Assessment of the cytoskeletal impact of beta-actin mutations leading to non-muscle actinopathies by means of dual laser optical tweezers (DLOT).

Abstract

Actin plays crucial roles in critical cellular processes, like the dynamic remodeling of the cytoskeleton. Non-muscle actinopathies (NMAs) are disorders caused by heterozygous mutations in the genes encoding for the β- and γ-isoforms of cytoskeletal actin, whose underlying mechanisms remain to be resolved. We used a Dual Laser Optical Tweezers (DLOT) to characterize the viscoelastic properties of the membrane-cytoskeleton system of neurons wild-type and expressing the β-actin mutation R196H associated with malformations of cortical development. Ramp-and-hold pulls to generate a tether were imposed on the membrane of neuron progenitor cells (NPC) and mature neurons (NC). Tether formation-elongation is characterized by a multiphase force response: an early rise to a peak associated with the formation of the tether and followed by a drop to a minimum; a continuous rise during the ramp at a rate that exponentially decreases to a steady rate; an exponential force relaxation to a constant value during the hold. The force responses were simulated by using a 2nd order Maxwell viscoelastic arrangement that provides the estimates of both undamped (k0) and damped elastic coefficients (k1 and k2) and the friction coefficients (η1 and η2) of tether formation. In both WT and R196H neurons the undamped elastic coefficient is not significantly affected by the maturation process. During maturation, friction and damped elastic coefficients show significant reductions in WT neurons, while in mutant neurons all the coefficients in NPCs are already reduced to values similar to those in NCs. The work provides the mechanical evidence that R196H mutation induces a faster differentiation of the NPC toward the greater connectivity of the mature neuron. Supported by EJP RD 2019.