scholarly journals In vivo single-molecule imaging of syntaxin1A reveals polyphosphoinositide- and activity-dependent trapping in presynaptic nanoclusters

2016 ◽  
Vol 7 (1) ◽  
Author(s):  
Adekunle T. Bademosi ◽  
Elsa Lauwers ◽  
Pranesh Padmanabhan ◽  
Lorenzo Odierna ◽  
Ye Jin Chai ◽  
...  
Keyword(s):  
Single Molecule ◽  
Neurotransmitter Release ◽  
Motor Nerve ◽  
Living Organism ◽  
Neurosecretory Cells ◽  
Single Particle Tracking ◽  
Snare Complex ◽  
Motor Nerve Terminal ◽  
Activity Dependent

Abstract Syntaxin1A is organized in nanoclusters that are critical for the docking and priming of secretory vesicles from neurosecretory cells. Whether and how these nanoclusters are affected by neurotransmitter release in nerve terminals from a living organism is unknown. Here we imaged photoconvertible syntaxin1A-mEos2 in the motor nerve terminal of Drosophila larvae by single-particle tracking photoactivation localization microscopy. Opto- and thermo-genetic neuronal stimulation increased syntaxin1A-mEos2 mobility, and reduced the size and molecular density of nanoclusters, suggesting an activity-dependent release of syntaxin1A from the confinement of nanoclusters. Syntaxin1A mobility was increased by mutating its polyphosphoinositide-binding site or preventing SNARE complex assembly via co-expression of tetanus toxin light chain. In contrast, syntaxin1A mobility was reduced by preventing SNARE complex disassembly. Our data demonstrate that polyphosphoinositide favours syntaxin1A trapping, and show that SNARE complex disassembly leads to syntaxin1A dissociation from nanoclusters. Lateral diffusion and trapping of syntaxin1A in nanoclusters therefore dynamically regulate neurotransmitter release.

scholarly journals In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

10.3791/56952 ◽  
2018 ◽  
Author(s):  
Adekunle T. Bademosi ◽  
Elsa Lauwers ◽  
Rumelo Amor ◽  
Patrik Verstreken ◽  
Bruno van Swinderen ◽  
...  
Keyword(s):  
Single Molecule ◽  
Nerve Terminal ◽  
Motor Nerve ◽  
Motor Nerve Terminal ◽  
Single Molecule Tracking

scholarly journals Syncrip/hnRNP Q is required for activity-induced Msp300/Nesprin-1 expression and new synapse formation

2020 ◽  
Vol 219 (3) ◽  
Author(s):  
Joshua Titlow ◽  
Francesca Robertson ◽  
Aino Järvelin ◽  
David Ish-Horowicz ◽  
Carlas Smith ◽  
...  
Keyword(s):  
Single Molecule ◽  
Posttranscriptional Regulation ◽  
Rna Binding ◽  
Synapse Formation ◽  
Long Distance ◽  
Key Factor ◽  
Larval Neuromuscular Junction ◽  
Rnp Granules ◽  
Activity Dependent

Memory and learning involve activity-driven expression of proteins and cytoskeletal reorganization at new synapses, requiring posttranscriptional regulation of localized mRNA a long distance from corresponding nuclei. A key factor expressed early in synapse formation is Msp300/Nesprin-1, which organizes actin filaments around the new synapse. How Msp300 expression is regulated during synaptic plasticity is poorly understood. Here, we show that activity-dependent accumulation of Msp300 in the postsynaptic compartment of the Drosophila larval neuromuscular junction is regulated by the conserved RNA binding protein Syncrip/hnRNP Q. Syncrip (Syp) binds to msp300 transcripts and is essential for plasticity. Single-molecule imaging shows that msp300 is associated with Syp in vivo and forms ribosome-rich granules that contain the translation factor eIF4E. Elevated neural activity alters the dynamics of Syp and the number of msp300:Syp:eIF4E RNP granules at the synapse, suggesting that these particles facilitate translation. These results introduce Syp as an important early acting activity-dependent regulator of a plasticity gene that is strongly associated with human ataxias.

scholarly journals Polybasic patches in both C2 domains of Synaptotagmin-1 are required for evoked neurotransmitter release

2021 ◽  
Author(s):  
Zhenyong Wu ◽  
Lu Ma ◽  
Nicholas A Courtney ◽  
Jie Zhu ◽  
Yongli Zhang ◽  
...  
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
Calcium Binding ◽  
Neurotransmitter Release ◽  
Transmembrane Domain ◽  
Snare Complex ◽  
Critical Feature ◽  
C2 Domains ◽  
Electrophysiological Recordings ◽  
Synaptotagmin 1

Synaptotagmin-1 (Syt1) is a vesicular calcium sensor required for synchronous neurotransmitter release. It is composed of a single-pass transmembrane domain linked to two tandem C2 domains (C2A and C2B) that bind calcium, acidic lipids, and SNARE proteins that drive fusion of the synaptic vesicle with the plasma membrane. Despite its essential role, how Syt1 couples calcium entry to synchronous release is not well understood. Calcium binding to C2B, but not to C2A, is critical for synchronous release and C2B additionally binds the SNARE complex. The C2A domain is also required for Syt1 function, but it is not clear why. Here we asked what critical feature of C2A may be responsible for its functional role, and compared this to the analogous feature in C2B. We focused on highly conserved poly-lysine patches located on the sides of C2A (K189-192) and C2B (K324-327). We tested effects of charge-neutralization mutations in either region (Syt1K189-192A and Syt1K326-327A) side-by-side to determine their relative contributions to Syt1 function in cultured cortical mouse neurons and in single-molecule experiments. Combining electrophysiological recordings and optical tweezers measurements to probe dynamic single C2 domain-membrane interactions, we show that both C2A and C2B polybasic patches contribute to membrane binding, and both are required for evoked release. The readily releasable vesicle pool or spontaneous release were not affected, so both patches are specifically required for synchronization of release. We suggest these patches contribute to cooperative binding to membranes, increasing the overall affinity of Syt1 for negatively charged membranes and facilitating evoked release.

scholarly journals Complexin induces a conformational change at the membrane-proximal C-terminal end of the SNARE complex

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Ucheor B Choi ◽  
Minglei Zhao ◽  
Yunxiang Zhang ◽  
Ying Lai ◽  
Axel T Brunger
Keyword(s):  
Conformational Change ◽  
Single Molecule ◽  
Neurotransmitter Release ◽  
Plasma Membranes ◽  
Molecular Mechanisms ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Snare Complex ◽  
Spontaneous Release ◽  
Trans Conformation

Complexin regulates spontaneous and activates Ca2+-triggered neurotransmitter release, yet the molecular mechanisms are still unclear. Here we performed single molecule fluorescence resonance energy transfer experiments and uncovered two conformations of complexin-1 bound to the ternary SNARE complex. In the cis conformation, complexin-1 induces a conformational change at the membrane-proximal C-terminal end of the ternary SNARE complex that specifically depends on the N-terminal, accessory, and central domains of complexin-1. The complexin-1 induced conformation of the ternary SNARE complex may be related to a conformation that is juxtaposing the synaptic vesicle and plasma membranes. In the trans conformation, complexin-1 can simultaneously interact with a ternary SNARE complex via the central domain and a binary SNARE complex consisting of syntaxin-1A and SNAP-25A via the accessory domain. The cis conformation may be involved in activation of synchronous neurotransmitter release, whereas both conformations may be involved in regulating spontaneous release.

scholarly journals The Munc18-1 domain 3a hinge-loop controls syntaxin-1A nanodomain assembly and engagement with the SNARE complex during secretory vesicle priming

2016 ◽  
Vol 214 (7) ◽  
pp. 847-858 ◽  
Author(s):  
Ravikiran Kasula ◽  
Ye Jin Chai ◽  
Adekunle T. Bademosi ◽  
Callista B. Harper ◽  
Rachel S. Gormal ◽  
...  
Keyword(s):  
Single Molecule ◽  
Botulinum Neurotoxin ◽  
Secretory Vesicle ◽  
Neurosecretory Cells ◽  
Snare Complex ◽  
Secretory Vesicles ◽  
Deficient Mutant ◽  
Syntaxin 1A ◽  
Single Molecule Analysis ◽  
Snare Complex Formation

Munc18-1 and syntaxin-1A control SNARE-dependent neuroexocytosis and are organized in nanodomains on the plasma membrane of neurons and neurosecretory cells. Deciphering the intra- and intermolecular steps via which they prepare secretory vesicles (SVs) for fusion is key to understanding neuronal and hormonal communication. Here, we demonstrate that expression of a priming-deficient mutant lacking 17 residues of the domain 3a hinge-loop (Munc18-1Δ317-333) in PC12 cells engineered to knockdown Munc18-1/2 markedly prolonged SV docking. Single-molecule analysis revealed nonhomogeneous diffusion of Munc18-1 and syntaxin-1A in and out of partially overlapping nanodomains. Whereas Munc18-1WT mobility increased in response to stimulation, syntaxin-1A became less mobile. These Munc18-1 and syntaxin-1A diffusional switches were blocked by the expression of Munc18-1Δ317-333, suggesting that a conformational change in the Munc18-1 hinge-loop controls syntaxin-1A and subsequent SNARE complex assembly. Accordingly, syntaxin-1A confinement was prevented by expression of botulinum neurotoxin type E. The Munc18-1 domain 3a hinge-loop therefore controls syntaxin-1A engagement into SNARE complex formation during priming.

scholarly journals Clinical Safety and Tolerability of A2NTX, a Novel Low Molecular Weight Neurotoxin Derived From Botulinum Toxin Subtype A2, in Comparison with Subtype A1 Toxins

2021 ◽  
Author(s):  
Ryuji Kaji ◽  
Toshiaki Takeuchi ◽  
Takefumi Okuno ◽  
Ai Miyashiro ◽  
Tomoko Kohda ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Open Study ◽  
Motor Nerve ◽  
Tolerability Profile ◽  
Low Molecular Weight ◽  
Motor Nerve Terminal ◽  
Clinical Safety ◽  
Botulinum Type

All the available botulinum type A neurotoxins for clinical uses are of A1 subtype. We developed a subtype A2 low molecular weight (150kD) neurotoxin (A2NTX), with less spread and faster entry into the motor nerve terminal than A1 in vitro and in vivo. Preliminary clinical studies showed its efficacy superior to A1 toxins. We conducted an open study exploring its safety and tolerability profile in comparison with A1LL (onabotulinumtoxinA) and low molecular weight (150kD) A1 neurotoxin (A1NTX). Those who had been using A1LL (n=90; 50-360 mouse LD50 units) or A1NTX (n=30; 50-580 units) were switched to A2NTX (n=120; 25-600 units) from 2010 till 2018 (number of sessions ~ 27, cumulative doses ~11,640 units per patient). Adverse events for A2NTX included weakness (n=1, ascribed to alcoholic polyneuropathy), dysphagia (1), local weakness (4), spread to other muscles (1), whereas those for A1LL or A1NTX comprised weakness (n=2, A1NTX), dysphagia (8), ptosis (6), local weakness (7) and spread to other muscles (15). After injections, 89 out of 120 patients preferred A2NTX to A1 for the successive sessions. The present study demonstrated that A2NTX had the clinical safety up to the dose of 500 units, and was well tolerated compared to A1 toxins.

scholarly journals Syncrip/hnRNPQ is required for activity-induced Msp300/Nesprin-1 expression and new synapse formation

2019 ◽  
Author(s):  
Josh Titlow ◽  
Francesca Robertson ◽  
Aino Järvelin ◽  
David Ish-Horowicz ◽  
Carlas Smith ◽  
...  
Keyword(s):  
Synaptic Plasticity ◽  
Single Molecule ◽  
Rna Binding ◽  
Synapse Formation ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Long Distance ◽  
Key Factor ◽  
Activity Dependent

AbstractMemory and learning involve activity-driven expression of proteins and cytoskeletal reorganisation at new synapses, often requiring post-transcriptional regulation a long distance from corresponding nuclei. A key factor expressed early in synapse formation is Msp300/Nesprin-1, which organises actin filaments around the new synapse. How Msp300 expression is regulated during synaptic plasticity is not yet known. Here, we show that the local translation of msp300 is promoted during activity-dependent plasticity by the conserved RNA binding protein Syncrip/hnRNP Q, which binds to msp300 transcripts and is essential for plasticity. Single molecule imaging shows that Syncrip is associated in vivo with msp300 mRNA in ribosome-rich particles. Elevated neural activity alters the dynamics of Syncrip RNP granules at the synapse, suggesting a change in particle composition or binding that facilitates translation. These results introduce Syncrip as an important early-acting activity-dependent translational regulator of a plasticity gene that is strongly associated with human ataxias.Syncrip regulates synaptic plasticity via msp300Titlow et al. find that Syncrip (hnRNPQ RNA binding protein) acts directly on msp300 to modulate activity-dependent synaptic plasticity. In vivo biophysical experiments reveal activity-dependent changes in RNP complex sizes compatible with an increase in translation at the synapse.

scholarly journals Presynaptic Proteins as Markers of the Neurotoxic Activity of BmjeTX-I and BmjeTX-II Toxins fromBothrops marajoensis(Marajó Lancehead) Snake Venom

2016 ◽  
Vol 2016 ◽  
pp. 1-10
Author(s):  
Antonio Lisboa ◽  
Rodolfo Melaré ◽  
Junia R. B. Franco ◽  
Carolina V. Bis ◽  
Marta Gracia ◽  
...  
Keyword(s):  
Nerve Terminal ◽  
Neurotransmitter Release ◽  
Acetylcholine Receptors ◽  
Motor Nerve ◽  
Synaptic Proteins ◽  
Muscle Fibres ◽  
Motor Nerve Terminal ◽  
Transmission Electron ◽  
Skeletal Muscle Fibres

Neuromuscular preparations exposed toB. marajoensisvenom show increases in the frequency of miniature end-plate potentials and twitch tension facilitation followed by presynaptic neuromuscular paralysis, without evidences of muscle damage. Considering that presynaptic toxins interfere into the machinery involved in neurotransmitter release (synaptophysin, synaptobrevin, and SNAP25 proteins), the main objective of this communication is to analyze, by immunofluorescence and western blotting, the expression of the synaptic proteins, synaptophysin, synaptobrevin, and SNAP25 and by myography, light, and transmission electron microscopy the pathology of motor nerve terminals and skeletal muscle fibres of chick biventer cervicis preparations (CBC) exposedin vitroto BmjeTX-I and BmjeTX-II toxins fromB. marajoensisvenom. CBC incubated with toxins showed irreversible twitch tension blockade and unaffected KCl- and ACh-evoked contractures, and the positive colabelling of acetylcholine receptors confirmed that their action was primarily at the motor nerve terminal. Hypercontraction and loose myofilaments and synaptic vesicle depletion and motor nerve damage indicated that the toxins displayed both myotoxic and neurotoxic effect. The blockade resulted from interference on synaptophysin, synaptobrevin, and SNAP25 proteins leading to the conclusion that BmjeTX-I and BmjeTX-II affected neurotransmitter release machinery by preventing the docking of synaptic vesicles to the axolemma of the nerve terminal.

scholarly journals Nerve-independent formation of a topologically complex postsynaptic apparatus

2004 ◽  
Vol 164 (7) ◽  
pp. 1077-1087 ◽  
Author(s):  
Terrance T. Kummer ◽  
Thomas Misgeld ◽  
Jeff W. Lichtman ◽  
Joshua R. Sanes
Keyword(s):  
Normal Development ◽  
Motor Nerve ◽  
Time Lapse ◽  
Motor Nerve Terminal ◽  
Topological Complexity ◽  
A Cell ◽  
Muscle Specific Kinase ◽  
Branched Structures ◽  
Time Lapse Imaging

As the mammalian neuromuscular junction matures, its acetylcholine receptor (AChR)–rich postsynaptic apparatus is transformed from an oval plaque into a pretzel-shaped array of branches that precisely mirrors the branching pattern of the motor nerve terminal. Although the nerve has been believed to direct postsynaptic maturation, we report here that myotubes cultured aneurally on matrix-coated substrates form elaborately branched AChR-rich domains remarkably similar to those seen in vivo. These domains share several characteristics with the mature postsynaptic apparatus, including colocalization of multiple postsynaptic markers, clustering of subjacent myonuclei, and dependence on the muscle-specific kinase and rapsyn for their formation. Time-lapse imaging showed that branched structures arise from plaques by formation and fusion of AChR-poor perforations through a series of steps mirroring that seen in vivo. Multiple fluorophore imaging showed that growth occurs by circumferential, asymmetric addition of AChRs. Analysis in vivo revealed similar patterns of AChR addition during normal development. These results reveal the sequence of steps by which a topologically complex domain forms on a cell and suggest an unexpected nerve-independent role for the postsynaptic cell in generating this topological complexity.

scholarly journals Establishing Live-Cell Single-Molecule Localization Microscopy Imaging and Single-Particle Tracking in the Archaeon Haloferax volcanii

2020 ◽  
Vol 11 ◽  
Author(s):  
Bartosz Turkowyd ◽  
Sandra Schreiber ◽  
Julia Wörtz ◽  
Ella Shtifman Segal ◽  
Moshe Mevarech ◽  
...  
Keyword(s):  
Single Molecule ◽  
Fluorescent Proteins ◽  
Single Particle Tracking ◽  
Haloferax Volcanii ◽  
Microscopy Imaging ◽  
Localization Microscopy ◽  
Cellular Dna ◽  
Eukaryotic Organisms ◽  
Single Molecule Localization Microscopy

In recent years, fluorescence microscopy techniques for the localization and tracking of single molecules in living cells have become well-established and are indispensable tools for the investigation of cellular biology and in vivo biochemistry of many bacterial and eukaryotic organisms. Nevertheless, these techniques are still not established for imaging archaea. Their establishment as a standard tool for the study of archaea will be a decisive milestone for the exploration of this branch of life and its unique biology. Here, we have developed a reliable protocol for the study of the archaeon Haloferax volcanii. We have generated an autofluorescence-free H. volcanii strain, evaluated several fluorescent proteins for their suitability to serve as single-molecule fluorescence markers and codon-optimized them to work under optimal H. volcanii cultivation conditions. We found that two of them, Dendra2Hfx and PAmCherry1Hfx, provide state-of-the-art single-molecule imaging. Our strategy is quantitative and allows dual-color imaging of two targets in the same field of view (FOV) as well as DNA co-staining. We present the first single-molecule localization microscopy (SMLM) images of the subcellular organization and dynamics of two crucial intracellular proteins in living H. volcanii cells, FtsZ1, which shows complex structures in the cell division ring, and RNA polymerase, which localizes around the periphery of the cellular DNA.This work should provide incentive to develop SMLM strategies for other archaeal organisms in the near future.

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