Neuritogenesis: Neuritogenesis and axonal guidance are key cellular processes necessary for proper development of the adult nervous system and spinal cord regeneration. Directional neurite extension from the soma depends on precise cytoskeletal and adhesion dynamics induced by sensing of attractive and repulsive extracellular cues such as chemokines and extracellular matrix proteins (ECM). This process is similar to directional pseudopodial formation and chemostaxis involving regulated actin-mediated membrane protrusion and signal transduction processes. However, the inability to biochemically purify significant amounts of neurites for biochemical analysis has precluded large-scale spatial analysis of this structure. This has limited our ability to understand signaling mechanisms of neuronal polarity and neuritogenesis. Our laboratory has developed a microporous filter system that allows large-scale biochemical purification of extending or collapsing neurites from neurons polarized towards a directional ECM gradient (see Figures 1-3 below). We are currently using this model system and large-scale quantitative proteomics, phosphoproteomics, and bioinformatics to identify novel proteins that mediate this process and to map networks of complex signaling pathways that control neuritogenesis. We are investigating in detail the role of ERK and the Rho family of GTPases which are highly enriched and activated in the extending neurite. This work is important for understanding spatial signaling mechanisms that contribute to brain development as well as neuropathological conditions associated with neurodegenerative diseases and spinal cord injury.

Figure 1. Schematic showing the protrusion of a cell neurite through the microporus membrane to the lower surface in response to a chemottactrant. The attractant is placed in the lower chamber or coated on the lower membrane surface. Typical attractants include growth factors and extracellular matrix proteins. The cell body (soma) on upper surface and the neurite on the lower surface can then be mechanically separated for biochemical comparison or visualized by immunofluorescence.

Figure 2. Photomicrograph of an extending neurite from a NIE-115 cell stained with antibodies to actin (red) and β3-tubulin.

Figure 3. Confocal photomicrograph of the lower surface of a 3.0 micron porous filter showing neurites protruding through the small pores. The filter was coated on the lower surface with laminin which served as directional cue. Neurites (green) were allowed to extend for 24 hours then fixed and stained with antibodies to β3-tubulin.

Figure 4. Phase-contrast time-lapse movie of a NIE-115 neuroblastoma cell protruding neurites for 18 hours on a laminin coated glass coverslip. Click on image to left to trigger movie