Mechanical properties of in-vivo intermediate filaments and their interplay with microtubules and microfilaments.

MARIANO SMOLER; CARLA PALLAVICINI; VALERIA LEVI; BRUNO, LUCIANA

La Plata

Congreso; XLVII Reunión Anual de la Sociedad Argenitna de Biofísica; 2018

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 arecomposed of several members of a large family of cytoskeletal proteins, including nuclearlamins, which contributes to the structural integrity of the nucleus. IFs mechanicalproperties have been obtained mostly in in-vitro assays, however, key aspects of theorganization 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 cellswith a vimentin-GFP plasmid and obtained images of the IFs networks using confocalmicroscopy. We recovered the coordinates of individual fluorescent filaments with sub-pixel precision from these images, using an algorithm developed in our lab. By performinga Fourier analysis of the IFs shapes we determined the persistence length of thesefilaments (the length of the filament over which thermal bending becomes appreciableand a measure of the filament rigidity - it is proportional to the flexural rigidity of thepolymer-) and found a value of 2.1μm, in the order of the in-vitro values. We alsoanalyzed the effect of microtubules and microfilaments networks on the stiffness of IFs inliving cells. Whereas microtubules depletion induced by nocodazol did not affect thepersistence 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 subtledepolimerization, driven by latrunculin B, also enhanced IFs apparent stiffness. Theseresults suggest a high environmental dependence of the IFs mechanical properties. Thiswork contributes to the comprehension of the mechanical behavior of the cytoskeletalfilaments to get a better insight into cell mechanics and organization.