The molecular events that underlie the process of growth cone guidance and axonal pathfinding are one of the major challenges for Neurobiology in the future. Recently, research in developmental neurobiology has moved decisively into the molecular arena with the discovery and characterization of molecules that act as extracellular pathfinding cues and specific receptors on growth cones and axons for recognizing these cues. Some of these receptors interact with extracellular matrix components that may serve as spatial cues, still others recognize cell adhesion molecules (CAMs) on other cell surfaces. Although there has been intense interest in molecules involved in neuronal guidance, little is actually known about how adhesion protein or receptor occupation lead to the rapid alterations of structure and motility that underlie pathfinding decisions nor how the forces involved are transduced. The proposed research attempts to fill this gap in our knowledge: (1) by characterizing the cytoskeletal protein dynamics underlying growth cone motility (2) by characterizing signal transduction mechanisms and cell surface recognition processes involved in converting this motility into guidance. The results of this work have direct implications for clinical interpretation of developmental brain disorders involving aberrant neuronal pathway formation and will extend our understanding of nerve regeneration. To accomplish these aims the applicant has developed a model system for studying growth cone interactions with pseudotarget substrates that mimic stereotypic morphological alterations observed when growth cones interact with native targets. Pseudotargets are constructed by derivatizing latex or silica microbeads with test ligands such as cell adhesion proteins or antibodies. When placed on the growth cone surface, they react with targeted membrane proteins and elicit an array of ligand dependent responses that are hypothesized to be related to mechanisms of cell adhesion and target recognition. Single beam gradient optical traps (laser tweezers) are used to facilitate bead placement on the growth cone surface. High resolution video enhanced DIC imaging and digital image processing techniques allow the tracking of bead movements and the analysis of structural changes with high spatial fidelity and temporal resolution. To address molecular substrates underlying these structural changes, fluorescent cytoskeletal and regulatory protein analogs are made and injected into cells. Their movements are being assessed using several fluorescence imaging techniques as well as photoactivation of fluorescence.
Showing the most recent 10 out of 34 publications
