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 Assessing the Perturbing Effects of Drugs on Lipid Bilayers Using Gramicidin Channel-Based In Silico and In Vitro Assays

2020 ◽  
Vol 63 (20) ◽  
pp. 11809-11818
Author(s):  
Delin Sun ◽  
Thasin A. Peyear ◽  
W. F. Drew Bennett ◽  
Matthew Holcomb ◽  
Stewart He ◽  
...  
Keyword(s):  
Lipid Bilayers ◽  
In Silico ◽  
In Vitro Assays ◽  
Gramicidin Channel

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 A synthetic biology platform for the reconstitution and mechanistic dissection of LINC complex assembly

2018 ◽  
Author(s):  
Sagardip Majumder ◽  
Patrick T. Willey ◽  
Maxwell S. DeNies ◽  
Allen P. Liu ◽  
G.W. Gant Luxton
Keyword(s):  
Synthetic Biology ◽  
Lipid Bilayers ◽  
Transmembrane Domain ◽  
Experimental System ◽  
Free Expression ◽  
Supported Lipid Bilayers ◽  
Linc Complex ◽  
Complex Assembly ◽  
Complex Core

ABSTRACTThe linker of nucleoskeleton and cytoskeleton (LINC) is a conserved nuclear envelope-spanning molecular bridge that is responsible for the mechanical integration of the nucleus with the cytoskeleton. LINC complexes are formed by a transluminal interaction between the outer and inner nuclear membrane KASH and SUN proteins, respectively. Despite recent structural insights, our mechanistic understanding of LINC complex assembly remains limited by the lack of an experimental system for its in vitro reconstitution and manipulation. Here, we describe artificial nuclear membranes (ANMs) as a synthetic biology platform based on mammalian cell-free expression for the rapid reconstitution of SUN proteins in supported lipid bilayers. We demonstrate that SUN1 and SUN2 are oriented in ANMs with solvent-exposed C-terminal KASH-binding SUN domains. We also find that SUN2 possesses a single transmembrane domain, while SUN1 possesses three. Finally, SUN protein-containing ANMs bind synthetic KASH peptides, thereby reconstituting the LINC complex core. This work represents the first in vitro reconstitution of KASH-binding SUN proteins in supported lipid bilayers using cell-free expression, which will be invaluable for testing proposed models of LINC complex assembly and its regulation.

scholarly journals Transport efficiency of membrane-anchored kinesin-1 motors depends on motor density and diffusivity

2016 ◽  
Vol 113 (46) ◽  
pp. E7185-E7193 ◽  
Author(s):  
Rahul Grover ◽  
Janine Fischer ◽  
Friedrich W. Schwarz ◽  
Wilhelm J. Walter ◽  
Petra Schwille ◽  
...  
Keyword(s):  
Lipid Bilayer ◽  
Lipid Bilayers ◽  
Single Molecule ◽  
Gliding Motility ◽  
Supported Lipid Bilayers ◽  
Transport Efficiency ◽  
Collective Transport ◽  
Wide Range ◽  
Motor Density

In eukaryotic cells, membranous vesicles and organelles are transported by ensembles of motor proteins. These motors, such as kinesin-1, have been well characterized in vitro as single molecules or as ensembles rigidly attached to nonbiological substrates. However, the collective transport by membrane-anchored motors, that is, motors attached to a fluid lipid bilayer, is poorly understood. Here, we investigate the influence of motors’ anchorage to a lipid bilayer on the collective transport characteristics. We reconstituted “membrane-anchored” gliding motility assays using truncated kinesin-1 motors with a streptavidin-binding peptide tag that can attach to streptavidin-loaded, supported lipid bilayers. We found that the diffusing kinesin-1 motors propelled the microtubules in the presence of ATP. Notably, we found the gliding velocity of the microtubules to be strongly dependent on the number of motors and their diffusivity in the lipid bilayer. The microtubule gliding velocity increased with increasing motor density and membrane viscosity, reaching up to the stepping velocity of single motors. This finding is in contrast to conventional gliding motility assays where the density of surface-immobilized kinesin-1 motors does not influence the microtubule velocity over a wide range. We reason that the transport efficiency of membrane-anchored motors is reduced because of their slippage in the lipid bilayer, an effect that we directly observed using single-molecule fluorescence microscopy. Our results illustrate the importance of motor–cargo coupling, which potentially provides cells with an additional means of regulating the efficiency of cargo transport.

scholarly journals Formation of Supported Lipid Bilayers by Vesicle Fusion: Effect of Deposition Temperature

Langmuir ◽  
2014 ◽  
Vol 30 (25) ◽  
pp. 7259-7263 ◽  
Author(s):  
Tania Kjellerup Lind ◽  
Marité Cárdenas ◽  
Hanna Pauliina Wacklin
Keyword(s):  
Lipid Bilayers ◽  
Deposition Temperature ◽  
Vesicle Fusion ◽  
Supported Lipid Bilayers

AFM Studies of Solid-Supported Lipid Bilayers Formed at a Au(111) Electrode Surface Using Vesicle Fusion and a Combination of Langmuir−Blodgett and Langmuir−Schaefer Techniques

Langmuir ◽  
2008 ◽  
Vol 24 (18) ◽  
pp. 10313-10323 ◽  
Author(s):  
Ming Li ◽  
Maohui Chen ◽  
Erin Sheepwash ◽  
Christa L. Brosseau ◽  
Hongqiang Li ◽  
...  
Keyword(s):  
Lipid Bilayers ◽  
Electrode Surface ◽  
Vesicle Fusion ◽  
Supported Lipid Bilayers ◽  
Langmuir Blodgett

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.

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