Mass photometry enables label-free tracking and mass measurement of single proteins on lipid bilayers

AbstractThe quantification of membrane-associated biomolecular interactions is crucial to our understanding of various cellular processes. State-of-the-art single-molecule approaches rely largely on the addition of fluorescent labels, which complicates the quantification of the involved stoichiometries and dynamics because of low temporal resolution and the inherent limitations associated with labeling efficiency, photoblinking and photobleaching. Here, we demonstrate dynamic mass photometry, a method for label-free imaging, tracking and mass measurement of individual membrane-associated proteins diffusing on supported lipid bilayers. Application of this method to the membrane remodeling GTPase, dynamin-1, reveals heterogeneous mixtures of dimer-based oligomers, oligomer-dependent mobilities, membrane affinities and (dis)association of individual complexes. These capabilities, together with assay-based advances for studying integral membrane proteins, will enable the elucidation of biomolecular mechanisms in and on lipid bilayers.

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Label-free tracking and mass measurement of single proteins on lipid bilayers

10.1101/2021.04.08.438951 ◽

2021 ◽

Author(s):

Eric Dylan Benjamin Foley ◽

Manish Kushwah ◽

Gavin Young ◽

Philipp Kukura

Keyword(s):

Lipid Bilayers ◽

Single Molecule ◽

Mass Measurement ◽

Label Free ◽

Single Molecule Level ◽

Dynamic Mass ◽

Membrane Affinity ◽

Fundamental Building Block ◽

Quantitative Studies ◽

Associated Proteins

We introduce dynamic mass photometry, a method for label-free imaging, tracking and mass measurement of membrane-associated proteins. Our method enables quantitative studies of their mobility, membrane affinity and interactions at the single molecule level. Application to the membrane remodelling GTPase dynamin1 reveals heterogeneous mixtures of oligomers suggesting that the fundamental building block for oligomerisation is a dimer, challenging current tetramer-centric models. Dynamic mass photometry has the ability to transform our approach to studying biomolecular mechanisms in and on lipid bilayers.

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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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Phase separation and clustering of an ABC transporter in Mycobacterium tuberculosis

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1820683116 ◽

2019 ◽

Vol 116 (33) ◽

pp. 16326-16331 ◽

Cited By ~ 21

Author(s):

Florian Heinkel ◽

Libin Abraham ◽

Mary Ko ◽

Joseph Chao ◽

Horacio Bach ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Phase Separation ◽

Lipid Bilayers ◽

Abc Transporter ◽

Single Molecule ◽

Intracellular Signaling ◽

Intrinsically Disordered ◽

Regulatory Module ◽

Condensate Formation ◽

Associated Proteins

Phase separation drives numerous cellular processes, ranging from the formation of membrane-less organelles to the cooperative assembly of signaling proteins. Features such as multivalency and intrinsic disorder that enable condensate formation are found not only in cytosolic and nuclear proteins, but also in membrane-associated proteins. The ABC transporter Rv1747, which is important for Mycobacterium tuberculosis (Mtb) growth in infected hosts, has a cytoplasmic regulatory module consisting of 2 phosphothreonine-binding Forkhead-associated domains joined by an intrinsically disordered linker with multiple phospho-acceptor threonines. Here we demonstrate that the regulatory modules of Rv1747 and its homolog in Mycobacterium smegmatis form liquid-like condensates as a function of concentration and phosphorylation. The serine/threonine kinases and sole phosphatase of Mtb tune phosphorylation-enhanced phase separation and differentially colocalize with the resulting condensates. The Rv1747 regulatory module also phase-separates on supported lipid bilayers and forms dynamic foci when expressed heterologously in live yeast and M. smegmatis cells. Consistent with these observations, single-molecule localization microscopy reveals that the endogenous Mtb transporter forms higher-order clusters within the Mycobacterium membrane. Collectively, these data suggest a key role for phase separation in the function of these mycobacterial ABC transporters and their regulation via intracellular signaling.

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Single-Molecule Detection with Lightguiding Nanowires: Determination of Protein Concentration and Diffusivity in Supported Lipid Bilayers

Nano Letters ◽

10.1021/acs.nanolett.9b02226 ◽

2019 ◽

Vol 19 (9) ◽

pp. 6182-6191 ◽

Cited By ~ 2

Author(s):

Damiano Verardo ◽

Björn Agnarsson ◽

Vladimir P. Zhdanov ◽

Fredrik Höök ◽

Heiner Linke

Keyword(s):

Lipid Bilayers ◽

Single Molecule ◽

Protein Concentration ◽

Single Molecule Detection ◽

Supported Lipid Bilayers

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TrackArt: the user friendly interface for single molecule tracking data analysis and simulation applied to complex diffusion in mica supported lipid bilayers

BMC Research Notes ◽

10.1186/1756-0500-7-274 ◽

2014 ◽

Vol 7 (1) ◽

Cited By ~ 13

Author(s):

Artur Matysik ◽

Rachel S Kraut

Keyword(s):

Data Analysis ◽

Lipid Bilayers ◽

Single Molecule ◽

Supported Lipid Bilayers ◽

Tracking Data ◽

Single Molecule Tracking ◽

Complex Diffusion ◽

User Friendly

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Single-Molecule Fluorescence Imaging of Peptide Binding to Supported Lipid Bilayers

Analytical Chemistry ◽

10.1021/ac9007682 ◽

2009 ◽

Vol 81 (13) ◽

pp. 5130-5138 ◽

Cited By ~ 31

Author(s):

Christopher B. Fox ◽

Joshua R. Wayment ◽

Grant A. Myers ◽

Scott K. Endicott ◽

Joel M. Harris

Keyword(s):

Fluorescence Imaging ◽

Lipid Bilayers ◽

Single Molecule ◽

Peptide Binding ◽

Supported Lipid Bilayers ◽

Single Molecule Fluorescence

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Electrically Controlling and Optically Observing the Membrane Potential of Supported Lipid Bilayers

10.1101/2021.12.10.472087 ◽

2021 ◽

Author(s):

Shimon Yudovich ◽

Adan Marzouqe ◽

Joseph Kantorovitsch ◽

Eti Teblum ◽

Tao Chen ◽

...

Keyword(s):

Lipid Bilayers ◽

Surface Science ◽

Electrochemical Impedance ◽

Optical Probe ◽

Electrical Circuit ◽

Supported Lipid Bilayers ◽

Membrane Biology ◽

Supported Membrane ◽

Voltage Dependent ◽

Associated Proteins

Supported lipid bilayers are a well-developed model system for the study of membranes and their associated proteins, such as membrane channels, enzymes, and receptors. These versatile model membranes can be made from various components, ranging from simple synthetic phospholipids to complex mixtures of constituents, mimicking the cell membrane with its relevant physiochemical and molecular phenomena. In addition, the high stability of supported lipids bilayers allows for their study via a wide array of experimental probes. In this work, we describe a platform for supported lipid bilayers that is accessible both electrically and optically. We show that the polarization of the supported membrane can be electrically controlled and optically probed using voltage-sensitive dyes. Membrane polarization dynamics is understood through electrochemical impedance spectroscopy and the analysis of the equivalent electrical circuit. We also describe the effect of the conducting electrode layer on the fluorescence of the optical probe through metal-induced energy transfer. We conclude with a discussion on possible applications of this platform for the study of voltage-dependent membrane proteins and other processes in membrane biology and surface science.

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1611398113 ◽

2016 ◽

Vol 113 (46) ◽

pp. E7185-E7193 ◽

Cited By ~ 35

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.

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