Tandem C2 Domains Research Articles

Pancreatic β-cells package double C2-like domain beta protein into extracellular vesicles via tandem C2 domains.

Double C2-like domain beta (DOC2B) is a vesicle priming protein critical for glucose-stimulated insulin secretion in β-cells. Individuals with type 1 diabetes (T1D) have lower levels of DOC2B in their residual functional β-cell mass and platelets, a phenotype also observed in a mouse model of T1D. Thus, DOC2B levels could provide important information on β-cell dys(function). Our objective was to evaluate the DOC2B secretome of β-cells. In addition to soluble extracellular protein, we assessed DOC2B localized within membrane-delimited nanoparticles - extracellular vesicles (EVs). Moreover, in rat clonal β-cells, we probed domains required for DOC2B sorting into EVs. Using Single Extracellular VEsicle Nanoscopy, we quantified EVs derived from clonal β-cells (human EndoC-βH1, rat INS-1 832/13, and mouse MIN6); two other cell types known to regulate glucose homeostasis and functionally utilize DOC2B (skeletal muscle rat myotube L6-GLUT4myc and human neuronal-like SH-SY5Y cells); and human islets sourced from individuals with no diabetes (ND). EVs derived from ND human plasma, ND human islets, and cell lines were isolated with either size exclusion chromatography or differential centrifugation. Isolated EVs were comprehensively characterized using dotblots, transmission electron microscopy, nanoparticle tracking analysis, and immunoblotting. DOC2B was present within EVs derived from ND human plasma, ND human islets, and INS-1 832/13 β-cells. Compared to neuronal-like SH-SY5Y cells and L6-GLUT4myc myotubes, clonal β-cells (EndoC-βH1, INS-1 832/13, and MIN6) produced significantly more EVs. DOC2B levels in EVs (over whole cell lysates) were higher in INS-1 832/13 β-cells compared to L6-GLUT4myc myotubes; SH-SY5Y neuronal-like cells did not release appreciable DOC2B. Mechanistically, we show that DOC2B was localized to the EV lumen; the tandem C2 domains were sufficient to confer sorting to INS-1 832/13 β-cell EVs. Clonal β-cells and ND human islets produce abundant EVs. In cell culture, appreciable DOC2B can be packaged into EVs, and a small fraction is excreted as a soluble protein. While DOC2B-laden EVs and soluble protein are present in ND plasma, further studies will be necessary to determine if DOC2B originating from β-cells significantly contributes to the plasma secretome.

Vesicle docking and fusion pore modulation by the neuronal calcium sensor Synaptotagmin-1.

Synaptotagmin-1 (Syt1) is a major calcium sensor for rapid neurotransmitter release in neurons and hormone release in many neuroendocrine cells. It possesses two tandem cytosolic C2 domains that bind calcium, negatively charged phospholipids, and the neuronal SNARE complex. Calcium binding to Syt1 triggers exocytosis, but how this occurs is not well understood. Syt1 has additional roles in docking dense core vesicles (DCV) and synaptic vesicles (SV) to the plasma membrane (PM) and in regulating fusion pore dynamics. Thus, Syt1 perturbations could affect release through vesicle docking, fusion triggering, fusion pore regulation, or a combination of these. Here, using a human neuroendocrine cell line, we show that neutralization of highly conserved polybasic patches in either C2 domain of Syt1 impairs both DCV docking and efficient release of serotonin from DCVs. Interestingly, the same mutations resulted in larger fusion pores and faster release of serotonin during individual fusion events. Thus, Syt1's roles in vesicle docking, fusion triggering, and fusion pore control may be functionally related.

Copines, a Family of Calcium Sensor Proteins and Their Role in Brain Function.

The Copines are a family of evolutionary conserved calcium-binding proteins found in most eukaryotic organisms from protists to humans. They share a unique architecture and contain tandem C2 domains and a Von Willebrand factor type A (VWA) domain. C2 domains in Copines bind calcium, phospholipids, and other proteins and mediate the transient association of these proteins with biological membranes at elevated calcium levels. The VWA domain also binds calcium and is involved in protein-protein interactions. Here, we provide a comprehensive review of the sequences, structures, expression, targeting, and function of the entire family of known Copine proteins (Copine 1-9 in mammals) with a particular emphasis on their functional roles in the mammalian brain. Neuronal Copines are implicated in a wide array of processes from cell differentiation to synaptic transmission and plasticity and are also linked to several pathological conditions from cancers to brain diseases. This review provides the most up-to-date insights into the structure and function of Copines, with an emphasis on their role in brain function.

The juxtamembrane linker of synaptotagmin 1 regulates Ca2+ binding via liquid-liquid phase separation

Synaptotagmin (syt) 1, a Ca2+ sensor for synaptic vesicle exocytosis, functions in vivo as a multimer. Syt1 senses Ca2+ via tandem C2-domains that are connected to a single transmembrane domain via a juxtamembrane linker. Here, we show that this linker segment harbors a lysine-rich, intrinsically disordered region that is necessary and sufficient to mediate liquid-liquid phase separation (LLPS). Interestingly, condensate formation negatively regulates the Ca2+-sensitivity of syt1. Moreover, Ca2+ and anionic phospholipids facilitate the observed phase separation, and increases in [Ca2+]i promote the fusion of syt1 droplets in living cells. Together, these observations suggest a condensate-mediated feedback loop that serves to fine-tune the ability of syt1 to trigger release, via alterations in Ca2+ binding activity and potentially through the impact of LLPS on membrane curvature during fusion reactions. In summary, the juxtamembrane linker of syt1 emerges as a regulator of syt1 function by driving self-association via LLPS.

A guide to selecting high-performing antibodies for Synaptotagmin-1 (Uniprot ID P21579) for use in western blot, immunoprecipitation, immunofluorescence and flow cytometry.

Synaptotagmin-1 is a synaptic vesicle transmembrane protein that senses calcium influx via its tandem C2-domains, triggering synchronous neurotransmitter release. Disruption to SYT1 is associated with neurodevelopmental disorders, highlighting the importance of identifying high-quality research reagents to enhance understanding of Synaptotagmin-1 in health and disease. Here we have characterized thirteen Synaptotagmin-1 commercial antibodies for western blot, immunoprecipitation, immunofluorescence and flow cytometry using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. These studies are part of a larger, collaborative initiative seeking to address antibody reproducibility issues by characterizing commercially available antibodies for human proteins and publishing the results openly as a resource for the scientific community. While use of antibodies and protocols vary between laboratories, we encourage readers to use this report as a guide to select the most appropriate antibodies for their specific needs.

TC2N Promotes Cell Proliferation and Metastasis in Hepatocellular Carcinoma by Targeting the Wnt/β-Catenin Signaling Pathway

Hepatocellular carcinoma (HCC), one of the most prevalent types of cancer worldwide, has an exceedingly poor prognosis. Tandem C2 domain nuclear protein (TC2N) has been implicated in tumorigenesis and serves as an oncogene or tumor suppressor in different types of cancer. Here, we explore the possible regulatory activities and molecular mechanisms of TC2N in HCC progression. However, TC2N expression was significantly upregulated in HCC tissues and hepatoma cell lines, and this upregulation was positively correlated with tumor progression in HCC patients. The ectopic overexpression of TC2N accelerated the proliferation, migration, and invasion of HCC cells, whereas its knockdown showed the opposite effects. Bioinformatics analysis showed that TC2N participates in the regulation of the Wnt/β-catenin signaling pathway. Mechanistically, TC2N activated the Wnt/β-catenin signaling pathway by regulating the expression levels of β-catenin and its downstream targets CyclinD1, MMP7, c-Myc, c-Jun, AXIN2, and glutamine synthase. Furthermore, the deletion of β-catenin effectively neutralized the regulation of TC2N in HCC proliferation and metastasis. Overall, this study showed that TC2N promotes HCC proliferation and metastasis by activating the Wnt/β-catenin signaling pathway, indicating that TC2N might be a potential molecular target for the treatment of HCC.

1770-P: Islet ß-Cells Shed DOC2B-Laden EVs via Sorting Signals Present in DOC2B Protein

Loss of β-cell function occurs early in type 1 diabetes (T1D). However, C-peptide is insufficient to detect early pre-type 1 diabetes-related β-cell dysfunction. Thus, the discovery of clinically accessible β-cell health biomarkers may enable early therapeutic interventions, mitigate T1D, and prevent complete loss of β-cell mass. The exocytosis regulatory protein double C2-containing protein β (DOC2B) abundance is closely related to β-cell health and function. Our recent publication demonstrated the correlation between the decline in DOC2B abundance in blood-derived platelets and the loss of β-cell function in early-onset T1D and pre-diabetic nonobese diabetic (NOD) mice. Indeed, preliminary data show that DOC2B reduction in plasma precedes C-peptide reduction in prediabetic mice, suggesting that reduced DOC2B levels in plasma may serve as an early biomarker of β-cell dysfunction. However, the mechanisms by which β-cells export DOC2B into the circulation remain unknown. We hypothesize that DOC2B is uniquely sorted into β-cell extracellular vesicles (EVs). Preliminary data show DOC2B is detected in EVs isolated from human plasma, and that DOC2B-laden EVs can stem from β-cells. Because DOC2B is a broadly expressed protein, we questioned whether DOC2B-laden EVs are predominantly released by β-cells, relative to that of other cell types known to express DOC2B protein. Biochemical analyses reveal that DOC2B largely exists as luminal cargo in β-cell-derived EVs and is largely absent from EVs released by liver, brain, and skeletal muscle cells. Mechanistically, the research has delineated that the sufficient structural domain enabling DOC2B sorting into β-cell-derived EVs is that which encompasses the tandem C2 domains at the C-terminus of DOC2B. Taken together, these data indicate that DOC2B in circulation could originate from DOC2B-laden EVs released from β-cells, and that the mechanism by which DOC2B is sorted into those EVs involves one or both C2 domains of the DOC2B protein. Disclosure D.Esparza: None. E.Oh: None. I.Ghaeli: None. T.Jovanovic-talisman: None. D.C.Thurmond: Consultant; Immutics, Inc. Funding National Institute of Diabetes and Digestive and Kidney Diseases (DK067912, DK112917, DK102233); Wanek Family Project for Type 1 Diabetes (to D.C.T); Ford Foundation (to D.E.)

Synaptotagmin 9 Modulates Spontaneous Neurotransmitter Release in Striatal Neurons by Regulating Substance P Secretion.

Synaptotagmin 9 (SYT9) is a tandem C2 domain Ca2+ sensor for exocytosis in neuroendocrine cells; its function in neurons remains unclear. Here, we show that, in mixed-sex cultures, SYT9 does not trigger rapid synaptic vesicle exocytosis in mouse cortical, hippocampal, or striatal neurons, unless it is massively overexpressed. In striatal neurons, loss of SYT9 reduced the frequency of spontaneous neurotransmitter release events (minis). We delved into the underlying mechanism and discovered that SYT9 was localized to dense-core vesicles that contain substance P (SP). Loss of SYT9 impaired SP release, causing the observed decrease in mini frequency. This model is further supported by loss of function mutants. Namely, Ca2+ binding to the C2A domain of SYT9 triggered membrane fusion in vitro, and mutations that disrupted this activity abolished the ability of SYT9 to regulate both SP release and mini frequency. We conclude that SYT9 indirectly regulates synaptic transmission in striatal neurons by controlling SP release.SIGNIFICANCE STATEMENT Synaptotagmin 9 (SYT9) has been described as a Ca2+ sensor for dense-core vesicle (DCV) exocytosis in neuroendocrine cells, but its role in neurons remains unclear, despite widespread expression in the brain. This article examines the role of SYT9 in synaptic transmission across cultured cortical, hippocampal, and striatal neuronal preparations. We found that SYT9 regulates spontaneous neurotransmitter release in striatal neurons by serving as a Ca2+ sensor for the release of the neuromodulator substance P from DCVs. This demonstrates a novel role for SYT9 in neurons and uncovers a new field of study into neuromodulation by SYT9, a protein that is widely expressed in the brain.

Expression Analysis of TREM2 and TC2N Genes in Human Breast Cancer Tissues

Background: Since breast cancer is the most common type of cancer in women around the world, finding new biomarkers for early diagnosis of breast cancer is invaluable. Objectives: This research assessed the mRNA expression of triggering receptors expressed on myeloid cell 2 (TREM2) and tandem C2 domains nuclear protein (TC2N) genes among Iranian patients with breast cancer. Methods: We acquired 50 samples of cancerous breast tumors and corresponding adjacent non-cancerous tissues from Iranian women. The gene expression of TREM2 and TC2N was measured by quantitative real-time polymerase chain reaction (q-RT-PCR). In addition, the association between TREM2 and TC2N levels with various clinicopathologic characteristics was also investigated. Results: The increased levels of TREM2 and TC2N mRNAs were shown in breast cancerous tissues in comparison with adjacent non-cancerous tissues (P < 0.05). Among the clinicopathological characteristics evaluated, tumor size, necrosis, and lymphatic tissue invasion were significantly associated with high TREM2 expression. A significant relationship was also seen between increased TC2N expression and tumor grade. Sensitivity and specificity were shown at 84% and 94%, respectively, for TREM2 and 72% and 100% for TC2N. Conclusions: The data suggest that TREM2 expression, but not TC2N, could be a suitable biomarker for breast cancer diagnosis.

Overexpression of Copines-1 is associated with clinicopathological parameters and poor outcome in gastric cancer.

Copines-1 (CPNE1) is a soluble membrane-binding protein that includes two tandem C2 domains at the N-terminus and a C terminal A domain. Importantly, it is associated with the prognosis of various tumors, but there are only a few studies regarding the role of CPNE1 in gastric cancer (GC). This study aimed to explore the clinicopathological significance and prognostic potential of CPNE1 expression in GC. Data from the TIMER2.0 and UALCAN were analyzed to assess CPNE1 mRNA levels in GC. The prognostic role of CPNE1 mRNA was examined via the Kaplan-Meier plotter. CPNE1 protein expression in tumor tissues was analyzed via immunohistochemistry of clinical samples from 99 GC patients. The relationship of CPNE1 expression with clinicopathological parameters and overall survival (OS) was evaluated using Cox proportional hazards regression models and Kaplan-Meier survival curves. Copines-1 mRNA levels were higher in GC tissues than in adjacent normal tissue (ANT) (p< 0.05). Further, high CPNE1 mRNA expression indicated poor OS (p= 9.4 e-10) and was significantly associated with first progression (FP) (p= 1.6 e-06) and post-progression survival (PPS) (p= 1.5 e-12). In addition, CPNE1 protein expression was higher in GC tissues than in ANT (p< 0.0001). Moreover, CPNE1 high expression was significantly related to advanced tumor-node-metastasis (TNM) stage (p= 0.004), lymph node metastasis (p= 0.003), and vascular invasion (p= 0.001). Kaplan-Meier analysis showed that GC patients with high expression CPNE1 group had worse OS than low expression group (p= 0.003). Univariate analysis showed that age (hazard ratio [HR]=1.992; 95% confidence interval [CI], 1.009-3.934; p= 0.047), advanced TNM stage (HR=4.941; 95% CI, 2.052-11.897; p= 0.000), tumor invasion (HR=3.472; 95% CI, 1.349-8.937; p= 0.010), lymph node metastasis (HR=8.846; 95% CI, 2.708-28.897; p= 0.000), vascular invasion (HR=3.237; 95% CI, 1.521-6.891; p= 0.002), nervous invasion (HR=2.324; 95% CI, 1.205-4.479; p= 0.012), and CPNE1 expression (HR=3.464; 95% CI, 1.440-8.334; p= 0.006) were correlated with OS. In the multivariate analysis, age (HR=2.514; 95% CI, 1.264-4.999; p= 0.009), lymph node metastasis (HR=8.441; 95% CI, 2.553-27.906; p< 0.05), and CPNE1 expression (HR=2.549; 95% CI, 1.051-6.186; p= 0.039) were significant prognostic predictors for GC. Copines-1 overexpression in GC is significantly associated with poor prognosis. Thus, CPNE1 levels may serve as a prognostic biomarker in GC patients.

Polybasic Patches in Both C2 Domains of Synaptotagmin-1 Are Required for Evoked Neurotransmitter Release.

Synaptotagmin-1 (Syt1) is a vesicular calcium sensor required for synchronous neurotransmitter release, composed of a single-pass transmembrane domain linked to two C2 domains (C2A and C2B) that bind calcium, acidic lipids, and SNARE proteins that drive fusion of the synaptic vesicle with the plasma membrane. Despite its essential role, how Syt1 couples calcium entry to synchronous release is poorly understood. Calcium binding to C2B is critical for synchronous release, and C2B additionally binds the SNARE complex. The C2A domain is also required for Syt1 function, but it is not clear why. Here, we asked what critical feature of C2A may be responsible for its functional role and compared this to the analogous feature in C2B. We focused on highly conserved poly-lysine patches located on the sides of C2A (K189-192) and C2B (K324-327). We tested effects of charge-neutralization mutations in either region (Syt1K189-192A and Syt1K326-327A) side by side to determine their relative contributions to Syt1 function in cultured cortical neurons from mice of either sex and in single-molecule experiments. Combining electrophysiological recordings and optical tweezers measurements to probe dynamic single C2 domain-membrane interactions, we show that both C2A and C2B polybasic patches contribute to membrane binding, and both are required for evoked release. The size of the readily releasable vesicle pool and the rate of spontaneous release were unaffected, so both patches are likely required specifically for synchronization of release. We suggest these patches contribute to cooperative membrane binding, increasing the overall affinity of Syt1 for negatively charged membranes and facilitating evoked release.SIGNIFICANCE STATEMENT Synaptotagmin-1 is a vesicular calcium sensor required for synchronous neurotransmitter release. Its tandem cytosolic C2 domains (C2A and C2B) bind calcium, acidic lipids, and SNARE proteins that drive fusion of the synaptic vesicle with the plasma membrane. How calcium binding to Synaptotagmin-1 leads to release and the relative contributions of the C2 domains are unclear. Combining electrophysiological recordings from cultured neurons and optical tweezers measurements of single C2 domain-membrane interactions, we show that conserved polybasic regions in both domains contribute to membrane binding cooperatively, and both are required for evoked release, likely by increasing the overall affinity of Synaptotagmin-1 for acidic membranes.

Tandem C2 domains mediate dynamic organelle targeting of a DOCK family guanine nucleotide exchange factor.

Multicellular organisms use dedicator of cytokinesis (DOCK) family guanine nucleotide exchange factors (GEFs) to activate Rac/Rho-of-plants small GTPases and coordinate cell shape change. In developing tissues, DOCK signals integrate cell-cell interactions with cytoskeleton remodeling, and the GEFs cluster reversibly at specific organelle surfaces to orchestrate cytoskeletal reorganization. The domain organizations among DOCK orthologs are diverse, and the mechanisms of localization control are poorly understood. Here, we use combinations of transgene complementation and live-cell imaging assays to uncover an evolutionarily conserved and essential localization determinant in the DOCK-GEF named SPIKE1. The SPIKE1-DHR3 domain is sufficient for organelle association in vivo, and displays a complicated lipid-binding selectivity for both phospholipid head groups and fatty acid chain saturation. SPIKE1-DHR3 is predicted to adopt a C2-domain structure and functions as part of a tandem C2 array that enables reversible clustering at the cell apex. This work provides mechanistic insight into how DOCK GEFs sense compositional and biophysical membrane properties at the interface of two organelle systems.

Polybasic patches in both C2 domains of synaptotagmin-1 are required for evoked neurotransmitter release

Synaptotagmin-1 (Syt1) is a vesicular calcium sensor required for synchronous neurotransmitter release. It is composed of a single-pass transmembrane domain linked to two tandem C2 domains (C2A and C2B) that bind calcium, acidic lipids, and SNARE proteins that drive fusion of the synaptic vesicle with the plasma membrane. Despite its essential role, how Syt1 couples calcium entry to synchronous release is not well understood. Calcium binding to C2B, but not to C2A, is critical for synchronous release and C2B additionally binds the SNARE complex.

TC2N: A Novel Vital Oncogene or Tumor Suppressor Gene In Cancers.

Several C2 domain-containing proteins play key roles in tumorigenesis, signal transduction, and mediating protein–protein interactions. Tandem C2 domains nuclear protein (TC2N) is a tandem C2 domain-containing protein that is differentially expressed in several types of cancers and is closely associated with tumorigenesis and tumor progression. Notably, TC2N has been identified as an oncogene in lung and gastric cancer but as a tumor suppressor gene in breast cancer. Recently, a large number of tumor-associated antigens (TAAs), such as heat shock proteins, alpha-fetoprotein, and carcinoembryonic antigen, have been identified in a variety of malignant tumors. Differences in the expression levels of TAAs between cancer cells and normal cells have led to these antigens being investigated as diagnostic and prognostic biomarkers and as novel targets in cancer treatment. In this review, we summarize the clinical characteristics of TC2N-positive cancers and potential mechanisms of action of TC2N in the occurrence and development of specific cancers. This article provides an exploration of TC2N as a potential target for the diagnosis and treatment of different types of cancers.

The neuronal calcium sensor Synaptotagmin-1 and SNARE proteins cooperate to dilate fusion pores.

All membrane fusion reactions proceed through an initial fusion pore, including calcium-triggered release of neurotransmitters and hormones. Expansion of this small pore to release cargo is energetically costly and regulated by cells, but the mechanisms are poorly understood. Here, we show that the neuronal/exocytic calcium sensor Synaptotagmin-1 (Syt1) promotes expansion of fusion pores induced by SNARE proteins. Pore dilation relied on calcium-induced insertion of the tandem C2 domain hydrophobic loops of Syt1 into the membrane, previously shown to reorient the C2 domain. Mathematical modelling suggests that C2B reorientation rotates a bound SNARE complex so that it exerts force on the membranes in a mechanical lever action that increases the height of the fusion pore, provoking pore dilation to offset the bending energy penalty. We conclude that Syt1 exerts novel non-local calcium-dependent mechanical forces on fusion pores that dilate pores and assist neurotransmitter and hormone release.

Ca2+ sensor proteins in spontaneous release and synaptic plasticity: Limited contribution of Doc2c, rabphilin-3a and synaptotagmin 7 in hippocampal glutamatergic neurons

Presynaptic neurotransmitter release is strictly regulated by SNARE proteins, Ca2+ and a number of Ca2+ sensors including synaptotagmins (Syts) and Double C2 domain proteins (Doc2s). More than seventy years after the original description of spontaneous release, the mechanism that regulates this process is still poorly understood. Syt-1, Syt7 and Doc2 proteins contribute predominantly, but not exclusively, to synchronous, asynchronous and spontaneous phases of release. The proteins share a conserved tandem C2 domain architecture, but are functionally diverse in their subcellular location, Ca2+-binding properties and protein interactions. In absence of Syt-1, Doc2a and -b, neurons still exhibit spontaneous vesicle fusion which remains Ca2+-sensitive, suggesting the existence of additional sensors. Here, we selected Doc2c, rabphilin-3a and Syt-7 as three potential Ca2+ sensors for their sequence homology with Syt-1 and Doc2b. We genetically ablated each candidate gene in absence of Doc2a and -b and investigated spontaneous and evoked release in glutamatergic hippocampal neurons, cultured either in networks or on microglial islands (autapses). The removal of Doc2c had no effect on spontaneous or evoked release. Syt-7 removal also did not affect spontaneous release, although it altered short-term plasticity by accentuating short-term depression. The removal of rabphilin caused an increased spontaneous release frequency in network cultures, an effect that was not observed in autapses. Taken together, we conclude that Doc2c and Syt-7 do not affect spontaneous release of glutamate in hippocampal neurons, while our results suggest a possible regulatory role of rabphilin-3a in neuronal networks. These findings importantly narrow down the repertoire of synaptic Ca2+ sensors that may be implicated in the spontaneous release of glutamate.

A novel dual Ca2+ sensor system regulates Ca2+-dependent neurotransmitter release.

Ca2+-dependent neurotransmitter release requires synaptotagmins as Ca2+ sensors to trigger synaptic vesicle (SV) exocytosis via binding of their tandem C2 domains-C2A and C2B-to Ca2+. We have previously demonstrated that SNT-1, a mouse synaptotagmin-1 (Syt1) homologue, functions as the fast Ca2+ sensor in Caenorhabditis elegans. Here, we report a new Ca2+ sensor, SNT-3, which triggers delayed Ca2+-dependent neurotransmitter release. snt-1;snt-3 double mutants abolish evoked synaptic transmission, demonstrating that C. elegans NMJs use a dual Ca2+ sensor system. SNT-3 possesses canonical aspartate residues in both C2 domains, but lacks an N-terminal transmembrane (TM) domain. Biochemical evidence demonstrates that SNT-3 binds both Ca2+ and the plasma membrane. Functional analysis shows that SNT-3 is activated when SNT-1 function is impaired, triggering SV release that is loosely coupled to Ca2+ entry. Compared with SNT-1, which is tethered to SVs, SNT-3 is not associated with SV. Eliminating the SV tethering of SNT-1 by removing the TM domain or the whole N terminus rescues fast release kinetics, demonstrating that cytoplasmic SNT-1 is still functional and triggers fast neurotransmitter release, but also exhibits decreased evoked amplitude and release probability. These results suggest that the fast and slow properties of SV release are determined by the intrinsically different C2 domains in SNT-1 and SNT-3, rather than their N-termini-mediated membrane tethering. Our findings therefore reveal a novel dual Ca2+ sensor system in C. elegans and provide significant insights into Ca2+-regulated exocytosis.

A Novel Two Ca2+ Sensor System in C. elegans

Ca2+-dependent neurotransmitter release requires synaptotagmins as Ca2+ sensors to trigger synaptic vesicle (SV) exocytosis through binding of their tandem C2 domains (C2A and C2B) to Ca2+. We have previously demonstrated that SNT-1, a mouse synaptotagmin-1 (Syt1) homolog, functions as the fast Ca2+ sensor in C. elegans. Here we report a new Ca2+ sensor, SNT-3, which triggers slow Ca2+-dependent neurotransmitter release. snt-1;snt-3 double mutants abolish evoked synaptic transmission, demonstrating that C. elegans NMJs use a dual Ca2+ sensor system. SNT-3 possesses canonical aspartate residues in both C2 domains, but lacks an N-terminal transmembrane (TM) domain. Biochemical evidence demonstrates that SNT-3 binds both Ca2+ and the plasma membrane. Functional analysis shows that SNT-3 is activated when SNT-1 function is impaired, triggering SV release that is loosely coupled to Ca2+ entry. Compared to SNT-1 which is tethered to SVs, SNT-3 is not SV associated. Eliminating the SV tethering of SNT-1 by removing the TM domain or the whole N terminus rescues fast release kinetics demonstrating that cytoplasmic SNT-1 is still functional and triggers fast neurotransmitter release, but exhibits decreased evoked amplitude and release probability. These results suggest that the fast and slow properties of SV release are determined by the intrinsically different C2 domains of the fast and slow Ca2+ sensors, rather than their N termini-mediated membrane tethering. Our findings therefore reveal a novel dual-Ca2+ sensor system in C. elegans and provide significant insights into Ca2+-regulated exocytosis.

Structure Prediction and Molecular Phylogenetic Analysis of Membrane Interactions in Synaptotagmin-Like Proteins

Membrane trafficking and exocytosis are evolutionarily conserved throughout eukaryotes and involve many interactions between proteins and membrane surfaces. In vertebrates, synaptotagmin-like proteins (Slp) play key roles in trafficking of large dense-core secretory vesicles. Slp family proteins promote secretory vesicle docking to the plasma membrane through their N-terminal Slp homology domains, which bind to vesicular Rab proteins, and their tandem C-terminal C2 domains (C2A and C2B), which bind plasma membrane lipids independently of calcium. Our previous experimental and computational studies showed that the C2A domain of Slp-4 (also called granuphilin) binds with high affinity to membrane surfaces containing phosphatidylinositol-(4,5)-bisphosphate (PIP2) and phosphatidylserine (PS). A conserved PIP2 binding site centered on three lysine residues in the beta3-beta4 region specifically anchors the domain to PIP2 lipids, while many other basic residues surrounding the Lys-cluster enhance interactions with PS. We hypothesize that the positive electrostatic potential of the membrane binding surface is a key determinant of its membrane affinity and function, and therefore the net charge on the protein surface is likely to be evolutionarily conserved. To test this hypothesis, the C2A domain sequences for Slp-4 and Slp-2 in vertebrates were compared to assess the evolution of their respective polybasic surfaces. Using a molecular phylogenetic approach, clades were identified and the net charges of individual and consensus sequences were calculated. Structural homology models were generated using a crystal structure of Slp-4 C2A, and Poisson-Boltzmann calculations were carried out to quantify the positive electrostatic surfaces. This approach may help characterize electrostatic interactions with charged membrane surfaces and identify conserved differences that govern affinity and function of membrane-binding proteins.

Multivalent lipid targeting by the calcium-independent C2A domain of synaptotagmin-like protein 4/granuphilin

Synaptotagmin-like protein 4 (Slp-4), also known as granuphilin, is a Rab effector responsible for docking secretory vesicles to the plasma membrane before exocytosis. Slp-4 binds vesicular Rab proteins via an N-terminal Slp homology domain, interacts with plasma membrane SNARE complex proteins via a central linker region, and contains tandem C-terminal C2 domains (C2A and C2B) with affinity for phosphatidylinositol-(4,5)-bisphosphate (PIP2). The Slp-4 C2A domain binds with low nanomolar apparent affinity to PIP2 in lipid vesicles that also contain background anionic lipids such as phosphatidylserine but much weaker when either the background anionic lipids or PIP2 is removed. Through computational and experimental approaches, we show that this high-affinity membrane binding arises from concerted interaction at multiple sites on the C2A domain. In addition to a conserved PIP2-selective lysine cluster, a larger cationic surface surrounding the cluster contributes substantially to the affinity for physiologically relevant lipid compositions. Although the K398A mutation in the lysine cluster blocks PIP2 binding, this mutated protein domain retains the ability to bind physiological membranes in both a liposome-binding assay and MIN6 cells. Molecular dynamics simulations indicate several conformationally flexible loops that contribute to the nonspecific cationic surface. We also identify and characterize a covalently modified variant that arises through reactivity of the PIP2-binding lysine cluster with endogenous bacterial compounds and binds weakly to membranes. Overall, multivalent lipid binding by the Slp-4 C2A domain provides selective recognition and high-affinity docking of large dense core secretory vesicles to the plasma membrane.