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Sellers - Takagi - Billington - 2022

Laboratory of Molecular Physiology (LMP)
NHLBI
Mentor Name
James R. Sellers, Ph.D.
Yasuharu “Harry” Takagi, Ph.D.
Neil Billington, Ph.D.
Mentor Email
Mentor Telephone
301-496-6887

Deciphering the chemo-mechanical properties of myosin-6 using scattering and fluorescence single molecule techniques

The Laboratory of Molecular Physiology (LMP) focuses on the understanding of cytoskeletal proteins, specifically myosins, a superfamily of molecular motors that convert chemical energy (ATP) to mechanical output (work/force/displacement). To understand the detailed mechanism of the myosin superfamily, we undertake a multidisciplinary approach, including techniques from molecular biology, cell biology, biophysics, biochemistry and engineering.

 

Recently in our lab, we have used a number of single molecule techniques: (1) single molecule total internal reflection fluorescence (TIRF) microscopy, (2) single molecule optical trapping and (3) interferometric scattering (iSCAT) microscopy. These techniques were used to determine information that could only be interrogated using single molecule approaches such as step-size, power-stroke size and stall force, but also information that could be compared to bulk solution studies such as the kinetics, or transition rates between specific states, of these motor proteins.

 

The projects in the LMP available to the Summer 2020 BESIP interns includes the following:

  • Use of the optical trap[1] or the TIRF microscope[2] to characterize the single molecule mechanics and kinetics of various myosins;
  • Use of the iSCAT microscopy[3] to determine the kinetics of macromolecular formation of  myosins[4];
  • And development of single molecule in vitro assays to simulate cellular environments[5] using micropatterning equipment.

We also have other instruments in the lab such as a stopped flow transient kinetic apparatus, spectrophotometers, fluorometers to perform biochemical measurements. Furthermore, the LMP is equipped with the Sf9/baculovirus expression system/technology to construct custom chimeric proteins.

Fixed/live cell imaging of myosins in a variety of cell lines using fluorescence microscopy techniques using the state-of-the-art light microscopy core facility of the NHLBI can also be used to study these myosins.

More information  about our laboratory and work can be found at this website: https://www.nhlbi.nih.gov/science/molecular-physiology

References:

  1. Takagi Y, Farrow RE, Billington N, Nagy A, Batters C, Yang Y, Sellers JR, Molloy JE. 2014. Myosin-10 produces its power-stroke in two phases and moves processively along a single actin filament under low load. PNAS. 6;111(18):E1833-42.
  2. Sakamoto T, Webb MR, Forgacs E, White HD, Sellers JR. 2008. Direct observation of the mechanochemical coupling in myosin Va during processive movement. Nature. 455(7209):128-32.
  3. Andrecka J, Ortega Arroyo J, Takagi Y, de Wit G, Fineberg A, MacKinnon L, Young G, Sellers JR, Kukura P. 2015. Structural dynamics of myosin 5 during processive motion revealed by interferometric scattering microscopy. Elife. 6;4. doi: 10.7554/eLife.05413.
  4. Young G, Hundt N, Cole D, Fineberg A, Andrecka J, Tyler A, Olerinyova A, Ansari A, Marklund EG, Collier MP, Chandler SA, Tkachenko O, Allen J, Crispin M, Billington N, Takagi Y, Sellers JR, Eichmann C, Selenko P, Frey L, Riek R, Galpin MR, Struwe WB, Benesch JLP, Kukura P. 2018. Quantitative mass imaging of single biological macromolecules. Science. 360(6387):423-427.
  5. Blanchoin L, Boujemaa-Paterski R, Sykes C, Plastino J. 2014. Actin dynamics, architecture, and mechanics in cell motility. Physiol Rev. 94(1):235-63.
BESIP Year