Two modes of exocytosis at hippocampal synapses revealed by rate of FM1-43 efflux from individual vesicles

We have examined the kinetics by which FM1-43 escapes from individual synaptic vesicles during exocytosis at hippocampal boutons. Two populations of exocytic events were observed; small amplitude events that lose dye slowly, which made up more than half of all events, and faster, larger amplitude events with a fluorescence intensity equivalent to single stained synaptic vesicles. These populations of destaining events are distinct in both brightness and kinetics, suggesting that they result from two distinct modes of exocytosis. Small amplitude events show tightly clustered rate constants of dye release, whereas larger events have a more scattered distribution. Kinetic analysis of the association and dissociation of FM1-43 with membranes, in combination with a simple pore permeation model, indicates that the small, slowly destaining events may be mediated by a narrow ∼1-nm fusion pore

The tandem C2 domains of synaptotagmin contain redundant Ca2+ binding sites that cooperate to engage t-SNAREs and trigger exocytosis

Real-time voltammetry measurements from cracked PC12 cells were used to analyze the role of synaptotagmin–SNARE interactions during Ca2+-triggered exocytosis. The isolated C2A domain of synaptotagmin I neither binds SNAREs nor inhibits norepinephrine secretion. In contrast, two C2 domains in tandem (either C2A-C2B or C2A-C2A) bind strongly to SNAREs, displace native synaptotagmin from SNARE complexes, and rapidly inhibit exocytosis. The tandem C2 domains of synaptotagmin cooperate via a novel mechanism in which the disruptive effects of Ca2+ ligand mutations in one C2 domain can be partially alleviated by the presence of an adjacent C2 domain. Complete disruption of Ca2+-triggered membrane and target membrane SNARE interactions required simultaneous neutralization of Ca2+ ligands in both C2 domains of the protein. We conclude that synaptotagmin–SNARE interactions regulate membrane fusion and that cooperation between synaptotagmin's C2 domains is crucial to its function

Synaptotagmin-Mediated Bending of the Target Membrane Is a Critical Step in Ca2+-Regulated Fusion

SummaryDecades ago it was proposed that exocytosis involves invagination of the target membrane, resulting in a highly localized site of contact between the bilayers destined to fuse. The vesicle protein synaptotagmin-I (syt) bends membranes in response to Ca2+, but whether this drives localized invagination of the target membrane to accelerate fusion has not been determined. Previous studies relied on reconstituted vesicles that were already highly curved and used mutations in syt that were not selective for membrane-bending activity. Here, we directly address this question by utilizing vesicles with different degrees of curvature. A tubulation-defective syt mutant was able to promote fusion between highly curved SNARE-bearing liposomes but exhibited a marked loss of activity when the membranes were relatively flat. Moreover, bending of flat membranes by adding an N-BAR domain rescued the function of the tubulation-deficient syt mutant. Hence, syt-mediated membrane bending is a critical step in membrane fusion

Exocytotic fusion pores are composed of both lipids and proteins.

During exocytosis, fusion pores form the first aqueous connection that allows escape of neurotransmitters and hormones from secretory vesicles. Although it is well established that SNARE proteins catalyze fusion, the structure and composition of fusion pores remain unknown. Here, we exploited the rigid framework and defined size of nanodiscs to interrogate the properties of reconstituted fusion pores, using the neurotransmitter glutamate as a content-mixing marker. Efficient Ca(2+)-stimulated bilayer fusion, and glutamate release, occurred with approximately two molecules of mouse synaptobrevin 2 reconstituted into ∼6-nm nanodiscs. The transmembrane domains of SNARE proteins assumed distinct roles in lipid mixing versus content release and were exposed to polar solvent during fusion. Additionally, tryptophan substitutions at specific positions in these transmembrane domains decreased glutamate flux. Together, these findings indicate that the fusion pore is a hybrid structure composed of both lipids and proteins.We thank Gerhard Wagner for providing the MSP∆1D1H4-H6 plasmid. This study was supported by a grant from the US National Institutes of Health (MH061876). H.B. is supported by a postdoctoral fellowship from Human Frontier Science Program. B.C. and M.P.G are supported by funding from the US National Institutes of Health (R01 GM084140). P.J. is supported by Kidney Research UK. J.M.E. is supported by the Biotechnology and Biological Sciences Research Council (BB/J018236/1) and Kidney Research UK. E.R.C. is supported as an Investigator of the Howard Hughes Medical Institute.This is the author accepted manuscript. The final version is available from NPG via http://dx.doi.org/10.1038/nsmb.314

Functional analysis of the interface between the tandem C2 domains of synaptotagmin-1.

C2 domains are widespread motifs that often serve as Ca(2+)-binding modules; some proteins have more than one copy. An open issue is whether these domains, when duplicated within the same parent protein, interact with one another to regulate function. In the present study, we address the functional significance of interfacial residues between the tandem C2 domains of synaptotagmin (syt)-1, a Ca(2+) sensor for neuronal exocytosis. Substitution of four residues, YHRD, at the domain interface, disrupted the interaction between the tandem C2 domains, altered the intrinsic affinity of syt-1 for Ca(2+), and shifted the Ca(2+) dependency for binding to membranes and driving membrane fusion in vitro. When expressed in syt-1 knockout neurons, the YHRD mutant yielded reductions in synaptic transmission, as compared with the wild-type protein. These results indicate that physical interactions between the tandem C2 domains of syt-1 contribute to excitation-secretion coupling.This study was supported by a grant from the NIH (MH061876). C.S.E. was supported by a PhRMA Foundation predoctoral fellowship and by a UW–Madison Molecular and Cellular Pharmacology Training Grant (5T32-GM008688). R.B.S. was supported by an NIH grant (AR063634). P.J. and J.M.E. were funded by Kidney Research UK, and J.M.E. was funded by the Biotechnology and Biological Sciences Research Council (Grant BB/J018236/1). E.R.C. is an investigator of the Howard Hughes Medical Institute.This is the final version of the article. It first appeared from the American Society for Cell Biology via http://dx.doi.org/10.1091/mbc.E15-07-050

Quotient inductive-inductive types

Higher inductive types (HITs) in Homotopy Type Theory (HoTT) allow the definition of datatypes which have constructors for equalities over the defined type. HITs generalise quotient types and allow to define types which are not sets in the sense of HoTT (i.e. do not satisfy uniqueness of equality proofs) such as spheres, suspensions and the torus. However, there are also interesting uses of HITs to define sets, such as the Cauchy reals, the partiality monad, and the internal, total syntax of type theory. In each of these examples we define several types that depend on each other mutually, i.e. they are inductive-inductive definitions. We call those HITs quotient inductive-inductive types (QIITs). Although there has been recent progress on the general theory of HITs, there isn't yet a theoretical foundation of the combination of equality constructors and induction-induction, despite having many interesting applications. In the present paper we present a first step towards a semantic definition of QIITs. In particular, we give an initial-algebra semantics and show that this is equivalent to the section induction principle, which justifies the intuitively expected elimination rules

Sar1 GTPase Activity Is Regulated by Membrane Curvature.

The majority of biosynthetic secretory proteins initiate their journey through the endomembrane system from specific subdomains of the endoplasmic reticulum. At these locations, coated transport carriers are generated, with the Sar1 GTPase playing a critical role in membrane bending, recruitment of coat components, and nascent vesicle formation. How these events are appropriately coordinated remains poorly understood. Here, we demonstrate that Sar1 acts as the curvature-sensing component of the COPII coat complex and highlight the ability of Sar1 to bind more avidly to membranes of high curvature. Additionally, using an atomic force microscopy-based approach, we further show that the intrinsic GTPase activity of Sar1 is necessary for remodeling lipid bilayers. Consistent with this idea, Sar1-mediated membrane remodeling is dramatically accelerated in the presence of its guanine nucleotide-activating protein (GAP), Sec23-Sec24, and blocked upon addition of guanosine-5'-[(β,γ)-imido]triphosphate, a poorly hydrolysable analog of GTP. Our results also indicate that Sar1 GTPase activity is stimulated by membranes that exhibit elevated curvature, potentially enabling Sar1 membrane scission activity to be spatially restricted to highly bent membranes that are characteristic of a bud neck. Taken together, our data support a stepwise model in which the amino-terminal amphipathic helix of GTP-bound Sar1 stably penetrates the endoplasmic reticulum membrane, promoting local membrane deformation. As membrane bending increases, Sar1 membrane binding is elevated, ultimately culminating in GTP hydrolysis, which may destabilize the bilayer sufficiently to facilitate membrane fission.This work was supported by grants from the NIH (GM110567 and GM088151 to AA). IM, RMH and JME were supported by a grant from the Biotechnology and Biological Sciences Research Council (BB/J018236/1). ERC is an Investigator of the Howard Hughes Medical Institute. We thank Elizabeth Miller for providing purified yeast COPII components, Subhanjan Mondal and Said Goueli at Promega Corporation for providing us access to the GTPase-Glo system ahead of release, and members of the Audhya lab for critically reading this manuscript.This is the final version of the article. It first appeared from the American Society for Biochemistry and Molecular Biology via http://dx.doi.org/10.1074/jbc.M115.67228

Synaptotagmins I and II mediate entry of botulinum neurotoxin B into cells

Botulinum neurotoxins (BoNTs) cause botulism by entering neurons and cleaving proteins that mediate neurotransmitter release; disruption of exocytosis results in paralysis and death. The receptors for BoNTs are thought to be composed of both proteins and gangliosides; however, protein components that mediate toxin entry have not been identified. Using gain-of-function and loss-of-function approaches, we report here that the secretory vesicle proteins, synaptotagmins (syts) I and II, mediate the entry of BoNT/B (but not BoNT/A or E) into PC12 cells. Further, we demonstrate that BoNT/B entry into PC12 cells and rat diaphragm motor nerve terminals was activity dependent and can be blocked using fragments of syt II that contain the BoNT/B-binding domain. Finally, we show that syt II fragments, in conjunction with gangliosides, neutralized BoNT/B in intact mice. These findings establish that syts I and II can function as protein receptors for BoNT/B