Biology subjects

Chapman, E. R.

Publications and source records attributed to Chapman, E. R..

At least 19 recordsLinked to original sources

bioRxiv2026

Munc18 reprograms the intrinsic neuronal SNARE complex assembly pathway

The SNARE proteins syntaxin/SNAP-25B (t-SNAREs) and synaptobrevin (v-SNARE) contain motifs that assemble into four-helix bundles to drive synaptic vesicle exocytosis; SNAP-25B contributes two helices, D1 and D2. The sequence in which these motifs interact remains unresolved. To address this, we used fluorescence anisotropy of SNARE motifs to conduct real-time order-of-addition experiments and found that the order in which components are mixed can determine whether on- or off-pathway complexes are formed. Beginning with soluble SNARE fragments alone, the first step in assembly is the binding of D1 to syntaxin, followed by the binding of synaptobrevin and D2, where the latter motif acts as a gatekeeper to control v-SNARE*t-SNARE interactions. We then examined the impact of two regulatory factors, Munc18 and the MUN domain of Munc13-1. Strikingly, in the presence of Munc18, all four isolated SNARE motifs must be present at the same time for assembly to occur, revealing a concerted mechanism, while the Munc13-1 fragment was without effect. We created C-SCORE, which reports assembly of the two SNARE motifs of SNAP-25B via FRET, and confirmed that in the presence of Munc18, SNARE assembly becomes concerted. Munc18 also disaggregated syntaxin, potentially contributing to its activation. Finally, our findings regarding SNARE motif folding were well-correlated with function using full-length SNAREs in in vitro lipid mixing assays. Hence, Munc18 acts as a molecular chaperone that directly promotes the concurrent assembly of SNARE proteins into functional fusion machines.

biochemistry↗

bioRxiv2026

Increased CA3 burst activity in Doc2α and Syt7 knockout mice

The hippocampal CA3 subfield is central to associative learning and memory consolidation. The principal cells of the CA3, pyramidal neurons, execute these functions by generating hypersynchronous bursts that feed forward to the CA1. Extensive recurrent collateral connections within the CA3 neuron population are crucial for the generation of this burst activity. Double C2 domain-containing protein (Doc2) and synaptotagmin 7 (Syt7) are high-affinity calcium sensors implicated in asynchronous synaptic vesicle (SV) release and in the exocytosis of dense-core vesicles (DCVs). Additionally, Doc2 is a sensor for miniature neurotransmission, whereas Syt7 is involved in synaptic facilitation and SV replenishment. Both Doc2 and Syt7 are expressed in the hippocampus, but their potential roles in spontaneous excitatory network activity remain unanswered. Using whole-cell recordings in disinhibited acute hippocampal slices obtained from juvenile Doc2- and Syt7- knockout (KO) mice (P15-21), we report increased CA3 burst generation without changes in spontaneous excitatory postsynaptic current (sEPSC) frequency or amplitude. Moreover, the intrinsic properties of CA3 pyramidal neurons, such as the resting membrane potential, firing rate and input resistance, are unchanged. We propose that this novel burst phenotype in Doc2- and Syt7- KO mice is unrelated to changes in SV release but might be mediated by changes in neuropeptide release from DCVs. Regardless of the underlying mechanisms, this work reveals that both proteins act to regulate network activity. Key pointsO_LICA3 burst activity is critical for associative learning and memory consolidation C_LIO_LIDoc2 and Syt7 are high-affinity Ca2+ sensors with similar kinetic properties and are expressed in the hippocampus C_LIO_LIDoc2 and Syt7 support various modes of neurotransmitter release and presynaptic plasticity C_LIO_LIBoth Doc2 and Syt7 are also involved in the exocytosis of dense-core vesicles in neuroendocrine cells C_LIO_LILoss of Doc2 and Syt7 results in increased CA3 burst activity without a change in the intrinsic properties of CA3 neurons C_LIO_LIOur results suggest a novel role for Doc2 and Syt7 in regulating network excitatory activity C_LI

neuroscience↗

bioRxiv2025

Alternative splicing of synaptotagmin 7 regulates oligomerization and short-term synaptic plasticity

Synaptic plasticity is crucial for learning and memory. The presynaptic calcium sensor synaptotagmin 7 (syt7) regulates aspects of short-term plasticity (STP), but the underlying mechanisms remain unclear. Here, we show that alternative splicing of the syt7 juxtamembrane linker acts as a molecular switch at both biochemical and functional levels. The and {beta} variants undergo liquid-liquid phase separation to form condensates, while the {gamma} variant forms aggregates. Using iGluSnFR imaging, we found that when expressed at equal levels, these three isoforms also diverge regarding their abilities to regulate two key aspects of STP: paired-pulse facilitation and synaptic depression. STED microscopy showed that all three isoforms form active zone-associated clusters that colocalize with syt1, while MINFLUX super-resolution microscopy resolved syt7 clusters within the active zone, well-positioned to directly control synaptic vesicle dynamics. Thus, alternative splicing might fine-tune STP by differentially impacting syt7 oligomerization.

neuroscience↗

bioRxiv2023

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.

neuroscience↗

bioRxiv2023

Label-free observation of individual solution phase molecules

The vast majority of chemistry and biology occurs in solution, and new label-free analytical techniques that can help resolve solution-phase complexity at the single-molecule level can provide new microscopic perspectives of unprecedented detail. Here, we use the increased light-molecule interactions in high-finesse fiber Fabry-Perot microcavities to detect individual biomolecules as small as 1.2 kDa with signal-to-noise ratios >100, even as the molecules are freely diffusing in solution. Our method delivers 2D intensity and temporal profiles, enabling the distinction of sub-populations in mixed samples. Strikingly, we observe a linear relationship between passage time and molecular radius, unlocking the potential to gather crucial information about diffusion and solution-phase conformation. Furthermore, mixtures of biomolecule isomers of the same molecular weight can also be resolved. Detection is based on a novel molecular velocity filtering and dynamic thermal priming mechanism leveraging both photo-thermal bistability and Pound-Drever-Hall cavity locking. This technology holds broad potential for applications in life and chemical sciences and represents a major advancement in label-free in vitro single-molecule techniques.

biophysics↗

bioRxiv2022

Synaptotagmin-9 and Tomosyn-1 molecular complex regulates Stx1A SNAREs to inhibit insulin secretion from pancreatic beta-cells

Stimulus-coupled insulin secretion from {beta}-cells involves the fusion of insulin granules to the plasma membrane (PM) via SNARE complex formation--a cellular process key for maintaining whole-body glucose homeostasis. Optimal insulin secretion depends on how the clamping of SNAREs is released, rendering granules fusogenic. We show that an insulin granule protein synaptotagmin-9 (Syt9) deletion in lean mice increased glucose clearance, random-fed plasma insulin levels, and insulin secretion (in vivo and ex vivo islets) without affecting insulin sensitivity. These outcomes demonstrate that Syt9 has an inhibitory function in insulin secretion. Moreover, Syt9 interacts with PM-Stx1A and soluble Tomosyn-1 proteins to form non-fusogenic complexes between PM and insulin granules, preventing Stx1A-SNARE formation and insulin secretion. Furthermore, Syt9 inhibits SNARE-complex formation by posttranscriptional regulation of Tomosyn-1. We conclude that Syt9 and Tomosyn-1 are endogenous inhibitors that modulate Stx1A availability to determine {beta}-cell secretory capacity. HighlightsO_LISynaptotagmin-9 inhibits biphasic insulin secretion from {beta}-cells. C_LIO_LISynaptotagmin-9, syntaxin-1A, and Tomosyn-1 forms a molecular complex that decreases the availability of syntaxin-1A to form SNARE complexes in insulin secretion. C_LIO_LISynaptotagmin-9-mediated inhibition of insulin secretion occurs through post-transcriptional regulation of Tomosyn-1. C_LI

cell biology↗

bioRxiv2022

Digital nanoreactors for control over absolute stoichiometry and spatiotemporal behavior of receptors within lipid bilayers

Interactions between membrane proteins are essential for cell survival and proper function, but the structural and mechanistic details of these interactions are often poorly understood. Even the biologically functional ratio of protein components within a multi-subunit membrane complex--the native stoichiometry--is difficult to establish. We have demonstrated digital nanoreactors that can control interactions between lipid-bound molecular receptors along three key dimensions: stoichiometric, spatial, and temporal. Each nanoreactor is based on a DNA origami ring, which both templates the synthesis of a liposome and provides tethering sites for DNA-based receptors. Receptors are released into the liposomal membrane using strand displacement and a DNA logic gate measures receptor heterodimer formation. High-efficiency tethering of receptors enables the kinetics of receptors in 1:1 and 2:2 absolute stoichiometries to be observed by bulk fluorescence in a plate reader which in principle is generalizable to any ratio. Similar single molecule in bulk experiments using DNA-linked membrane proteins could determine native stoichiometry and the kinetics of membrane protein interactions for applications ranging from signalling research to drug discovery.

bioengineering↗

bioRxiv2022

Highly fucosylated N-glycans at the synaptic vesicle and neuronal plasma membrane

At neuronal synapses, synaptic vesicles (SVs) require glycoproteins for normal trafficking, and N-linked glycosylation is required for delivery of the major SV glycoproteins synaptophysin and SV2A to SVs. The molecular compositions of SV N-glycans, which may drive important neurobiological processes, are largely unknown. In this study, we combined organelle isolation techniques, fluorescence detection of N-glycans, and high-resolution mass spectrometry to characterize N-glycosylation at synapses and SVs from mouse brain. Detecting over 2,500 unique glycopeptides from over 550 glycoproteins, we found that abundant SV proteins harbor N-glycans with fucose on their complex antennae, and we identify a highly fucosylated N-glycan enriched in SVs as compared to synaptosomes. Antennary fucosylation was also characteristic of plasma membrane proteins and cell adhesion molecules with established roles in synaptic function and development. Our results represent the first defined N-glycoproteome of a neuronal organelle and raise new questions in the glycobiology of synaptic pruning and neuroinflammation.

neuroscience↗

bioRxiv2022

Synaptotagmin 7 outperforms synaptotagmin 1 to open and stabilize nascent fusion pores via robust membrane penetration

Synaptotagmin-1 and synaptotagmin-7 are two prominent Ca2+ sensors that regulate exocytosis in neuronal and neuroendocrine cells. Upon binding Ca2+, both proteins partially penetrate lipid bilayers that bear anionic phospholipids, but the specific underlying mechanisms that enable them to trigger exocytosis remain controversial. Here, we examined the biophysical properties of these two synaptotagmin isoforms and compared their interactions with phospholipid membranes. We discovered that synaptotagmin-1*membrane interactions are greatly influenced by membrane order; tight packing of phosphatidylserine inhibits binding due to impaired membrane penetration. In contrast, synaptotagmin-7 exhibits robust membrane binding and penetration activity regardless of phospholipid acyl chain structure. Thus, synaptotagmin-7 is a "super-penetrator". We exploited these observations to specifically isolate and examine the role of membrane penetration in synaptotagmin function. Using nanodisc-black lipid membrane electrophysiology, we demonstrate that membrane penetration is a critical component that underlies how synaptotagmin proteins regulate reconstituted, exocytic fusion pores in response to Ca2+.

neuroscience↗

bioRxiv2022

Mitochondrial complementation occurs in rat hippocampal axons and supports the synaptic vesicle cycle.

Mitochondria exert powerful control over cellular physiology, contributing to ion homeostasis, energy production, and metabolite biosynthesis. Mitochondrial trafficking and function are vital to neurons, with organelle impairment or altered morphology observed in every neurodegenerative disorder studied. While this organelle and its biosynthetic products are critical for cellular function, decreased output and/or byproducts (e.g., free radicals), can be harmful, and organelle quality control (QC) mechanisms are required to maintain function and prevent a cascade of damage. Owing to its length and general lack of biosynthetic machinery, the axon is particularly sensitive to damage and there is little consensus regarding the details of mitochondrial QC mechanisms in this compartment. Here we investigate the basal, unstressed behavior of axonal mitochondria, focusing on mitochondrial trafficking and fusion to better understand potential QC mechanisms. We observed size and redox asymmetry of mitochondrial traffic in the axon, suggestive of an active QC mechanism. Importantly, we demonstrate, in detail, biochemical complementation of axonal mitochondria. Upon disruption of mitochondrial fusion, we observed an altered synaptic proteome, presynaptic calcium dyshomeostasis, decreased levels of exocytosis, and a reduction in synaptic vesicle recruitment from the reserve pool during extended stimulation. These results support an active mitochondrial trafficking and fusion related QC process that supports presynaptic physiology. O_LIAnterograde trafficked mitochondria are larger and relatively more reduced than retrograde trafficked mitochondria C_LIO_LIAnterograde mitochondria fuse with and complement resident, stationary, axonal mitochondria C_LIO_LILoss of mitofusin 2 (MFN2) mediated mitochondrial fusion leads to alterations in the synaptic vesicle cycle and decreased reserve pool mobilization C_LI

neuroscience↗

bioRxiv2022

Multifunctional fluorophores for live-cell imaging and affinity capture of proteins

Enzyme-based self-labeling tags enable covalent attachment of synthetic molecules to proteins inside living cells. A frontier of this field is designing multifunctional ligands that contain both fluorophores and affinity tags or pharmacological agents and can still efficiently enter cells. Self-labeling tag ligands with short linkers can enter cells readily but often show less activity due to steric issues; ligands with long linkers can be more potent but show lower cell permeability. Here, we overcome this tug-of-war between efficacy and cell-permeability by devising a rational strategy for making cell permeable multifunctional ligands for labeling HaloTag fusions. We found that the lactone-zwitterion equilibrium sconstant (KL-Z) of rhodamines inversely correlates with their distribution coefficients (logD7.4), suggesting that ligands based on dyes exhibiting low KL-Z and high logD7.4 values, such as Si-rhodamines, would efficiently enter cells. We designed cell-permeable multifunctional HaloTag ligands with a biotin moiety to purify mitochondria or a JQ1 appendage to translocate BRD4 from euchromatin to the nucleolus or heterochromatin. We discovered that translocation of BRD4 to constitutive heterochromatin in cells expressing HaloTag-HP1a fusion proteins can lead to apparent increases in transcriptional activity. These new reagents enable affinity capture and translocation of intracellular proteins in living cells and the use of Si-rhodamines and other low KL- Z/high logD7.4 dye scaffolds will facilitate the design of new multifunctional chemical tools for biology. SIGNIFICANCE STATEMENTUnderstanding cellular processes requires tools to measure and manipulate proteins in living cells. Self- labeling tags, such as the HaloTag and SNAP-tag, enable modification of cellular proteins with synthetic molecules. Creating ligands for these systems that have more than one chemical motif remains challenging, however, due to competing demands between cell permeability and functionality. We discovered that multifunctional ligands based on Si-rhodamines efficiently entered cells and enabled affinity purification of mitochondria or translocation of nuclear proteins; the performance of these molecules could be verified by fluorescence microscopy. These compounds should be useful for a variety of biological experiments and our general framework will allow the design of other multifunctional ligands to study living systems.

cell biology↗

bioRxiv2022

Membrane compression by synaptic vesicle exocytosis triggers ultrafast endocytosis

Compensatory endocytosis keeps the surface area of secretory cells constant following exocytosis. At chemical synapses, clathrin-independent ultrafast endocytosis maintains such homeostasis. This endocytic pathway is temporally and spatially coupled to exocytosis, initiating within 50 ms at the region immediately next to where vesicles fuse: the active zone. How synaptic vesicle exocytosis induces ultrafast endocytosis is unknown. Here, we demonstrate that actin filaments are enriched in the region surrounding active zone at mouse hippocampal synapses and that the membrane area conservation due to this actin corral is necessary for exo-endocytic coupling. Simulations suggest that flattening of fused vesicles exerts lateral membrane pressure in the plasma membrane against the actin corral, resulting in rapid formation of endocytic pits at the border between the active zone and the surrounding actin-enriched region. Consistent with our simulations, ultrafast endocytosis does not initiate when actin organization is disrupted, either pharmacologically or by ablation of the actin-binding protein Epsin1. These data suggest that endocytosis is mechanically coupled to exocytosis at synapses.

cell biology↗

bioRxiv2022

Synaptotagmin 7 docks synaptic vesicles for Doc2α-triggered asynchronous neurotransmitter release

Despite decades of intense study, the molecular basis of asynchronous neurotransmitter release remains enigmatic. Synaptotagmin (syt) 7 and Doc2 have both been proposed as Ca2+ sensors that trigger this mode of exocytosis, but conflicting findings have led to controversy. Here, we demonstrate that at excitatory mouse hippocampal synapses, Doc2 is the major Ca2+ sensor for asynchronous release, while syt7 supports this process through activity-dependent docking of synaptic vesicles. In synapses lacking Doc2, asynchronous release after single action potentials is strongly reduced, while deleting syt7 has no effect. However, in the absence of syt7, docked vesicles cannot be replenished on millisecond timescales. Consequently, both synchronous and asynchronous release depress from the second pulse onward during repetitive activity. By contrast, synapses lacking Doc2 have normal activity-dependent docking, but continue to exhibit decreased asynchronous release after multiple stimuli. Moreover, disruption of both Ca2+ sensors is non-additive. These findings result in a new model whereby syt7 drives activity-dependent docking, thus providing synaptic vesicles for synchronous (syt1) and asynchronous (Doc2 and other unidentified sensors) release during ongoing transmission.

neuroscience↗

bioRxiv2022

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 endogenous SYT9 does not trigger rapid synaptic vesicle exocytosis in cultured cortical, hippocampal, or striatal neurons; rather, synaptotagmin 1 (SYT1) fulfills this function. SYT9 is able to regulate evoked synaptic vesicle exocytosis, but only when massively over-expressed. In striatal neurons, loss of SYT9 reduced the rate of spontaneous miniature neurotransmitter release events (minis). To delve into the underlying mechanism, we localized SYT9 within these neurons and found that it is targeted to dense core vesicles, where it regulates the release of substance P (SP), a neuropeptide known to modulate mini frequency. Exogenous SP, but not other striatal peptide hormones, completely rescued the Syt9 KO mini phenotype. Biochemical experiments revealed that Ca2+-binding to the C2A domain of SYT9 triggers membrane fusion in vitro, and mutations that disrupt this activity abolished the ability of SYT9 to regulate both SP release and mini frequency in striatal neurons. We conclude that SYT9 indirectly regulates synaptic transmission in striatal neurons by controlling SP release.

neuroscience↗

bioRxiv2022

Control of contextual memory through interneuronal α5-GABAA receptors

{gamma}-aminobutyric acid type A receptors that incorporate 5 subunits (5-GABAARs) are highly enriched in the hippocampus and are strongly implicated in control of learning and memory. Receptors located on pyramidal neuron dendrites have long been considered responsible, but here we report that their selective knockout from either interneurons (5-i-KO) or pyramidal neurons (5-pyr-KO) interferes with the ability of the general anesthetic etomidate to suppress contextual conditioning. Using Ca2+ imaging of CA1 pyramidal neuron activity in freely exploring mice to assess hippocampal function directly, we found that etomidate blocked the development of place cells and spatial engrams in wild type (WT) and 5-pyr-KO mice, but not in 5-i-KO mice. In addition, 5-i-KO mice developed weaker spatial engrams than WT mice under control conditions. These findings show that interneuronal 5-GABAARs serve a physiological role in promoting spatial learning, and that they mediate the suppression of hippocampus-dependent memory by etomidate.

neuroscience↗

bioRxiv2022

Synaptic vesicle proteins are selectively delivered to axons in mammalian neurons

Neurotransmitter-filled synaptic vesicles (SV) mediate synaptic transmission and are a hallmark specialization in neuronal axons. Yet, how SV proteins are sorted to presynaptic nerve terminals remains the subject of debate. The leading models posit that these proteins are randomly trafficked throughout neurons and are selectively retained in presynaptic boutons. Here, we used the RUSH system, in conjunction with HaloTag labeling approaches, to study the egress of two distinct SV proteins from the soma of cultured neurons. In sharp contrast to the selective retention model, both proteins selectively and specifically entered axons and did not traffic through dendrites; only upon overexpression do SV proteins spillover into other compartments. Moreover, we observed that SV constituents were first delivered to the presynaptic plasma membrane before incorporation into SVs. These experiments reveal a new-found membrane trafficking pathway in classically polarized mammalian neurons and provide a glimpse at the first steps of SV biogenesis.

neuroscience↗

bioRxiv2021

Synaptotagmin 1 oligomerization via the juxtamembrane linker regulates spontaneous and evoked neurotransmitter release

Synaptotagmin-1 (syt1) is a Ca2+ sensor that regulates synaptic vesicle exocytosis. Cell-based experiments suggest that syt1 functions as a multimer, however biochemical and electron microscopy studies have yielded contradictory findings regarding putative self-association. Here, we performed dynamic light scattering on syt1 in solution, followed by electron microscopy, and used atomic force microscopy to study syt1 self-association on supported lipid bilayers under aqueous conditions. Ring-like multimers were clearly observed. Multimerization was enhanced by Ca2+ and required anionic phospholipids. Large ring-like structures ([~]180 nm) were reduced to smaller rings ([~]30 nm) upon neutralization of a cluster of juxtamembrane lysine residues; further substitution of residues in the second C2-domain completely abolished self-association. When expressed in neurons, syt1 mutants with graded reductions in self-association activity exhibited concomitant reductions in: a) clamping spontaneous release, and b) triggering and synchronizing evoked release. Thus, the juxtamembrane linker of syt1 plays a crucial role in exocytosis by mediating multimerization.

neuroscience↗

bioRxiv2021

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. 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 mouse neurons 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 readily releasable vesicle pool or spontaneous release were not affected, so both patches are specifically required for synchronization of release. We suggest these patches contribute to cooperative binding to membranes, increasing the overall affinity of Syt1 for negatively charged membranes and facilitating evoked release. Significance StatementSynaptotagmin-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 two C2 domains is not clear: unlike C2B, calcium-binding to C2A is not critical for evoked release, yet both domains are needed for Syt1 function. Combining electrophysiological recordings from cultured neurons and optical tweezers measurements that probe 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 negatively charged membranes.

neuroscience↗