Summary Synaptotagmin-1 and neuronal SNARE proteins play key roles in evoked synchronous neurotransmitter release. However, it is unknown how they cooperate to trigger synaptic vesicle fusion. Here we report atomic-resolution crystal structures of Ca2+- and Mg2+-bound complexes between synaptotagmin-1 and the neuronal SNARE complex, one of which was determined with diffraction data from an X-ray free electron laser, leading to an atomic-resolution structure with accurate rotamer assignments for many sidechains. The structures revealed several interfaces, including a large, specific, Ca2+-independent, and conserved interface. Tests of this interface by mutagenesis suggest that it is essential for Ca2+-triggered neurotransmitter release in neuronal synapses and for Ca2+-triggered vesicle fusion in a reconstituted system. We propose that this interface forms prior to Ca2+-triggering, and moves en bloc as Ca2+ influx promotes the interactions between synaptotagmin-1 and the plasma membrane, and consequently remodels the membrane to promote fusion, possibly in conjunction with other interfaces.

In forebrain neurons, knockout of synaptotagmin-1 blocks fast Ca2+-triggered synchronous neurotransmitter release, but enables manifestation of slow Ca2+-triggered asynchronous release. Here, we show using single-cell PCR that individual hippocampal neurons abundantly co-express two Ca2+-binding synaptotagmin isoforms, synaptotagmin-1 and synaptotagmin-7. In synaptotagmin-1 deficient synapses of excitatory and inhibitory neurons, loss-of-function of synaptotagmin-7 suppressed asynchronous release. This phenotype was rescued by wild-type but not mutant synaptotagmin-7 lacking functional Ca2+-binding sites. Even in synaptotagmin-1 containing neurons, synaptotagmin-7 ablation partly impaired asynchronous release induced by extended high-frequency stimulus trains. Synaptotagmins bind Ca2+ via two C2-domains, the C2A- and C2B-domains. Surprisingly, synaptotagmin-7 function selectively required its C2A-domain Ca2+-binding sites, whereas synaptotagmin-1 function required its C2B-domain Ca2+-binding sites. Our data show that nearly all Ca2+-triggered release at a synapse is due to synaptotagmins, with synaptotagmin-7 mediating a slower form of Ca2+-triggered release that is normally occluded by faster synaptotagmin-1-induced release, but becomes manifest upon synaptotagmin-1 deletion.

Sensory organs are composed of neurons, which convert environmental stimuli to electrical signals, and glia-like cells, whose functions are not well-understood. To decipher glial roles in sensory organs, we ablated the sheath glial cell of the major sensory organ of Caenorhabditis elegans. We found that glia-ablated animals exhibit profound sensory deficits and that glia provide activities that affect neuronal morphology, behavior generation, and neuronal uptake of lipophilic dyes. To understand the molecular bases of these activities, we identified 298 genes whose mRNAs are glia-enriched. One gene, fig-1, encodes a labile protein with conserved thrombospondin TSP1 domains. FIG-1 protein functions extracellularly, is essential for neuronal dye uptake, and also affects behavior. Our results suggest that glia are required for multiple aspects of sensory organ function.Glia, the largest cell population in vertebrate nervous systems, are implicated in processes governing nervous system development and function (1). However, the functions of few glial proteins are characterized. Astrocytic glia are often positioned near synapses, and can respond to and participate in synaptic activity (2,3), influencing the response of postsynaptic cells to presynaptic stimulation (4).Sensory neurons convert environmental stimuli into neuronal activity, and their receptive endings are often associated with glia, such as retinal pigmented epithelial cells and Müller glia or olfactory ensheathing cells. Because sensory neurons are postsynaptic to the environment, their associated glia may impact sensory activity in ways analogous to synaptic astrocytes.Sensory organs are conserved structures, exhibiting morphological, functional, and molecular similarities among diverged species (5). To understand glial contributions to sensory neuron functions, we studied the largest sensory organ of the nematode C. elegans, the amphid. This organ mediates responses to chemical, thermal, and tactile stimuli, promoting attractive and repulsive behaviors that are easily assayed. Each of the bilateral amphids comprises twelve neurons extending ciliated dendrites to the anterior tip (5). These neurons can be grouped based on association with the single amphid sheath glial cell: the dendritic receptive endings of four neurons are entirely surrounded by this glial cell in a hand-in-glove configuration, while remaining cilia are encased in a channel formed by the same glial cell, and are exposed, through a pore, to the outside environment (5,6) ( fig. S1).We ablated sheath glia in first-stage larvae, after the amphid had formed, by either using a laser microbeam (7), or expressing the diphtheria toxin A gene from a sheath-glia-specific promoter (8). Ablation success was monitored by disappearance of a glia-specific GFP reporter, and by electron microscopy (EM) reconstruction of amphid sensory endings. We first examined the †To whom correspondence should be addressed.