The role of actin-binding proteins in mediating growth cone guidance to neurotrophins.

Abstract

Development of neuronal circuits rely on growth cones, the dynamic tips of growing neurites, to detect and respond to molecular cues expressed in the developing organism. Guidance cues steer growth cones by activating intracellular signaling cascades that culminate in cytoskeletal remodeling. Although many of the guidance cues, receptors and signaling molecules have been identified, the role of actin-binding proteins involved in mediating growth cone guidance are poorly understood. The content of this thesis present evidence for the regulation of two actin-binding proteins crucial in mediating attractive axon guidance to nerve growth factor (NGF) and characterizes the changes in actin filament dynamics that produce attractive growth cone turning.

The first section characterizes a mechanism by which activation of the actin-binding protein ADF/cofilin (AC) mediates attractive guidance of sensory neuronal growth cones to NGF and retinal ganglion cell growth cones to netrin-1 by locally increasing actin filament (F-actin) polymerization. The presented research demonstrates that NGF or netrin-1 induces an increase in F-actin and free F-actin barbed ends in dorsal root ganglion (DRG) or retinal growth cones, respectively, and a gradient of these cues also induce a local F-actin increase toward the cue source. Netrin-1 (retina) and NGF (DRG) also activate AC in both a global and local manner, and directly elevating AC activity mimics the guidance cue-induced F-actin and free F-actin barbed end increase. A cell-permeable gradient of active AC proteins locally elevate growth cone F-actin levels and induce attractive growth cone turning. Disrupting AC function blocks NGF (DRG) and netrin-1 (retina) membrane protrusion and attractive turning.

The next section identifies the membrane-cytoskeletal linker proteins of the ezrin/radixin/moesin (ERM) family as mediators of attractive growth cone guidance to NGF in sensory DRGs. Data are presented that demonstrate a global and local increase in active phospho-ERM proteins at the growth cone leading margin in response to NGF. Disrupting ERM function (with a dominant-negative ERM; DNERM) results in disorganized actin filaments, and DNERM growth cones do not exhibit increased F-actin after addition of NGF or cell-permeable active AC proteins. Moreover, localization of active AC and F-actin barbed ends are displaced from the growth cone periphery in DNERM growth cones. Active phospho-ERM levels increase when AC activity is elevated, and the NGF-induced phospho-ERM increase is blocked in growth cones with reduced AC activity. Attractive growth cone turning to NGF is reduced in growth cones with disrupted ERM function or lowered ERM protein levels.

The final section demonstrates that local protein synthesis is not required for all growth cone guidance. Data is shown to demonstrate that although protein synthesis does occur in response to many guidance cues, attractive growth cone turning to NGF occurs in conditions where local protein synthesis is inhibited.