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RESEARCH PROJECTS Despite a century of investigation, the question of how axons grow remains unsolved. This question is interesting for at least two reasons: Firstly, if we understand the mechanism of axonal elongation we can assign specific roles to the proteins and molecules important for wiring the brain. Secondly, if we understand how axons grow we can devise rational strategies to overcome intrinsic limitations to regrowth and to accelerate regeneration following injury. Enormous gains have recently been made in our understanding of the cell biology of axonal growth: force generation by molecular motors and microtubule dynamics have emerged as crucial processes for both axonal guidance and lengthening. Yet most studies to date have used simple read-outs, such as measuring changes in the rates of axonal elongation, rather than characterizing the underlying behavior of axonal components. Our approach is to quantitatively analyze the relationship between forces acting on neurons, the bulk movement of material in response to forces inside living neurons, and microtubule dynamics to develop a mechanistic understanding of growth cone motility.Project 1: Do axons grow by stretching or microtubule polymerization at the growth cone in vivo? Forces cause axons to stretch without thinning, but without direct observation of subcellular markers it is difficult to deduce sites of stretching. Previous studies suggest it could occur along the axon, in the growth cone neck, or not at all. Our approach here is to examine the movement of multiple types of organelles and cytoskeletal elements in vitro and in vivo in Drosophila embryos. Our goals are to (1) determine whether guidance and elongation occur by bulk movement of the growth cone or directed assembly of microtubules, (2) if axonal elongation is conserved between species, and (3) to determine how axons grow in vivo. Project 2: Identification of the Molecular Motors that Power Growth Cone Motility Molecular motors are known to be critical for generating the forces needed for axonal elongation, yet the mechanics of growth cone advance remain poorly understood. We have recently developed a new model of axonal elongation that proposes forces generated in the growth cone drive elongation by stretching the axon. Extending this model to accommodate conflicting experimental observations on the role of myosin II in elongation, we plan to test if a myosin II based antagonist force balance between the growth cone and axon regulates the rate of growth cone translocation. Since establishing my lab, we have developed sophisticated biophysical approaches using chick sensory neurons to study axonal elongation. In addition, we have recently refined methods for studying the elongation of Drosophila neurons both in vitro and in vivo. Our approach will be to combine biophysical tools and Drosophila molecular genetics to gain insights into the mechanics of axonal elongation and growth cone guidance. We believe that a better understanding of neuronal biomechanics will lead to new insights into the initial wiring of the nervous system and will aid in the rational development of therapeutics to augment axonal regeneration. |