests and can be achieved through a fine interplay of the influence on the amphiphilic property of molecules (66), choice of counteranions (67, 68), or pH values of the media (69), which would govern the self-assembly of molecules into desirable aggregates of helical ribbons or nanotube scaffolds. However, a precise control of supramolecular morphology between helical ribbons and nanotubes remains challenging, particularly for the polycyclic aromatics in the field of molecular assembly (64–69). Oligo(para-phenylene ethynylene)s (OPEs) with solely π−π stacking interactions are well-recognized to self-assemble into supramolecular system of various nanostructures but rarely result in the formation of tubular scaffolds (70–73). In view of the rich photophysical properties of square-planar d⁸ platinum(II) systems and their propensity toward formation of directional Pt···Pt interactions in distinctive morphologies (27–31, 39–45), it is anticipated that such directional and noncovalent metal−metal interactions might be capable of directing or dictating molecular ordering and alignment to give desirable nanostructures of helical ribbons or nanotubes in a precise and controllable manner.Herein, we report the design and synthesis of mono- and dinuclear alkynylplatinum(II) terpyridine complexes containing hydrophilic OPEs with two 3,6,9-trioxadec-1-yloxy chains. The mononuclear alkynylplatinum(II) terpyridine complex with amphiphilic property is found to show a strong tendency toward the formation of supramolecular structures on diffusion of diethyl ether in dichloromethane or dimethyl sulfoxide (DMSO) solution. Interestingly, additional end-capping with another platinum(II) terpyridine moiety of various steric bulk at the terminal alkyne would result in nanotubes or helical ribbons in the self-assembly process. To the best of our knowledge, this finding represents the first example of the utilization of the steric bulk of the moieties, which modulates the formation of directional metal−metal interactions to precisely control the formation of nanotubes or helical ribbons in the self-assembly process. Application of the nucleation–elongation model into this assembly process by UV-visible (UV-vis) absorption spectroscopic studies has elucidated the nature of the molecular self-assembly, and more importantly, it has revealed the role of metal−metal interactions in the formation of these two types of nanostructures.