P16 − Using Optical Tweezers to Study the Elementary Events Underlying Force Generation in Neuronal Lamellipodia

The propulsion of the leading edge of neuronal lamellipodia is a complex process in which the polymerization of actin filaments towards the cell membrane is a major component (1,2). This process is at the origin of force generation in neurons. By using optical tweezers, we have characterized the dynamics by which lamellipodia of Dorsal Root Ganglia neurons exerted force on encountered obstacles such as silica beads. To determine the displacements produced by elementary events behind force generation, the stiffness of the optical trap was kept as low as 0.015 pN·nm-1, so that the underlying motion could occur in an unhindered fashion (3). At such a low stiffness the bead held in the optical tweezers necessarily fluctuate with large amplitude possibly masking the underlying biological events. Because of the presence of adhesion forces, beads in close contact with a lamellipodium could seal on its membrane so that the standard deviation of Brownian fluctuations could be reduced by 10 times. In several experiments, the bead remained within 300 nm from the center of the optical trap where the voltage sensitivity of the detector and the trap stiffness is constant. Under these conditions, if the lamellipodium pushed the bead, discrete jumps could be detected. Jumps were detected using an algorithm based on nonlinear diffusion filtering (4). Briefly, the original signal was smoothed in order to obtain a smooth piece-wise (regularized) trace where the discrete jumps were enhanced and then detected. These jumps occurred within 1 ms and had an amplitude varying from 5 to 20 nm. When the lamellipodium retracted, pulling the beads with it, no discrete events were observed. These discrete events were not observed in the presence of Latrunculin A, a blocker of actin polymerization or when neurons were fixed with paraformaldehyde. These jumps show that force generation in lamellipodia is a discontinuous process in which bursts of actin polymerization and depolymerization alternate continuously. In future we will explore changes in the characteristics of these jumps by pharmacologically altering the membrane rigidity to understand the role of the membrane in this process.