Forces, Kinetics, and Fusion Efficiency Altered by the Full-Length Synaptotagmin-1 -PI(4,5)P2 Interaction in Constrained Geometries

AbstractSynaptotagmin 1 acts as the Ca2+-sensor for synchronous neurotransmitter release; however, the mechanism by which it functions is not understood and is presently a topic of considerable interest. Here we describe measurements on full-length membrane reconstituted synaptotagmin 1 using site-directed spin labeling where we characterize the linker region as well as the cis (vesicle membrane) and trans (cytoplasmic membrane) binding of its two C2 domains. In the full-length protein, the C2A domain does not undergo membrane insertion in the absence of Ca2+; however, the C2B domain will bind to and penetrate in trans to a membrane containing phosphatidylinositol 4,5 bisphosphate (PIP2), even if phosphatidylserine (PS) is present in the cis membrane. In the presence of Ca2+, the Ca2+-binding loops of C2A and C2B both insert into the membrane interface; moreover, C2A preferentially inserts into PS containing bilayers and will bind in a cis configuration to membranes containing PS even if a PIP2 membrane is presented in trans. The data are consistent with a bridging activity for Syt1 where the two domains bind to opposing vesicle and plasma membranes. The failure of C2A to bind membranes in the absence of Ca2+ and the long unstructured segment linking C2A to the vesicle membrane indicates that synaptotagmin 1 could act to significantly shorten the vesicle-plasma membrane distance with increasing levels of Ca2+.

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A Calcium-Independent Oligomerization of Full-Length Synaptotagmin 1 is Mediated by its Juxta-Membrane Linker

Biophysical Journal ◽

10.1016/j.bpj.2013.11.1109 ◽

2014 ◽

Vol 106 (2) ◽

pp. 189a-190a

Author(s):

Bin Lu ◽

Volker Kiessling ◽

Lukas Tamm ◽

David Cafiso

Keyword(s):

Full Length ◽

Synaptotagmin 1

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The role of PI(4,5)P2 and synaptotagmin in membrane fusion - an in vitro study

10.1101/2021.05.15.444276 ◽

2021 ◽

Author(s):

Joern Dietz ◽

Marieelen Oelkers ◽

Raphael Hubrich ◽

Angel Perez-Lara ◽

Reinhard Jahn ◽

...

Keyword(s):

Membrane Fusion ◽

Membrane Lipids ◽

In Vitro Study ◽

Snare Proteins ◽

Large Unilamellar Vesicles ◽

Intermediate States ◽

Synaptotagmin 1 ◽

Fusion Efficiency

Synaptotagmin-1 (syt-1) is known to trigger fusion of neuronal synaptic vesicles with the presynaptic membrane by recognizing acidic membrane lipids. In particular, binding to PI(4,5)P2 is believed to be crucial for its function as a calcium sensor. We propose a mechanism for syt-1 to interact with anionic bilayers and promote fusion in the presence of SNARE proteins. We found that in the absence of Ca2+ the binding of syt-1 to membranes depends on the PI(4,5)P2 content. Addition of Ca2+ switches the interaction forces from weak to strong eventually exceeding the cohesion of the C2A domain, while the interaction between PI(4,5)P2 and the C2B domain was preserved even in the absence of Ca2+ or phosphatidylserine. Fusion of large unilamellar vesicles equipped with syt-1 and synaptobrevin with freestanding target membranes composed of PS/PI(4,5)P2 show an increased fusion speed, and by effective suppression of stalled intermediate states, a larger number of full fusion events. Fusion efficiency could be maximized when irreversible docking is additionally prevented by addition of multivalent anions. The picture that emerges is that syt-1 remodels the membrane in the presence of calcium and PIP2, thereby substantially increasing the efficiency of membrane fusion by avoiding stalled intermediate states.

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Interactions of Full-Length Synaptotagmin 1 with Ca2+ and Membrane Surfaces

Biophysical Journal ◽

10.1016/j.bpj.2012.11.1259 ◽

2013 ◽

Vol 104 (2) ◽

pp. 223a

Author(s):

Bin Lu ◽

Volker Kiessling ◽

Lukas K. Tamm ◽

David S. Cafiso

Keyword(s):

Full Length ◽

Membrane Surfaces ◽

Synaptotagmin 1

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Synaptotagmin-1–, Munc18-1–, and Munc13-1–dependent liposome fusion with a few neuronal SNAREs

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2019314118 ◽

2021 ◽

Vol 118 (4) ◽

pp. e2019314118

Author(s):

Karolina P. Stepien ◽

Josep Rizo

Keyword(s):

Neurotransmitter Release ◽

Plasma Membranes ◽

Snare Complex ◽

Complex Assembly ◽

Synaptotagmin 1 ◽

Low Levels ◽

Liposome Fusion ◽

Membrane Anchoring ◽

Fusion Efficiency

Neurotransmitter release is governed by eight central proteins among other factors: the neuronal SNAREs syntaxin-1, synaptobrevin, and SNAP-25, which form a tight SNARE complex that brings the synaptic vesicle and plasma membranes together; NSF and SNAPs, which disassemble SNARE complexes; Munc18-1 and Munc13-1, which organize SNARE complex assembly; and the Ca2+ sensor synaptotagmin-1. Reconstitution experiments revealed that Munc18-1, Munc13-1, NSF, and α-SNAP can mediate Ca2+-dependent liposome fusion between synaptobrevin liposomes and syntaxin-1–SNAP-25 liposomes, but high fusion efficiency due to uncontrolled SNARE complex assembly did not allow investigation of the role of synaptotagmin-1 on fusion. Here, we show that decreasing the synaptobrevin-to-lipid ratio in the corresponding liposomes to very low levels leads to inefficient fusion and that synaptotagmin-1 strongly stimulates fusion under these conditions. Such stimulation depends on Ca2+ binding to the two C2 domains of synaptotagmin-1. We also show that anchoring SNAP-25 on the syntaxin-1 liposomes dramatically enhances fusion. Moreover, we uncover a synergy between synaptotagmin-1 and membrane anchoring of SNAP-25, which allows efficient Ca2+-dependent fusion between liposomes bearing very low synaptobrevin densities and liposomes containing very low syntaxin-1 densities. Thus, liposome fusion in our assays is achieved with a few SNARE complexes in a manner that requires Munc18-1 and Munc13-1 and that depends on Ca2+ binding to synaptotagmin-1, all of which are fundamental features of neurotransmitter release in neurons.

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