Visualizing Telokin-Dependent Regulation of Myosin Filaments by Electron Microscopy

Smooth muscle myosin (SmM) is the primary force-generating motor protein in smooth muscle cells. It undergoes conformational transitions between a compact, inactive folded state (10S) and an extended, filament-forming state (6S). Telokin (Tkn), the noncatalytic C-terminal domain of myosin light chain kinase (MLCK), has been implicated in regulating myosin filament stability. However, the molecular basis of its interaction with SmM remains incompletely understood. In this study, we have employed recombinant human Tkn to investigate its role in SmM filament assembly. Circular dichroism spectroscopy, fluorescence spectroscopy, and chromatographic studies were used to confirm the structural integrity and functional activity of the purified proteins. Remarkably, the presence of Tkn promoted myosin filament assembly even with ATP, a condition that typically favors the folded 10S conformation and inhibits filament formation. Transmission Electron Microscope (TEM) micrographs of the resulting filaments showed predominantly non-bipolar filaments that closely resembled the native side-polar smooth muscle thick filaments. Despite sequence variations within the acidic C-terminal region, human Tkn efficiently induced filament assembly of avian SmM, indicating conservation of the underlying filament-promoting mechanism across species. Collectively, these findings provide direct experimental evidence that Tkn acts as a structural regulator of filament assembly and stability. This offers new insights into the mechanisms governing smooth muscle contractility.