CONICET | Buscador de Institutos y Recursos Humanos

Intermediate filaments (IFs) are one of the main components of the eukaryoticcytoskeleton. IFs are crosslinked with the stiffer microtubules and actin networks, contributing to the viscoelasticity of the cytoplasm and cell mechanical integrity. IFs are composed of several members of a large family of cytoskeletal proteins, including nuclear lamins, which contributes to the structural integrity of the nucleus. IFs mechanical properties have been obtained mostly in in-vitro assays, however, key aspects of the organization of these filaments in the intracellular environment remain elusive. In order toexplore mechanical properties of IFs in living cells, we transfected BHK and MEF 3T3 cells with a vimentin-GFP plasmid and obtained images of the IFs networks using confocal microscopy. We recovered the coordinates of individual fluorescent filaments with sub-pixel precision from these images, using an algorithm developed in our lab. By performing a Fourier analysis of the IFs shapes we determined the persistence length of these filaments (the length of the filament over which thermal bending becomes appreciable and a measure of the filament rigidity - it is proportional to the flexural rigidity of the polymer-) and found a value of 2.1μm, in the order of the in-vitro values. We also analyzed the effect of microtubules and microfilaments networks on the stiffness of IFs in living cells. Whereas microtubules depletion induced by nocodazol did not affect the persistence length of IFs, vinblastin treatment ?a drug that stabilizes MTs dynamics-resulted in a 2-fold increase of IFs apparent stiffness. On the other hand, actin subtle depolimerization, driven by latrunculin B, also enhanced IFs apparent stiffness. These results suggest a high environmental dependence of the IFs mechanical properties. This work contributes to the comprehension of the mechanical behavior of the cytoskeletal filaments to get a better insight into cell mechanics and organization.