Highly-parallel microfluidics-based force spectroscopy on single cytoskeletal motors

AbstractCytoskeletal motors transform chemical energy into mechanical work to drive essential cellular functions. Optical trapping experiments have provided crucial insights into the operation of these molecular machines under load. However, the throughput of such force spectroscopy experiments is typically limited to one measurement at a time. Here, we describe an alternative, highly-parallel, microfluidics-based method that allows for rapid collection of force-dependent motility parameters of cytoskeletal motors. We applied tunable hydrodynamic forces to stepping kinesin-1 motors via DNA-tethered beads and utilized a large field-of-view to simultaneously track the velocities, run lengths and interaction times of hundreds of individual kinesin-1 molecules under varying resisting and assisting loads. Importantly, the 16-μm long DNA tethers between the motors and the beads significantly reduced the vertical component of the applied force pulling the motors away from the microtubule. Our approach is readily applicable to other molecular systems and constitutes a new methodology for parallelized single-molecule force studies on cytoskeletal motors.

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The influence of optical aberrations in large field-of-view single-molecule localization microscopy

Single Molecule Spectroscopy and Superresolution Imaging XIV ◽

10.1117/12.2584546 ◽

2021 ◽

Author(s):

Lei Xu ◽

Jun Lu ◽

Biqin Dong

Keyword(s):

Single Molecule ◽

Field Of View ◽

Large Field ◽

Optical Aberrations ◽

Localization Microscopy ◽

Single Molecule Localization Microscopy

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Versatile, High-Resolution, and Large Field-of-View Single-Molecule Imaging by Oblique Plane Microscopy

Biophysical Journal ◽

10.1016/j.bpj.2020.11.1250 ◽

2021 ◽

Vol 120 (3) ◽

pp. 179a

Author(s):

Peter Brown ◽

Rory Kruithoff ◽

Lei Zhou ◽

Yoshihiko Kobayashi ◽

Purushothama Rao Tata ◽

...

Keyword(s):

High Resolution ◽

Single Molecule ◽

Field Of View ◽

Large Field ◽

Single Molecule Imaging

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Polymer sequencing by molecular machines: a framework for predicting the resolving power of a sliding contact force spectroscopy sequencing method

Nanoscale ◽

10.1039/c7nr03358c ◽

2017 ◽

Vol 9 (39) ◽

pp. 15089-15097

Author(s):

Alex Dunlop ◽

Kate Bowman ◽

Olav Aarstad ◽

Gudmund Skjåk-Bræk ◽

Bjørn T. Stokke ◽

...

Keyword(s):

Contact Force ◽

Single Molecule ◽

Force Spectroscopy ◽

Molecular Machines ◽

Sliding Contact ◽

Spectroscopy Method ◽

Resolving Power ◽

Single Molecule Force Spectroscopy ◽

Sequencing Method

An AFM-based single molecule force spectroscopy method for polymer sequencing distinguishes between different monomers on the basis of their size and hydrophobicity.

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ChemInform Abstract: Force Spectroscopy of Molecular Systems - Single Molecule Spectroscopy of Polymers and Biomolecules

ChemInform ◽

10.1002/chin.200048292 ◽

2000 ◽

Vol 31 (48) ◽

pp. no-no

Author(s):

Andreas Janshoff ◽

Marcus Neitzert ◽

York Oberdoerfer ◽

Harald Fuchs

Keyword(s):

Single Molecule ◽

Force Spectroscopy ◽

Single Molecule Spectroscopy ◽

Molecular Systems

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Force Spectroscopy of Molecular Systems—Single Molecule Spectroscopy of Polymers and Biomolecules

Angewandte Chemie International Edition ◽

10.1002/1521-3773(20000915)39:18<3212::aid-anie3212>3.0.co;2-x ◽

2000 ◽

Vol 39 (18) ◽

pp. 3212-3237 ◽

Cited By ~ 270

Author(s):

Andreas Janshoff ◽

Marcus Neitzert ◽

York Oberdörfer ◽

Harald Fuchs

Keyword(s):

Single Molecule ◽

Force Spectroscopy ◽

Single Molecule Spectroscopy ◽

Molecular Systems

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Calculating the force-dependent unbinding rate of biological macromolecular bonds from the force-ramp optical trapping assays

10.1101/2021.09.20.461030 ◽

2021 ◽

Author(s):

Apurba Paul ◽

Joshua Alper

Keyword(s):

Optical Tweezers ◽

Single Molecule ◽

Force Spectroscopy ◽

Optical Tweezer ◽

Stochastic Simulations ◽

Cumulative Distribution ◽

Cellular Functions ◽

Single Molecule Force Spectroscopy ◽

Protein Ligand Interactions ◽

Ligand Interactions

AbstractThe non-covalent biological bonds that constitute protein-protein or protein-ligand interactions play crucial roles in many cellular functions, including mitosis, motility, and cell-cell adhesion. The effect of external force (F) on the unbinding rate (koff(F)) of macromolecular interactions is a crucial parameter to understanding the mechanisms behind these functions. Optical tweezer-based single-molecule force spectroscopy is frequently used to obtain quantitative force-dependent dissociation data on slip, catch, and ideal bonds. However, analyses of this data using dissociation time or dissociation force histograms often quantitatively compare bonds without fully characterizing their underlying biophysical properties. Additionally, the results of histogram-based analyses can depend on the rate at which force was applied during the experiment and the experiment’s sensitivity. Here, we present an analytically derived cumulative distribution function-like approach to analyzing force-dependent dissociation force spectroscopy data. We demonstrate the benefits and limitations of the technique using stochastic simulations of various bond types. We show that it can be used to obtain the detachment rate and force sensitivity of biological macromolecular bonds from force spectroscopy experiments by explicitly accounting for loading rate and noisy data. We also discuss the implications of our results on using optical tweezers to collect force-dependent dissociation data.

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Calculating the force-dependent unbinding rate of biological macromolecular bonds from force-ramp optical trapping assays

10.21203/rs.3.rs-933021/v1 ◽

2021 ◽

Author(s):

Apurba Paul ◽

Joshua Alper

Keyword(s):

Optical Tweezers ◽

Single Molecule ◽

Force Spectroscopy ◽

Optical Tweezer ◽

Stochastic Simulations ◽

Cumulative Distribution ◽

Cellular Functions ◽

Single Molecule Force Spectroscopy ◽

Protein Ligand Interactions ◽

Ligand Interactions

Abstract The non-covalent biological bonds that constitute protein-protein or protein-ligand interactions play crucial roles in many cellular functions, including mitosis, motility, and cell-cell adhesion. The effect of external force (𝐹) on the unbinding rate (𝑘off(𝐹)) of macromolecular interactions is a crucial parameter to understanding the mechanisms behind these functions. Optical tweezer-based single-molecule force spectroscopy is frequently used to obtain quantitative force-dependent dissociation data on slip, catch, and ideal bonds. However, analyses of this data using dissociation time or dissociation force histograms often quantitatively compare bonds without fully characterizing their underlying biophysical properties. Additionally, the results of histogram-based analyses can depend on the rate at which force was applied during the experiment and the experiment’s sensitivity. Here, we present an analytically derived cumulative distribution function-like approach to analyzing force-dependent dissociation force spectroscopy data. We demonstrate the benefits and limitations of the technique using stochastic simulations of various bond types. We show that it can be used to obtain the detachment rate and force sensitivity of biological macromolecular bonds from force spectroscopy experiments by explicitly accounting for loading rate and noisy data. We also discuss the implications of our results on using optical tweezers to collect force-dependent dissociation data.

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Calculating the force-dependent unbinding rate of biological macromolecular bonds from force-ramp optical trapping assays

Scientific Reports ◽

10.1038/s41598-021-03690-1 ◽

2022 ◽

Vol 12 (1) ◽

Author(s):

Apurba Paul ◽

Joshua Alper

Keyword(s):

Optical Tweezers ◽

Single Molecule ◽

Force Spectroscopy ◽

Optical Tweezer ◽

Stochastic Simulations ◽

Cumulative Distribution ◽

Cellular Functions ◽

Single Molecule Force Spectroscopy ◽

Protein Ligand Interactions ◽

Ligand Interactions

AbstractThe non-covalent biological bonds that constitute protein–protein or protein–ligand interactions play crucial roles in many cellular functions, including mitosis, motility, and cell–cell adhesion. The effect of external force ($$F$$ F ) on the unbinding rate ($${k}_{\text{off}}\left(F\right)$$ k off F ) of macromolecular interactions is a crucial parameter to understanding the mechanisms behind these functions. Optical tweezer-based single-molecule force spectroscopy is frequently used to obtain quantitative force-dependent dissociation data on slip, catch, and ideal bonds. However, analyses of this data using dissociation time or dissociation force histograms often quantitatively compare bonds without fully characterizing their underlying biophysical properties. Additionally, the results of histogram-based analyses can depend on the rate at which force was applied during the experiment and the experiment’s sensitivity. Here, we present an analytically derived cumulative distribution function-like approach to analyzing force-dependent dissociation force spectroscopy data. We demonstrate the benefits and limitations of the technique using stochastic simulations of various bond types. We show that it can be used to obtain the detachment rate and force sensitivity of biological macromolecular bonds from force spectroscopy experiments by explicitly accounting for loading rate and noisy data. We also discuss the implications of our results on using optical tweezers to collect force-dependent dissociation data.

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