Deciphering the mechanism of macromolecular formation of non-muscle myosin-2 filaments 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) on actin filament “tracks”. 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.

In recent years, the focus of our laboratory has been on understanding the structure, function and regulation of non-muscle myosin-2s using different  techniques. Non-muscle myosin-2 isoforms are one of the most abundant molecular motors in a cell, which forms an organized filamentous structure as its physiological significant “unit”. It is directly associated with many physiological processes including cell migration and adhesion, morphogenesis, cytokinesis and vesicular transport and mutations in nonmuscle myosin-2 isoforms are associated with diseases such as thrombocytopenia, kidney disease, blindness and squamous cell cancers  

 

In our lab, we have specifically used several single molecule techniques: (1) single molecule total internal reflection fluorescence (TIRF) microscopy1,2, (2) single molecule optical trapping3 and (3) interferometric scattering (iSCAT) microscopy4, and (4) a modified iSCAT technique, called mass photometry5 (MP). 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 2024 BESIP intern includes the following:

• Use of the mass photometry technique to characterize the macromolecular formation, or polymerization process, of non-muscle myosins and its dynamics on actin filaments suspended on lipid bilayers; 
• Use of the TIRF and iSCAT microscopy to characterize the single molecule mechanics and kinetics of various myosins under different conditions mimicking cellular conditions. 

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

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

References:

1. Melli L, Billington N, Sun SA, Bird JE, Nagy A, Friedman TB, Takagi Y, Sellers JR. 2018. Bipolar filaments of human nonmuscle myosin 2-A and 2-B have distinct motile and mechanical properties.  Elife 7:e32871. doi: 10.7554/eLife.32871.
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. 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.
4. 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. 4:e05413. doi: 10.7554/eLife.05413.
5. 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.