Role of Myosin II and Arp 2/3 in the motility and force generation of Neuronal Growth Cones

Differentiating neurons have to find chemical cues to form the correct synaptic connections with the other neurons so that they can create a functional neuronal network. During their development differentiating neurons project neurites, at the distal part of which there is a growth cone (GCs). The growth cone has highly motile structures, referred as lamellipodia and filopodia. Lamellipodia and filopodia sense the environment and process the mechanical and chemical stimulus and also exert forces. During my work for the completion of my PhD thesis, I used Optical Tweezers, video imaging and immunocytochemistry to quantify the motility and the force exerted by lamellipodia and filopodia from Dorsal Ganglion (DRG) neurons. I have also precisely quantified the role of some proteins and signaling pathways which regulate the motility of the DRG GCs. The first part of my results entitled, “The role of myosin-II in force generation of DRG filopodia and lamellipodia”, characterizes the role of Myosin II in growth cone dynamics. Myosin II has been shown to control the retrograde flow of actin polymers, to be involved in the orchestration of actin and microtubules (MTs) dynamics and to possess contractile activity. GCs advance due to combined effects of the adhesion of lamellipodia and filopodia on the substrate and the contractile activity of Myosin II. Therefore, I probed the functional role of Myosin II on GCs dynamics by using its specific inhibitor, Blebbistatin. I show that the force exerted by lamellipodia decreased but surprisingly the force exerted by filopodia increased upon treatment with Blebbistatin. Moreover I show that the well organized and distributed structures of lamellipodia and filopodia of the GCs depend on the activity of Myosin II and confirmed the coupling between actin and microtubule dynamics. The next chapter, “The role of Rac1 in force generation of DRG neurons”, describes the function of Rac1 and its downstream effector Arp2/3 in lamellipodia and filopodia formation and dynamics. It is well known that Rac1 Rho-GTPase acts as a switch between GTP bound active state and GDP bound inactive state. I observed that GCs retract following partial inhibition of Arp2/3 but recover their usual motility within 5-10 minutes. I found that this recovery is caused by the activation of Rac1. This indicates that Rac1 acts as switch and activates upon Arp2/3 inhibition, possibly through integrin pathways. I also confirmed that the activity of Arp2/3 not only regulates the formation of lamellipodia but also controls the dynamics and formation of filopodia.