scholarly journals A new in vitro hemagglutinin inhibitor screening system based on a single-vesicle fusion assay

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Hanki Lee ◽  
Wook Jin ◽  
Byeong-Chul Jeong ◽  
Joo-Won Suh
Keyword(s):  
Vesicle Fusion ◽  
Screening System ◽  
Inhibitor Screening ◽  
Single Vesicle

scholarly journals A single vesicle-vesicle fusion assay for in vitro studies of SNAREs and accessory proteins

2012 ◽  
Vol 7 (5) ◽  
pp. 921-934 ◽  
Author(s):  
Jiajie Diao ◽  
Yuji Ishitsuka ◽  
Hanki Lee ◽  
Chirlmin Joo ◽  
Zengliu Su ◽  
...  
Keyword(s):  
Vesicle Fusion ◽  
In Vitro Studies ◽  
Accessory Proteins ◽  
Single Vesicle

scholarly journals Molecular Determinants of Complexin Clamping in Reconstituted Single-Vesicle Fusion

2021 ◽  
Author(s):  
Manindra Bera ◽  
Sathish Ramakrishnan ◽  
Jeff Coleman ◽  
Shyam S Krishnakumar ◽  
James E Rothman
Keyword(s):  
Specific Interaction ◽  
Vesicle Fusion ◽  
Assembly Process ◽  
Inhibitory Function ◽  
Physiological Relevance ◽  
Vesicular Release ◽  
Molecular Determinants ◽  
Synaptotagmin 1 ◽  
Single Vesicle

Previously we reported that Synaptotagmin-1 and Complexin synergistically clamp the SNARE assembly process to generate and maintain a pool of docked vesicles that fuse rapidly and synchronously upon Ca2+ influx (Ramakrishnan et al. 2020). Here using the same in vitro single-vesicle fusion assay, we establish the molecular details of the Complexin clamp and its physiological relevance. We find that a delay in fusion kinetics, likely imparted by Synaptotagmin-1, is needed for Complexin to block fusion. Systematic truncation/mutational analyses reveal that continuous alpha-helical accessory-central domains of Complexin are essential for its inhibitory function and specific interaction of the accessory helix with the SNAREpins, analogous to the trans clamping model, enhances this functionality. The c-terminal domain promotes clamping by locally elevating Complexin concentration through interactions with the membrane. Further, we find that Complexin likely contributes to rapid Ca2+-synchronized vesicular release by preventing un-initiated fusion rather than by directly facilitating vesicle fusion.

scholarly journals In vitro single vesicle fusion assays based on pore-spanning membranes: merits and drawbacks

2020 ◽  
Author(s):  
Peter Mühlenbrock ◽  
Merve Sari ◽  
Claudia Steinem
Keyword(s):  
Lipid Bilayers ◽  
Vesicle Fusion ◽  
In Vitro Assays ◽  
Supported Lipid Bilayers ◽  
Protein Receptors ◽  
Membrane Components ◽  
Interconnected Structure ◽  
Single Vesicle ◽  
Fusion Efficiency

AbstractNeuronal fusion mediated by soluble N-ethylmaleimide-sensitive-factor attachment protein receptors (SNAREs) is a fundamental cellular process by which two initially distinct membranes merge resulting in one interconnected structure to release neurotransmitters into the presynaptic cleft. To get access to the different stages of the fusion process, several in vitro assays have been developed. In this review, we provide a short overview of the current in vitro single vesicle fusion assays. Among those assays, we developed a single vesicle assay based on pore-spanning membranes (PSMs) on micrometre-sized pores in silicon, which might overcome some of the drawbacks associated with the other membrane architectures used for investigating fusion processes. Prepared by spreading of giant unilamellar vesicles with reconstituted t-SNAREs, PSMs provide an alternative tool to supported lipid bilayers to measure single vesicle fusion events by means of fluorescence microscopy. Here, we discuss the diffusive behaviour of the reconstituted membrane components as well as that of the fusing synthetic vesicles with reconstituted synaptobrevin 2 (v-SNARE). We compare our results with those obtained if the synthetic vesicles are replaced by natural chromaffin granules under otherwise identical conditions. The fusion efficiency as well as the different fusion states observable in this assay by means of both lipid mixing and content release are illuminated.

scholarly journals The Caenorhabditis elegans unc-64 Locus Encodes a Syntaxin That Interacts Genetically with Synaptobrevin

1998 ◽  
Vol 9 (6) ◽  
pp. 1235-1252 ◽  
Author(s):  
Owais Saifee ◽  
Liping Wei ◽  
Michael L. Nonet
Keyword(s):  
Caenorhabditis Elegans ◽  
Synaptic Vesicle ◽  
Acetylcholinesterase Inhibitor ◽  
Coiled Coil ◽  
Vesicle Fusion ◽  
Loss Of Function ◽  
Hydrophobic Membrane ◽  
Coiled Coil Domain ◽  
Synaptic Vesicle Fusion

We describe the molecular cloning and characterization of theunc-64 locus of Caenorhabditis elegans. unc-64 expresses three transcripts, each encoding a molecule with 63–64% identity to human syntaxin 1A, a membrane- anchored protein involved in synaptic vesicle fusion. Interestingly, the alternative forms of syntaxin differ only in their C-terminal hydrophobic membrane anchors. The forms are differentially expressed in neuronal and secretory tissues; genetic evidence suggests that these forms are not functionally equivalent. A complete loss-of-function mutation in unc-64 results in a worm that completes embryogenesis, but arrests development shortly thereafter as a paralyzed L1 larva, presumably as a consequence of neuronal dysfunction. The severity of the neuronal phenotypes of C. elegans syntaxin mutants appears comparable to those ofDrosophila syntaxin mutants. However, nematode syntaxin appears not to be required for embryonic development, for secretion of cuticle from the hypodermis, or for the function of muscle, in contrast to Drosophila syntaxin, which appears to be required in all cells. Less severe viable unc-64 mutants exhibit a variety of behavioral defects and show strong resistance to the acetylcholinesterase inhibitor aldicarb. Extracellular physiological recordings from pharyngeal muscle of hypomorphic mutants show alterations in the kinetics of transmitter release. The lesions in the hypomorphic alleles map to the hydrophobic face of the H3 coiled-coil domain of syntaxin, a domain that in vitro mediates physical interactions with similar coiled-coil domains in SNAP-25 and synaptobrevin. Furthermore, the unc-64 syntaxin mutants exhibit allele-specific genetic interactions with mutants carrying lesions in the coiled-coil domain of synaptobrevin, providing in vivo evidence for the significance of these domains in regulating synaptic vesicle fusion.

scholarly journals A Dual-Sensor-Based Screening System for In Vitro Selection of TDP1 Inhibitors

Sensors ◽  
2021 ◽  
Vol 21 (14) ◽  
pp. 4832
Author(s):  
Ann-Katrine Jakobsen ◽  
Josephine Geertsen Keller ◽  
María Gonzalez ◽  
Endika Martin-Encinas ◽  
Francisco Palacios ◽  
...  
Keyword(s):  
Dna Damage ◽  
In Vitro Selection ◽  
Main Function ◽  
Screening System ◽  
Drug Candidates ◽  
Topoisomerase 1 ◽  
High Throughput Drug Screening ◽  
Dual Sensor ◽  
Selection Of

DNA sensors can be used as robust tools for high-throughput drug screening of small molecules with the potential to inhibit specific enzymes. As enzymes work in complex biological pathways, it is important to screen for both desired and undesired inhibitory effects. We here report a screening system utilizing specific sensors for tyrosyl-DNA phosphodiesterase 1 (TDP1) and topoisomerase 1 (TOP1) activity to screen in vitro for drugs inhibiting TDP1 without affecting TOP1. As the main function of TDP1 is repair of TOP1 cleavage-induced DNA damage, inhibition of TOP1 cleavage could thus reduce the biological effect of the TDP1 drugs. We identified three new drug candidates of the 1,5-naphthyridine and 1,2,3,4-tetrahydroquinolinylphosphine sulfide families. All three TDP1 inhibitors had no effect on TOP1 activity and acted synergistically with the TOP1 poison SN-38 to increase the amount of TOP1 cleavage-induced DNA damage. Further, they promoted cell death even with low dose SN-38, thereby establishing two new classes of TDP1 inhibitors with clinical potential. Thus, we here report a dual-sensor screening approach for in vitro selection of TDP1 drugs and three new TDP1 drug candidates that act synergistically with TOP1 poisons.

scholarly journals Simultaneous lipid and content mixing assays for in vitro reconstitution studies of synaptic vesicle fusion

2017 ◽  
Vol 12 (9) ◽  
pp. 2014-2028 ◽  
Author(s):  
Xiaoxia Liu ◽  
Alpay Burak Seven ◽  
Junjie Xu ◽  
Victoria Esser ◽  
Lijing Su ◽  
...  
Keyword(s):  
Synaptic Vesicle ◽  
Vesicle Fusion ◽  
In Vitro Reconstitution ◽  
Synaptic Vesicle Fusion

scholarly journals Studying calcium-triggered vesicle fusion in a single vesicle-vesicle content and lipid-mixing system

2012 ◽  
Vol 8 (1) ◽  
pp. 1-16 ◽  
Author(s):  
Minjoung Kyoung ◽  
Yunxiang Zhang ◽  
Jiajie Diao ◽  
Steven Chu ◽  
Axel T Brunger
Keyword(s):  
Vesicle Fusion ◽  
Vesicle Content ◽  
Single Vesicle
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