Fluorescence microscopic imaging of single desmin intermediate filaments elongated by the presence of divalent cations in vitro

The formation of intermediate filaments (IFs), a paradigmatic assembly system in biological macromolecules, depends on cations. Herein, to explore the combined effect of ionic strength and divalent cations, we used fluorescence microscopy and examined the in vitro effects of MgCl 2, CaCl 2, and SrCl 2 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 at high resolution via 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.