Open AccessVisualizing the in vitro assembly of tropomyosin/actin filaments using TIRF microscopy
Author(s) -
Miro Janco,
Irina Dedova,
Nicole S. Bryce,
Edna C. Hardeman,
Peter W. Gunning
Publication year - 2020
Publication title -
biophysical reviews
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.766
H-Index - 39
eISSN - 1867-2469
pISSN - 1867-2450
DOI - 10.1007/s12551-020-00720-6
Subject(s) - total internal reflection fluorescence microscope , tropomyosin , protein filament , actin , actin remodeling , microbiology and biotechnology , biophysics , actin binding protein , cytoskeleton , mdia1 , in vitro , chemistry , biology , actin cytoskeleton , biochemistry , cell , membrane
Tropomyosins are elongated alpha-helical proteins that form co-polymers with most actin filaments within a cell and play important roles in the structural and functional diversification of the actin cytoskeleton. How the assembly of tropomyosins along an actin filament is regulated and the kinetics of tropomyosin association with an actin filament is yet to be fully determined. A recent series of publications have used total internal reflection fluorescence (TIRF) microscopy in combination with advanced surface and protein chemistry to visualise the molecular assembly of actin/tropomyosin filaments in vitro. Here, we review the use of the in vitro TIRF assay in the determination of kinetic data on tropomyosin filament assembly. This sophisticated approach has enabled generation of real-time single-molecule data to fill the gap between in vitro bulk assays and in vivo assays of tropomyosin function. The in vitro TIRF assays provide a new foundation for future studies involving multiple actin-binding proteins that will more accurately reflect the physiological protein-protein interactions in cells.