The long-term objectives of the project are to understand at the subcellular and molecular levels a) how events in the growth cone (the specialized ending of a growing neuronal process) lead to neurite growth and pathfinding and b) how substratum-bindug factors influence these events to promote and guide growth. The main technique used is video microscopic (VEC-DIC and fluorescence) examination of cells in culture and our approach is based upon breaking the process of neurite growth into a sequence of a few morphologically defined steps which are then analyzed separately. The proposed work is aimed at clarifying aspects of the organization of microtubules and actin filaments in the growth cone and providing insight into how these cytoskeletal elements participate in the steps of the growth sequence. A possible role for Ca++ as a signal for eliciting protrusion of filopodia will also be examined. In addition, work will be directed towards determining the nature of large particles transported in filopodia newly formed after transection of an axon and determining whether this transport is actin- or microtubule-based. A major group of experiments will examine how laminin (a protein of the extracellular matrix) rapidly affects the growth cone to stimulate growth of neonatal rat sympathetic neurons. Questions to be addressed are: a) which step(s) in the growth sequence is(are) promoted by laminin, b) whether laminin must be bound to an immobile substratum to exert its effects, c) where laminin must bind to the growth cone to exert its effects, and d) whether rapid chariges in [Ca++]i or pHi might mediate the response to laminin. The research is potentially useful for designing therapies for the intractable paralysis stemming from injury to the spinal cord as well as, perhaps, for other neurodegenerative conditions. This is because recovery in these situations depends on axon growth and because growth-promoting proteins of the type studied here are likely to be important in determining which axons will regenerate after injury.
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