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

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.

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

10.1101/064246 ◽

2016 ◽

Cited By ~ 1

Author(s):

Rahul Grover ◽

Janine Fischer ◽

Friedrich W. Schwarz ◽

Wilhelm J. Walter ◽

Petra Schwille ◽

...

Keyword(s):

Lipid Bilayer ◽

Lipid Bilayers ◽

Gliding Motility ◽

Supported Lipid Bilayers ◽

Transport Efficiency ◽

Collective Transport ◽

Wide Range ◽

Biological Substrates ◽

Motor Density

AbstractIn 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 non-biological substrates. However, the collective transport by membrane-anchored motors, i.e. 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 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 which we directly observed using singlemolecule fluorescence microscopy. Our results illustrate the importance of the motor-cargo coupling, which potentially provides cells with an additional means of regulating the efficiency of cargo transport.

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Nanoarchitectured air-stable supported lipid bilayer incorporating sucrose–bicelle complex system

Nano Convergence ◽

10.1186/s40580-021-00292-5 ◽

2022 ◽

Vol 9 (1) ◽

Author(s):

Hyunhyuk Tae ◽

Soohyun Park ◽

Gamaliel Junren Ma ◽

Nam-Joon Cho

Keyword(s):

Complex System ◽

Lipid Bilayer ◽

Lipid Bilayers ◽

Supported Lipid Bilayers ◽

Air Exposure ◽

Supported Lipid Bilayer ◽

Wide Range ◽

Aqueous Environments ◽

One Step ◽

Self Assembled Layer

AbstractCell-membrane-mimicking supported lipid bilayers (SLBs) provide an ultrathin, self-assembled layer that forms on solid supports and can exhibit antifouling, signaling, and transport properties among various possible functions. While recent material innovations have increased the number of practically useful SLB fabrication methods, typical SLB platforms only work in aqueous environments and are prone to fluidity loss and lipid-bilayer collapse upon air exposure, which limits industrial applicability. To address this issue, herein, we developed sucrose–bicelle complex system to fabricate air-stable SLBs that were laterally mobile upon rehydration. SLBs were fabricated from bicelles in the presence of up to 40 wt% sucrose, which was verified by quartz crystal microbalance-dissipation (QCM-D) and fluorescence recovery after photobleaching (FRAP) experiments. The sucrose fraction in the system was an important factor; while 40 wt% sucrose induced lipid aggregation and defects on SLBs after the dehydration–rehydration process, 20 wt% sucrose yielded SLBs that exhibited fully recovered lateral mobility after these processes. Taken together, these findings demonstrate that sucrose–bicelle complex system can facilitate one-step fabrication of air-stable SLBs that can be useful for a wide range of biointerfacial science applications.

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A Close-To-Native System to Study Membrane Protein Dynamics by Single-Molecule Optical Microscopy: an Application to Bacteriorhodopsin

Microscopy and Microanalysis ◽

10.1017/s1431927600026222 ◽

2001 ◽

Vol 7 (S2) ◽

pp. 30-31

Author(s):

Nicoletta Kahya ◽

Eve I. Pécheur ◽

Douwe A. Wiersma ◽

Dick Hoekstra

Keyword(s):

Optical Microscopy ◽

Lipid Bilayers ◽

Single Molecule ◽

Transmembrane Protein ◽

Passive Component ◽

Signal Transducers ◽

Wide Range ◽

Minimum Number

Biological membranes are not just a passive component of the cells, they actively support their functioning as the imbedded protein machineries carry out a wide range of crucial biochemical processes. Although studies in vivo are becoming more and more accessible to single-molecule optical microscopy, in vitro studies are still very much informative for the understanding of individual biological machineries. One of the major goals is to define the minimum number of components of a machinery that is necessary for a particular step, thereby allowing detailed studies of the mechanism of action. Reconstitution of a transmembrane protein system in artificial membranes (liposomes) is the main method for such a strategy, which thus may provide the option to investigate the functioning of transport proteins, ion channels, fusion machineries, and signal transducers in relation to their environment.We present a novel procedure in order to reconstitute transmembrane proteins in chemically well-defined and close-to-native lipid bilayers, providing an in vitro system for single-molecule optical microscopy. Furthermore, an application of this technique is shown in the case of a single-molecule study of protein-protein and protein-lipid interactions for the light-induced proton pump bacteriorhodopsin.In this study, Giant Unilamellar Vesicles (GUV), 10-100 μm sized, are used as lipid bilayer models for several reasons.

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Tracking Single Particles for Hours via Continuous DNA-mediated Fluorophore Exchange

10.1101/2020.05.17.100354 ◽

2020 ◽

Author(s):

Johannes Stein ◽

Florian Stehr ◽

Julian Bauer ◽

Christian Niederauer ◽

Ralf Jungmann ◽

...

Keyword(s):

Lipid Bilayers ◽

Single Molecule ◽

Single Particle Tracking ◽

Supported Lipid Bilayers ◽

Single Particles ◽

Major Bottleneck ◽

Labeling Approach ◽

Fluorescently Labeled Oligonucleotides ◽

Observation Times

AbstractFluorophores are commonly used to covalently label biomolecules for monitoring their motion in single particle tracking experiments. However, photobleaching is still a major bottleneck in these experiments, as the fluorophores’ finite photon budget typically limits observation times to merely a few seconds. Here, we overcome this inherent constraint via continuous fluorophore exchange based on DNA-PAINT, whereby fluorescently-labeled oligonucleotides bind to a 54 bp single-stranded DNA handle attached to the molecule of interest. When we assayed our approach in vitro by tracking single DNA origami, first surface-immobilized and subsequently diffusing on supported lipid bilayers, we were able to observe these origami for up to hours without losing their fluorescence signals. Our versatile and easily implemented labeling approach allows monitoring single-molecule motion and interactions over unprecedented observation periods, opening the doors to advanced quantitative studies.

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Lipid bilayer degradation induced by SARS-CoV-2 spike protein as revealed by neutron reflectometry

Scientific Reports ◽

10.1038/s41598-021-93996-x ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Alessandra Luchini ◽

Samantha Micciulla ◽

Giacomo Corucci ◽

Krishna Chaithanya Batchu ◽

Andreas Santamaria ◽

...

Keyword(s):

Lipid Bilayer ◽

Lipid Bilayers ◽

Membrane Lipids ◽

Lipid Extraction ◽

Specific Interaction ◽

Structural Features ◽

Supported Lipid Bilayers ◽

Fusion Event ◽

Angiotensin Converting Enzyme 2 ◽

Neutron Reflection

AbstractSARS-CoV-2 spike proteins are responsible for the membrane fusion event, which allows the virus to enter the host cell and cause infection. This process starts with the binding of the spike extramembrane domain to the angiotensin-converting enzyme 2 (ACE2), a membrane receptor highly abundant in the lungs. In this study, the extramembrane domain of SARS-CoV-2 Spike (sSpike) was injected on model membranes formed by supported lipid bilayers in presence and absence of the soluble part of receptor ACE2 (sACE2), and the structural features were studied at sub-nanometer level by neutron reflection. In all cases the presence of the protein produced a remarkable degradation of the lipid bilayer. Indeed, both for membranes from synthetic and natural lipids, a significant reduction of the surface coverage was observed. Quartz crystal microbalance measurements showed that lipid extraction starts immediately after sSpike protein injection. All measurements indicate that the presence of proteins induces the removal of membrane lipids, both in the presence and in the absence of ACE2, suggesting that sSpike molecules strongly associate with lipids, and strip them away from the bilayer, via a non-specific interaction. A cooperative effect of sACE2 and sSpike on lipid extraction was also observed.

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Correlated diffusion in lipid bilayers

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2113202118 ◽

2021 ◽

Vol 118 (48) ◽

pp. e2113202118

Author(s):

Rafael L. Schoch ◽

Frank L. H. Brown ◽

Gilad Haran

Keyword(s):

Lipid Bilayers ◽

Single Molecule ◽

Lipid Membranes ◽

Supported Lipid Bilayers ◽

Single Molecule Tracking ◽

Black Lipid Membranes ◽

Molecular Tracking ◽

Physical Materials ◽

Multiple Molecules ◽

Major Target

Lipid membranes are complex quasi–two-dimensional fluids, whose importance in biology and unique physical/materials properties have made them a major target for biophysical research. Recent single-molecule tracking experiments in membranes have caused some controversy, calling the venerable Saffman–Delbrück model into question and suggesting that, perhaps, current understanding of membrane hydrodynamics is imperfect. However, single-molecule tracking is not well suited to resolving the details of hydrodynamic flows; observations involving correlations between multiple molecules are superior for this purpose. Here dual-color molecular tracking with submillisecond time resolution and submicron spatial resolution is employed to reveal correlations in the Brownian motion of pairs of fluorescently labeled lipids in membranes. These correlations extend hundreds of nanometers in freely floating bilayers (black lipid membranes) but are severely suppressed in supported lipid bilayers. The measurements are consistent with hydrodynamic predictions based on an extended Saffman–Delbrück theory that explicitly accounts for the two-leaflet bilayer structure of lipid membranes.

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