Phosphatidylinositol 4,5-bisphosphate drives Ca2+-independent membrane penetration by the tandem C2 domain proteins synaptotagmin-1 and Doc2β

Abstract

Exocytosis mediates the release of neurotransmitters and hormones from neurons and neuroendocrine cells. Tandem C2 domain proteins in the synaptotagmin (syt) and double C2 domain (Doc2) families regulate exocytotic membrane fusion via direct interactions with Ca2+ and phospholipid bilayers. Syt1 is a fast-acting, low-affinity Ca2+ sensor that penetrates membranes upon binding Ca2+ to trigger synchronous vesicle fusion. The closely related Doc2β is a slow-acting, high-affinity Ca2+ sensor that triggers spontaneous and asynchronous vesicle fusion, but whether it also penetrates membranes is unknown. Both syt1 and Doc2β bind the dynamically regulated plasma membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2), but it is unclear whether PIP2 serves only as a membrane contact or enables specialized membrane-binding modes by these Ca2+ sensors. Furthermore, it has been shown that PIP2 uncaging can trigger rapid, syt1-dependent exocytosis in the absence of Ca2+ influx, suggesting that current models for the action of these Ca2+ sensors are incomplete. Here, using a series of steady-state and time-resolved fluorescence measurements, we show that Doc2β, like syt1, penetrates membranes in a Ca2+-dependent manner. Unexpectedly, we observed that PIP2 can drive membrane penetration by both syt1 and Doc2β in the absence of Ca2+, providing a plausible mechanism for Ca2+-independent, PIP2-dependent exocytosis. Quantitative measurements of penetration depth revealed that, in the presence of Ca2+, PIP2 drives Doc2β, but not syt1, substantially deeper into the membrane, defining a biophysical regulatory mechanism specific to this high-affinity Ca2+ sensor. Our results provide evidence of a novel role for PIP2 in regulating, and under some circumstances triggering, exocytosis.

SNARE Complex Alters the Interactions of the Ca2+sensor Synaptotagmin 1 with Lipid Bilayers

“…These residues interchange in forming salt bridges with several PIP2 molecules, enabling dynamic and reliable anchoring to a PIP2 cluster. In agreement with [63], the simulations showed that such attachment of the C2B domain to a PIP2 cluster occurs even in the absence of Ca 2+ . The simulations also showed that upon Ca 2+ binding the C2B module tilts and its Ca 2+ -bound tip immerses deeper into the bilayer.…”

“…The importance of PIP2 in anchoring Syt1 to lipid bilayers has been already established [25,[63][64][65][66]. Our MD simulations identified the Syt1 residues interacting with PIP2 and determined the 3D configurations for the attachment of each of the C2 modules to lipid bilayers.…”

Phosphatidylinositol(4,5)bisphosphate: diverse functions at the plasma membrane

“…Subsequent work in several types of secretory cells found PI(4,5P) 2 necessary for exocytosis [ 107 , 108 ]. Several PI(4,5P) 2 -binding proteins have been identified with important functions in SNARE complex assembly, including C2-domain-containing proteins, synaptotagmin-1 and Munc13-1 [ 109 ], PH- and C2-domain-containing protein CAPS (Ca 2+ -dependent activator protein for secretion), and syntaxin-1 [ 110 ]. Synaptotagmin-1 is a synaptic vesicle-associated membrane protein whilst Munc13-1 and CAPS are cytosolic protein recruited by PI(4,5)P 2 .…”

Plasma membrane phosphatidylinositol (4,5)-bisphosphate promotes Weibel–Palade body exocytosis

“…A contribution of PI(4,5)P 2 had been reported before in other types of exocytotic fusion events where the lipid appears to participate in protein-lipid interactions and protein activation at a pre-fusion stage ( Holz, 2006 ; Martin, 2015 ; Lauwers et al, 2016 ; Gawden-Bone et al, 2018 ). In neurons and neuroendocrine cells, the two best studied cell types in this respect, PI(4,5)P 2 has been shown to interact with components of the protein machinery involved in docking, priming and PM fusion of synaptic vesicles and dense core granules, for example, to facilitate the ability of Munc13 to induce SNARE complex formation, to increase syntaxin clustering and to regulate the membrane interaction of synaptotagmin-1, CAPS, or Doc2β and thus their role in vesicle docking and fusion ( Kabachinski et al, 2014 ; Park et al, 2015 ; Petrie et al, 2016 ; Walter et al, 2017 ; Bradberry et al, 2019 ). By interacting with proteins of the (clathrin-mediated) endocytosis machinery, PI(4,5)P 2 can also function in membrane/protein internalization and probably contributes to the maintenance of a homeostatic balance between PM expansion by exocytosis and compensatory retrieval by endocytosis ( Mettlen et al, 2018 ).…”