Fluorescence Microscopic Imaging of Single Desmin Intermediate Filaments Elongated by the Presence of Divalent Cations in Vitro
23 Pages Posted: 21 Mar 2022
Abstract
The formation of intermediate filaments (IFs), a paradigmatic assembly system in biological macromolecules, depends on cations. Herein, with a view to explore the combined effect of ionic strength and divalent cations, we used fluorescence microscopy to examine the in vitro effects of MgCl2, CaCl2, and SrCl2 on the KCl concentration-dependent growth of desmin IFs. Fluorescently labeled desmin IF assembly initiated by KCl and 5 mM divalent cations led to the formation of single desmin IFs in the KCl concentration range of 25–50 mM. Addition of divalent cations resulted in increased fluorescence intensity in the filament images. KCl concentrations lower or higher than the aforementioned range resulted in the induction of networks of entangled IFs, which were visualized with high resolution by direct stochastic optical reconstruction microscopy. These findings provide insights into the versatility of the IF assembly mechanism and the optimization of fluorescence microscopy of single desmin IFs.
Keywords: intermediate filament, cytoskeleton, self-assembly, electrostatic interaction, super-resolution microscopy
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