Jihong Bai

PIP2 increases the speed of response of synaptotagmin and steers its membrane-penetration activity toward the plasma membrane.

Synaptotagmin-1 (syt), the putative Ca2+ sensor for exocytosis, is anchored to the membrane of secretory organelles. Its cytoplasmic domain is composed of two Ca2+-sensing modules, C2A and C2B. Syt binds phosphatidylinositol 4,5-bisphosphate (PIP2), a plasma membrane lipid with an essential role in exocytosis and endocytosis. We resolved two modes of PIP2 binding that are mediated by distinct surfaces on the C2B domain of syt. A novel Ca2+-independent mode of binding predisposes syt to penetrate PIP2-harboring target membranes in response to Ca2+ with submillisecond kinetics. Thus, PIP2 increases the speed of response of syt and steers its membrane-penetration activity toward the plasma membrane. We propose that syt-PIP2 interactions are involved in exocytosis by facilitating the close apposition of the vesicle and target membrane on rapid time scales in response to Ca2+.

Different domains of synaptotagmin control the choice between kiss-and-run and full fusion

Exocytosis—the release of the contents of a vesicle—proceeds by two mechanisms. Full fusion occurs when the vesicle and plasma membranes merge. Alternatively, in what is termed kiss-and-run, vesicles can release transmitter during transient contacts with the plasma membrane. Little is known at the molecular level about how the choice between these two pathways is regulated. Here we report amperometric recordings of catecholamine efflux through individual fusion pores. Transfection with synaptotagmin (Syt) IV increased the frequency and duration of kiss-and-run events, but left their amplitude unchanged. Endogenous Syt IV, induced by forskolin treatment, had a similar effect. Full fusion was inhibited by mutation of a Ca2+ ligand in the C2A domain of Syt I; kiss-and-run was inhibited by mutation of a homologous Ca2+ ligand in the C2B domain of Syt IV. The Ca2+ sensitivity for full fusion was 5-fold higher with Syt I than Syt IV, but for kiss-and-run the Ca2+ sensitivities differed by a factor of only two. Syt thus regulates the choice between full fusion and kiss-and-run, with Ca2+ binding to the C2A and C2B domains playing an important role in this choice.

Fusion pore dynamics are regulated by synaptotagmin*t-SNARE interactions

Exocytosis involves the formation of a fusion pore that connects the lumen of secretory vesicles with the extracellular space. Exocytosis from neurons and neuroendocrine cells is tightly regulated by intracellular [Ca2+] and occurs rapidly, but the molecular events that mediate the opening and subsequent dilation of fusion pores remain to be determined. A putative Ca2+ sensor for release, synaptotagmin I (syt), binds directly to syntaxin and SNAP-25, which are components of a conserved membrane fusion complex. Here, we show that Ca2+-triggered syt*SNAP-25 interactions occur rapidly. The tandem C2 domains of syt cooperate to mediate binding to syntaxin/SNAP-25; lengthening the linker that connects C2A and C2B selectively disrupts this interaction. Expression of the linker mutants in PC12 cells results in graded reductions in the stability of fusion pores. Thus, the final step of Ca2+-triggered exocytosis is regulated, at least in part, by direct contacts between syt and SNAP-25/syntaxin.

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.

C2A activates a cryptic Ca2+-triggered membrane penetration activity within the C2B domain of synaptotagmin I

Synaptotagmin (syt) I, an integral membrane protein localized to secretory vesicles, is a putative Ca2+ sensor for exocytosis. Its N terminus spans the membrane once, and its cytoplasmic domain contains two conserved C2 domains, designated C2A and C2B. The isolated C2A domain penetrates membranes in response to Ca2+; isolated C2B does not. Here, we have addressed the function of each C2 domain, but in the context of the intact cytoplasmic domain (C2A-C2B), by using fluorescent reporters placed in the Ca2+-binding loops of either C2A or C2B. Surprisingly, these reporters revealed that, analogous to C2A, a Ca2+-binding loop in C2B directly penetrates into lipid bilayers. Penetration of each C2 domain was very rapid (kon ≈1010 M−1⋅s−1) and resulted in high affinity C2A-C2B–liposome complexes (Kd ≈13–14 nM). C2B-bilayer penetration strictly depended on the presence, but not the membrane binding activity, of an adjacent C2A domain, severing C2A from C2B after protein synthesis abolished the ability of C2B to dip into bilayers in response to Ca2+. The activation of C2B by C2A was also displayed by the C2 domains of syt III but not the C2 domains of syt IV. A number of proteins contain more than one C2 domain; the findings reported here suggest these domains may harbor cryptic activities that are not detected when they are studied in isolation.

Identification of synaptotagmin effectors via acute inhibition of secretion from cracked PC12 cells

T he synaptotagmins (syts) are a family of membrane proteins proposed to regulate membrane traffic in neuronal and nonneuronal cells. In neurons, the Ca2+-sensing ability of syt I is critical for fusion of docked synaptic vesicles with the plasma membrane in response to stimulation. Several putative Ca2+–syt effectors have been identified, but in most cases the functional significance of these interactions remains unknown. Here, we have used recombinant C2 domains derived from the cytoplasmic domains of syts I–XI to interfere with endogenous syt–effector interactions during Ca2+-triggered exocytosis from cracked PC12 cells. Inhibition was closely correlated with syntaxin–SNAP-25 and phosphatidylinositol 4,5-bisphosphate (PIP2)–binding activity. Moreover, we measured the expression levels of endogenous syts in PC12 cells; the major isoforms are I and IX, with trace levels of VII. As expected, if syts I and IX function as Ca2+ sensors, fragments from these isoforms blocked secretion. These data suggest that syts trigger fusion via their Ca2+-regulated interactions with t-SNAREs and PIP2, target molecules known to play critical roles in exocytosis.

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 synaptotagmins C2 domains is crucial to its function.

Synaptotagmin-Ca2+ triggers two sequential steps in regulated exocytosis in rat PC12 cells : fusion pore opening and fusion pore dilation

Synaptotagmin I (Syt I), the putative Ca2+ sensor in regulated exocytosis, has two Ca2+‐binding modules, the C2A and C2B domains, and a number of putative effectors to which Syt I binds in a Ca2+‐dependent fashion. The role of Ca2+ binding to these domains remains unclear, as efforts to address questions about Ca2+‐triggered effector interactions have led to conflicting results. We have studied the effects of Ca2+ on fusion pores using amperometry to follow the exocytosis of single vesicles in real time and analyse the kinetics of fusion pore transitions. Elevating [Ca2+] in permeabilized cells reduced the fusion pore lifetime, indicating an action of Ca2+ during the actual fusion process. Analysing the Ca2+ dependence of the fusion pore lifetime, together with the frequency of pore openings and the proportion of openings that close without dilating (kiss‐and‐run events) enabled us to resolve exocytosis into a sequence of kinetic steps representing functional transitions in the fusion pore. Fusion pore opening and dilation were both accelerated by Ca2+, indicating separate Ca2+ control over each of these steps. Ca2+ ligand mutations in either the C2A or C2B domains of Syt I reduced fusion pore opening, but had opposite actions on the rate of fusion pore closure. These studies resolve two separate and distinct Ca2+‐triggered steps during regulated exocytosis. The C2A and C2B domains of Syt I have different actions during these steps, and these actions may be linked to their distinctive effector interactions.

Membrane-embedded Synaptotagmin Penetrates cis or trans Target Membranes and Clusters via a Novel Mechanism*

The synaptic vesicle protein synaptotagmin I has been proposed to serve as a Ca2+ sensor for rapid exocytosis. Synaptotagmin spans the vesicle membrane once and possesses a cytoplasmic domain largely comprised of two C2 domains designated C2A and C2B. We have determined how deep the Ca2+-binding loops of Ca2+·C2A penetrate into the lipid bilayer and report mutations in synaptotagmin that can uncouple membrane penetration from Ca2+-triggered interactions with the SNARE complex. To determine whether C2A penetrates into the vesicle (“cis”) or plasma (“trans”) membrane, we reconstituted a fragment of synaptotagmin that includes the membrane-spanning and C2A domain (C2A-TMR) into proteoliposomes. Kinetics experiments revealed that cis interactions are rapid (≤500 μs). Binding in the trans mode was distinguished by the slow diffusion of trans target vesicles. Both modes of binding were observed, indicating that the linker between the membrane anchor and C2A domain functions as a flexible tether. C2A-TMR assembled into oligomers via a novel N-terminal oligomerization domain suggesting that synaptotagmin may form clusters on the surface of synaptic vesicles. This novel mode of clustering may allow for rapid Ca2+-triggered oligomerization of the protein via the membrane distal C2B domain.

Visualization of synaptotagmin I oligomers assembled onto lipid monolayers

Neuronal exocytosis is mediated by Ca2+-triggered rearrangements between proteins and lipids that result in the opening and dilation of fusion pores. Synaptotagmin I (syt I) is a Ca2+-sensing protein proposed to regulate fusion pore dynamics via Ca2+-promoted binding of its cytoplasmic domain (C2A-C2B) to effector molecules, including anionic phospholipids and other copies of syt. Functional studies indicate that Ca2+-triggered oligomerization of syt is a critical step in excitation–secretion coupling; however, this activity has recently been called into question. Here, we show that Ca2+ does not drive the oligomerization of C2A-C2B in solution. However, analysis of Ca2+⋅C2A-C2B bound to lipid monolayers, using electron microscopy, revealed the formation of ring-like heptameric oligomers that are ≈11 nm long and ≈11 nm in diameter. In some cases, C2A-C2B also assembled into long filaments. Oligomerization, but not membrane binding, was disrupted by neutralization of two lysine residues (K326,327) within the C2B domain of syt. These data indicate that Ca2+ first drives C2A-C2B⋅membrane interactions, resulting in conformational changes that trigger a subsequent C2B-mediated oligomerization step. Ca2+-mediated rearrangements between syt subunits may regulate the opening or dilation kinetics of fusion pores or may play a role in endocytosis after fusion.