scholarly journals C-terminal domain of mammalian complexin-1 localizes to highly curved membranes

2016 ◽  
Vol 113 (47) ◽  
pp. E7590-E7599 ◽  
Author(s):  
Jihong Gong ◽  
Ying Lai ◽  
Xiaohong Li ◽  
Mengxian Wang ◽  
Jeremy Leitz ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Neurotransmitter Release ◽  
Membrane Curvature ◽  
Neuronal Cultures ◽  
Spontaneous Release ◽  
Vesicle Membrane ◽  
Curvature Sensing ◽  
Postsynaptic Currents ◽  
Terminal Domain

In presynaptic nerve terminals, complexin regulates spontaneous “mini” neurotransmitter release and activates Ca2+-triggered synchronized neurotransmitter release. We studied the role of the C-terminal domain of mammalian complexin in these processes using single-particle optical imaging and electrophysiology. The C-terminal domain is important for regulating spontaneous release in neuronal cultures and suppressing Ca2+-independent fusion in vitro, but it is not essential for evoked release in neuronal cultures and in vitro. This domain interacts with membranes in a curvature-dependent fashion similar to a previous study with worm complexin [Snead D, Wragg RT, Dittman JS, Eliezer D (2014) Membrane curvature sensing by the C-terminal domain of complexin. Nat Commun 5:4955]. The curvature-sensing value of the C-terminal domain is comparable to that of α-synuclein. Upon replacement of the C-terminal domain with membrane-localizing elements, preferential localization to the synaptic vesicle membrane, but not to the plasma membrane, results in suppression of spontaneous release in neurons. Membrane localization had no measurable effect on evoked postsynaptic currents of AMPA-type glutamate receptors, but mislocalization to the plasma membrane increases both the variability and the mean of the synchronous decay time constant of NMDA-type glutamate receptor evoked postsynaptic currents.

scholarly journals Membrane curvature sensing by the C-terminal domain of complexin

2014 ◽  
Vol 5 (1) ◽  
Author(s):  
David Snead ◽  
Rachel T. Wragg ◽  
Jeremy S. Dittman ◽  
David Eliezer
Keyword(s):  
Membrane Curvature ◽  
Curvature Sensing ◽  
Terminal Domain

scholarly journals An amphipathic helix enables septins to sense micrometer-scale membrane curvature

2019 ◽  
Vol 218 (4) ◽  
pp. 1128-1137 ◽  
Author(s):  
Kevin S. Cannon ◽  
Benjamin L. Woods ◽  
John M. Crutchley ◽  
Amy S. Gladfelter
Keyword(s):  
Membrane Curvature ◽  
Amphipathic Helix ◽  
Curvature Sensing ◽  
C Terminus ◽  
Number Of Binding Sites ◽  
Curved Membranes ◽  
Necessary And Sufficient ◽  
Micrometer Scale

Cell shape is well described by membrane curvature. Septins are filament-forming, GTP-binding proteins that assemble on positive, micrometer-scale curvatures. Here, we examine the molecular basis of curvature sensing by septins. We show that differences in affinity and the number of binding sites drive curvature-specific adsorption of septins. Moreover, we find septin assembly onto curved membranes is cooperative and show that geometry influences higher-order arrangement of septin filaments. Although septins must form polymers to stay associated with membranes, septin filaments do not have to span micrometers in length to sense curvature, as we find that single-septin complexes have curvature-dependent association rates. We trace this ability to an amphipathic helix (AH) located on the C-terminus of Cdc12. The AH domain is necessary and sufficient for curvature sensing both in vitro and in vivo. These data show that curvature sensing by septins operates at much smaller length scales than the micrometer curvatures being detected.

scholarly journals Structural elements that underlie Doc2β function during asynchronous synaptic transmission

2015 ◽  
Vol 112 (31) ◽  
pp. E4316-E4325 ◽  
Author(s):  
Renhao Xue ◽  
Jon D. Gaffaney ◽  
Edwin R. Chapman
Keyword(s):  
Plasma Membrane ◽  
Synaptic Transmission ◽  
Neurotransmitter Release ◽  
Lipid Binding ◽  
Slow Phase ◽  
Structural Elements ◽  
Excitatory Postsynaptic Current ◽  
Synaptotagmin 1 ◽  
Asynchronous Release

Double C2-like domain-containing proteins alpha and beta (Doc2α and Doc2β) are tandem C2-domain proteins proposed to function as Ca2+ sensors for asynchronous neurotransmitter release. Here, we systematically analyze each of the negatively charged residues that mediate binding of Ca2+ to the β isoform. The Ca2+ ligands in the C2A domain were dispensable for Ca2+-dependent translocation to the plasma membrane, with one exception: neutralization of D220 resulted in constitutive translocation. In contrast, three of the five Ca2+ ligands in the C2B domain are required for translocation. Importantly, translocation was correlated with the ability of the mutants to enhance asynchronous release when overexpressed in neurons. Finally, replacement of specific Ca2+/lipid-binding loops of synaptotagmin 1, a Ca2+ sensor for synchronous release, with corresponding loops from Doc2β, resulted in chimeras that yielded slower kinetics in vitro and slower excitatory postsynaptic current decays in neurons. Together, these data reveal the key determinants of Doc2β that underlie its function during the slow phase of synaptic transmission.

scholarly journals A new life for an old pump: V-ATPase and neurotransmitter release

2014 ◽  
Vol 205 (1) ◽  
pp. 7-9 ◽  
Author(s):  
Stefano Vavassori ◽  
Andreas Mayer
Keyword(s):  
Plasma Membrane ◽  
Complex Formation ◽  
Membrane Fusion ◽  
Synaptic Vesicle ◽  
Neurotransmitter Release ◽  
Synaptic Vesicles ◽  
Snare Complex ◽  
Spontaneous Release ◽  
Synaptic Vesicle Fusion ◽  
Snare Complex Formation

Neurons fire by releasing neurotransmitters via fusion of synaptic vesicles with the plasma membrane. Fusion can be evoked by an incoming signal from a preceding neuron or can occur spontaneously. Synaptic vesicle fusion requires the formation of trans complexes between SNAREs as well as Ca2+ ions. Wang et al. (2014. J. Cell Biol. http://dx.doi.org/jcb.201312109) now find that the Ca2+-binding protein Calmodulin promotes spontaneous release and SNARE complex formation via its interaction with the V0 sector of the V-ATPase.

scholarly journals Complexin inhibits spontaneous release and synchronizes Ca2+-triggered synaptic vesicle fusion by distinct mechanisms

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Ying Lai ◽  
Jiajie Diao ◽  
Daniel J Cipriano ◽  
Yunxiang Zhang ◽  
Richard A Pfuetzner ◽  
...  
Keyword(s):  
Lipid Content ◽  
Point Contact ◽  
Vesicle Fusion ◽  
Neuronal Cultures ◽  
Spontaneous Release ◽  
Terminal Domain ◽  
Synaptotagmin 1 ◽  
Free Membrane ◽  
Synaptic Vesicle Fusion ◽  
Single Vesicle

Previously we showed that fast Ca2+-triggered vesicle fusion with reconstituted neuronal SNAREs and synaptotagmin-1 begins from an initial hemifusion-free membrane point contact, rather than a hemifusion diaphragm, using a single vesicle–vesicle lipid/content mixing assay (<xref ref-type="bibr" rid="bib5">Diao et al., 2012</xref>). When complexin-1 was included, a more pronounced Ca2+-triggered fusion burst was observed, effectively synchronizing the process. Here we show that complexin-1 also reduces spontaneous fusion in the same assay. Moreover, distinct effects of several complexin-1 truncation mutants on spontaneous and Ca2+-triggered fusion closely mimic those observed in neuronal cultures. The very N-terminal domain is essential for synchronization of Ca2+-triggered fusion, but not for suppression of spontaneous fusion, whereas the opposite is true for the C-terminal domain. By systematically varying the complexin-1 concentration, we observed differences in titration behavior for spontaneous and Ca2+-triggered fusion. Taken together, complexin-1 utilizes distinct mechanisms for synchronization of Ca2+-triggered fusion and inhibition of spontaneous fusion.

scholarly journals Comparative study of curvature sensing mediated by F-BAR domain and an intrinsically disordered region of FBP17

2020 ◽  
Author(s):  
Maohan Su ◽  
Yinyin Zhuang ◽  
Xinwen Miao ◽  
Yongpeng Zeng ◽  
Weibo Gao ◽  
...  
Keyword(s):  
Lipid Bilayer ◽  
Membrane Trafficking ◽  
Membrane Curvature ◽  
Wave System ◽  
Common Belief ◽  
Bar Domain ◽  
Curvature Sensing ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Region

Membrane curvature has emerged as an intriguing physical organization principle underlying biological signaling and membrane trafficking. FBP17 of the CIP4/FBP17/Toca-1 F-BAR family is unique in the BAR family because its structurally folded F-BAR domain does not contain any hydrophobic motifs that insert into lipid bilayer. While it has been widely assumed so, whether the banana-shaped F-BAR domain alone can sense curvature has never been experimentally demonstrated. Using a nanopillar-supported lipid bilayer system, we found that the F-BAR domain of FBP17 displayed minimal curvature sensing in vitro. We further identified an alternatively spliced intrinsically disordered region (IDR) of FBP17 next to its F-BAR domain that is conserved in sequence across species. The IDR senses membrane curvature and its sensing ability greatly exceeds that of F-BAR domain alone. In living cells, presence of the IDR domain changed the dynamics of FBP17 recruitment in a curvature-coupled cortical wave system. Collectively, we propose that FBP17 does sense curvature but contrary to the common belief, its curvature sensing capability largely originates from its disordered region, not F-BAR domain itself.

scholarly journals Control of neurotransmitter release by two distinctmembrane-binding faces of the Munc13-1 C­1C2B region

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Marcial Camacho ◽  
Bradley Quade ◽  
Thorsten Trimbuch ◽  
Junjie Xu ◽  
Levent Sari ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Neurotransmitter Release ◽  
Synaptic Vesicles ◽  
Point Mutations ◽  
Short Term ◽  
In Vitro Reconstitution ◽  
Presynaptic Plasticity ◽  
Hippocampal Cultures

Munc13-1 plays a central role in neurotransmitter release through its conserved C-terminal region, which includes a diacyglycerol (DAG)-binding C1 domain, a Ca2+/PIP2-binding C2B domain, a MUN domain and a C2C domain. Munc13-1 was proposed to bridge synaptic vesicles to the plasma membrane through distinct interactions of the C­1C2B region with the plasma membrane: i) one involving a polybasic face that is expected to yield a perpendicular orientation of Munc13-1 and hinder release; and ii) another involving the DAG-Ca2+-PIP2-binding face that is predicted to result in a slanted orientation and facilitate release. Here we have tested this model and investigated the role of the C­1C2B region in neurotransmitter release. We find that K603E or R769E point mutations in the polybasic face severely impair Ca2+-independent liposome bridging and fusion in in vitro reconstitution assays, and synaptic vesicle priming in primary murine hippocampal cultures. A K720E mutation in the polybasic face and a K706E mutation in the C2B domain Ca2+-binding loops have milder effects in reconstitution assays and do not affect vesicle priming, but enhance or impair Ca2+-evoked release, respectively. The phenotypes caused by combining these mutations are dominated by the K603E and R769E mutations. Our results show that the C1-C2B region of Munc13-1 plays a central role in vesicle priming and support the notion that two distinct faces of this region control neurotransmitter release and short-term presynaptic plasticity.

scholarly journals Acute disruption of the synaptic vesicle membrane protein synaptotagmin 1 using knockoff in mouse hippocampal neurons

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Jason D Vevea ◽  
Edwin R Chapman
Keyword(s):  
Membrane Protein ◽  
Synaptic Vesicle ◽  
Protein Function ◽  
Neurotransmitter Release ◽  
Cell Biology ◽  
Hippocampal Neurons ◽  
Cell Types ◽  
Spontaneous Release ◽  
Vesicle Membrane ◽  
Synaptotagmin 1

The success of comparative cell biology for determining protein function relies on quality disruption techniques. Long-lived proteins, in postmitotic cells, are particularly difficult to eliminate. Moreover, cellular processes are notoriously adaptive; for example, neuronal synapses exhibit a high degree of plasticity. Ideally, protein disruption techniques should be both rapid and complete. Here, we describe knockoff, a generalizable method for the druggable control of membrane protein stability. We developed knockoff for neuronal use but show it also works in other cell types. Applying knockoff to synaptotagmin 1 (SYT1) results in acute disruption of this protein, resulting in loss of synchronous neurotransmitter release with a concomitant increase in the spontaneous release rate, measured optically. Thus, SYT1 is not only the proximal Ca2+ sensor for fast neurotransmitter release but also serves to clamp spontaneous release. Additionally, knockoff can be applied to protein domains as we show for another synaptic vesicle protein, synaptophysin 1.

scholarly journals Membrane curvature sensing of the lipid-anchored K-Ras small GTPase

2019 ◽  
Vol 2 (4) ◽  
pp. e201900343 ◽  
Author(s):  
Hong Liang ◽  
Huanwen Mu ◽  
Frantz Jean-Francois ◽  
Bindu Lakshman ◽  
Suparna Sarkar-Banerjee ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Growth ◽  
Cell Shape ◽  
Membrane Curvature ◽  
Small Gtpase ◽  
Live Cells ◽  
Mitogenic Signaling ◽  
Curvature Sensing ◽  
Intracellular Organelle ◽  
Cell Property

Plasma membrane (PM) curvature defines cell shape and intracellular organelle morphologies and is a fundamental cell property. Growth/proliferation is more stimulated in flatter cells than the same cells in elongated shapes. PM-anchored K-Ras small GTPase regulates cell growth/proliferation and plays key roles in cancer. The lipid-anchored K-Ras form nanoclusters selectively enriched with specific phospholipids, such as phosphatidylserine (PS), for efficient effector recruitment and activation. K-Ras function may, thus, be sensitive to changing lipid distribution at membranes with different curvatures. Here, we used complementary methods to manipulate membrane curvature of intact/live cells, native PM blebs, and synthetic liposomes. We show that the spatiotemporal organization and signaling of an oncogenic mutant K-RasG12V favor flatter membranes with low curvature. Our findings are consistent with the more stimulated growth/proliferation in flatter cells. Depletion of endogenous PS abolishes K-RasG12V PM curvature sensing. In cells and synthetic bilayers, only mixed-chain PS species, but not other PS species tested, mediate K-RasG12V membrane curvature sensing. Thus, K-Ras nanoclusters act as relay stations to convert mechanical perturbations to mitogenic signaling.

scholarly journals An intramolecular t-SNARE complex functions in vivo without the syntaxin NH2-terminal regulatory domain

2006 ◽  
Vol 172 (2) ◽  
pp. 295-307 ◽  
Author(s):  
Jeffrey S. Van Komen ◽  
Xiaoyang Bai ◽  
Brenton L. Scott ◽  
James A. McNew
Keyword(s):  
Plasma Membrane ◽  
Complex Formation ◽  
Secretory Pathway ◽  
Regulatory Element ◽  
Snare Complex ◽  
Regulatory Domain ◽  
Vesicle Membrane ◽  
Snare Complex Formation

Membrane fusion in the secretory pathway is mediated by SNAREs (located on the vesicle membrane [v-SNARE] and the target membrane [t-SNARE]). In all cases examined, t-SNARE function is provided as a three-helix bundle complex containing three ∼70–amino acid SNARE motifs. One SNARE motif is provided by a syntaxin family member (the t-SNARE heavy chain), and the other two helices are contributed by additional t-SNARE light chains. The syntaxin family is the most conformationally dynamic group of SNAREs and appears to be the major focus of SNARE regulation. An NH2-terminal region of plasma membrane syntaxins has been assigned as a negative regulatory element in vitro. This region is absolutely required for syntaxin function in vivo. We now show that the required function of the NH2-terminal regulatory domain (NRD) of the yeast plasma membrane syntaxin, Sso1p, can be circumvented when t-SNARE complex formation is made intramolecular. Our results suggest that the NRD is required for efficient t-SNARE complex formation and does not recruit necessary scaffolding factors.

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