Ward C. Tucker

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+.

Ca 2+ –synaptotagmin directly regulates t-SNARE function during reconstituted membrane fusion

In nerve terminals, exocytosis is mediated by SNARE proteins and regulated by Ca2+ and synaptotagmin-1 (syt). Ca2+ promotes the interaction of syt with anionic phospholipids and the target membrane SNAREs (t-SNAREs) SNAP-25 and syntaxin. Here, we have used a defined reconstituted fusion assay to determine directly whether syt–t-SNARE interactions couple Ca2+ to membrane fusion by comparing the effects of Ca2+–syt on neuronal (SNAP-25, syntaxin and synaptobrevin) and yeast (Sso1p, Sec9c and Snc2p) SNAREs. Ca2+–syt aggregated neuronal and yeast SNARE liposomes to similar extents via interactions with anionic phospholipids. However, Ca2+–syt was able to bind and stimulate fusion mediated by only neuronal SNAREs and had no effect on yeast SNAREs. Thus, Ca2+–syt regulates fusion through direct interactions with t-SNAREs and not solely through aggregation of vesicles. Ca2+–syt drove assembly of SNAP-25 onto membrane-embedded syntaxin, providing direct evidence that Ca2+–syt alters t-SNARE structure.

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.

Role of synaptotagmin in Ca2+-triggered exocytosis

The Ca(2+)-binding synaptic-vesicle protein synaptotagmin I has attracted considerable interest as a potential Ca(2+) sensor that regulates exocytosis from neurons and neuroendocrine cells. Recent studies have shed new light on the structure, biochemical/biophysical properties and function of synaptotagmin, and the emerging view is that it plays an important role in both exocytosis and endocytosis. At least a dozen additional isoforms exist, some of which are expressed outside of the nervous system, suggesting that synaptotagmins might regulate membrane traffic in a variety of cell types. Here we provide an overview of the members of this gene family, with particular emphasis on the question of whether and how synaptotagmin I functions during the final stages of membrane fusion: does it regulate the Ca(2+)-triggered opening and dilation of fusion pores?

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.

Molecular Regulation of Membrane Resealing in 3T3 Fibroblasts

Membrane resealing in mammalian cells after injury depends on Ca2+-dependent fusion of intracellular vesicles with the plasma membrane. When cells are wounded twice, the subsequent resealing is generally faster. Physiological and biochemical studies have shown the initiation of two different repair signaling pathways, which are termed facilitated and potentiated responses. The facilitated response is dependent on the generation and recruitment of new vesicles, whereas the potentiated response is not. Here, we report that the two responses can be differentially defined molecularly. Using recombinant fragments of synaptobrevin-2 and synaptotagmin C2 domains we were able to dissociate the molecular requirements of vesicle exocytosis for initial membrane resealing and the facilitated and potentiated responses. The initial resealing response was blocked by fragments of synaptobrevin-2 and the C2B domain of synaptotagmin VII. Both the facilitated and potentiated responses were also blocked by the C2B domain of synaptotagmin VII. Although the initial resealing response was not blocked by the C2AB domain of synaptotagmin I or the C2A domain of synaptotagmin VII, recruitment of new vesicles for the facilitated response was inhibited. We also used Ca2+ binding mutant studies to show that the effects of synaptotagmins on membrane resealing are Ca2+-dependent. The pattern of inhibition by synaptotagmin C2 fragments that we observed cannot be used to specify a vesicle compartment, such as lysosomes, in membrane repair.