Purdue University Calumet, Biological Sciences, Hammond, IN 46323
Directed neuronal growth cone motility is essential in axon pathfinding of developing embryos and regeneration of damaged nerves. A growth cone with a lamellipodium and numerous filopodia is a highly dynamic structure at the tip of an axon. Lamellipodium is the sheeth-like region which provides the major site of adhesion or point of contact. The filopodia are the finger-like protrusions that function as sensors and respond to the environment. Protein motors have been shown to drive and control growth cone motility. In this study, a myosin specific inhibitor - blebbistatin is used to probe the role of non-muscle myosin II in neurite outgrowth, morphology of growth cones, and movement of filopodia. Dorsal root ganglion neurons obtained from day 11 – 13 chick embryos were treated with 100 µM blebbistatin and observed using a Zeiss Axiovert 100 STV inverted microscope equipment with a cool CCD camera controlled by the MetaView software. Our data shows that blebbistatin reduced the rate of neurite extension significantly and led to an observable reduction of lamellipodial size and a dramatic increase of the average filopodial length (88.04 µm ± 3.47 treated vs. 47.03 µm ± 2.20 control). Further analysis indicates that drug treatment increased the rate of filopodial extension (0.198 µm/s ± 0.026 treated vs. 0.131 µm/s ± 0.008 control) and did not alter the rate of filopodial retraction (0.090 µm/s ± 0.007 treated vs. 0.091 µm/s ± 0.011 control). Our studies demonstrate non-muscle myosins function in neurite outgrowth, maintaining growth cone morphology, and filopodial extension.
[Abstract (DOC)]