A Study on Molecular Mechanisms underlying Force Generation in Neuronal Growth Cones using Optical Tweezers

Understanding the dynamical and molecular properties of force generation in neuronal growth cones is fundamental in elucidating how neurons sense the environment and process mechanical information. In this study and in order to address this issue, I used optical tweezers to measure the force exerted by filopodia and lamellipodia of Dorsal Root Ganglia (DRG) and hippocampal neurons. I have investigated in detail the roles of several important players in force generation such as actin turnover, membrane stiffness and myosin II. Therefore, my PhD thesis provides precise characterization of the molecular mechanism underlying force generation in growth cones. In the first chapter of my result dynamical properties of force generation in neuronal lamellipodia are presented. Force-velocity (Fv) relationship has been measured with millisecond (ms) temporal resolution and picoNewton (pN) sensitivity. My results show that force generation is a probabilistic process and the fast growth of lamellipodia leading edge alternates with local retractions. The results of the second part of this study show that force generation in neuronal lamellipodia of DRG neurons is composed of elementary events corresponding to forward and backward jumps of bead displacement. These jumps have an amplitude ranging from 2 to 20 nm suggesting that force generation occurs at different rates. A detailed statistical analysis of these jumps and their importance in characterizing the force generation are discussed. In order to understand the role of actin turnover and membrane stiffness on force generation, I analyzed the effect of jasplakinolide and cyclodextrin on force exerted by neuronal growth cones. I found that 25 nM of jasplakinolide, which slows down the actin filament turnover, reduced both the maximal exerted force and the maximal velocity during lamellipodia leading edge protrusion. On the contrary, lamellipodia treated with 2.5 mM of cyclodextrin could advance with a higher velocity. The amplitude and frequency of elementary jumps underlying force generation were reduced by jasplakinolide but not by cyclodextrin. Using atomic force microscopy, I verified that cyclodextrin decreases the membrane stiffness of DRG neurons. The results of this part of my thesis indicate that membrane stiffness provides a selective pressure that shapes force generation and confirm the fundamental role of actin turnover during protrusion. Studying the details of the inhibition of myosin II and its effect on the morphology, kinetics and dynamics of lamellipodia and filopodia emerging from the growth cones of DRG neurons is the subject of next part of my thesis. Treatment with Blebbistatin, inhibitor of myosin II, had the opposite effect on the force generated by lamellipodia and filopodia. My results suggest a possible role of myosin II in force generation and in particular during lamellipodia retractions and confirm a coupling between actin and microtubule dynamics. At the end, the comparison of force generation in growth cones of the central nervous system (hippocampal) and peripheral nervous system (DRG) are presented. I found that filopodia and lamellipodia of DRG and hippocampal growth cones can exert forces with amplitudes varying from 1 to 20 pN developing with a similar time course. At a more quantitative level two main differences appear: firstly, filopodia from hippocampal growth cones exert a force larger than from DRG growth cones; secondly, lamellipodia from DRG growth cones exert a larger force and can move up at a higher speed in axial direction.