Growth cones turn and migrate up an immobilized gradient of the laminin IKVAV peptide

Abstract

Growth cone navigation is guided by extrinsic environmental proteins, called guidance cues. Many in vitro studies have characterized growth cone turning up and down gradients of soluble guidance cues. Although previous studies have shown that axonal elongation rates can be regulated by gradients of surface-bound molecules, there are no convincing demonstrations of growth cones turning to migrate up a surface-bound gradient of an adhesive ligand or guidance cue. In order to test this mode of axonal guidance, we used a photo-immobilization technique to create grids and gradients of an adhesive laminin peptide on polystyrene culture dish surfaces. Chick embryo dorsal root ganglia (DRGs) were placed on peptide grid patterns containing surface-bound gradients of the IKVAV-containing peptide. DRG growth cones followed a path of surface-bound peptide to the middle of a perpendicularly oriented gradient with a 25% concentration difference across 30 microm. The majority of growth cones turned and migrated up the gradient, turning until they were oriented directly up the gradient. Growth cones slowed their migration when they encountered the gradient, but growth cone velocity returned to the previous rate after turning up or down the gradient. This resembles in vivo situations where growth cones slow at a choice point before changing the direction of axonal extension. Thus, these results support the hypothesis that mechanisms of axonal guidance include growth cone orientation by gradients of surface-bound adhesive molecules and guidance cues.

Crk-associated substrate (Cas) signaling protein functions with integrins to specify axon guidance during development

“…Disruption of axon-substrate attachment in vitro with integrin function-blocking antibodies encourages axon-axon adhesive interactions (fasciculation) in place of axon-substrate adhesion (Bozyczko and Horwitz, 1986). Furthermore, contact with integrin ligands can slow axon elongation, as axons encountering an increasing gradient of laminin peptide exhibit reduced velocity, but growth cone velocity returns to previous rates when axons turn down the gradient (Adams et al, 2005). This in vitro observation resembles in vivo situations in which growth cones slow at a choice point, exhibit increased morphological complexity and then extend along distinct pathways (Godement et al, 1994;Gomez and Spitzer, 1999;Tosney and Landmesser, 1985).…”

“…Genetic analyses in C. elegans, Drosophila and mice reveal that axon extension in integrin mutants in vivo is not compromised during development; however, defects in axon guidance result from loss of integrin function (Baum and Garriga, 1997;Billuart et al, 2001;Hoang and Chiba, 1998;Pietri et al, 2004;Stevens and Jacobs, 2002). In combination with in vitro and in vivo observations addressing the effects integrins and their ligands exert on growing axons (Adams et al, 2005;Bonner and O'Connor, 2001;Garcia-Alonso et al, 1996;Gomez et al, 1996;Hopker et al, 1999;Kuhn et al, 1995), these studies define functions for integrin signaling in guiding growing axons, strongly suggesting that integrins are essential for axon guidance.…”

Bioactive Nanomaterials for Neural Engineering

“…Functionality of these peptides has been assessed in various applications. Adams et al showed that DRG neurons, that were grown on gradients of photoimmobilized IKVAV bound polystyrene grids, preferentially directed their neurites toward higher concentration of IKVAV containing surface (Adams et al 2005 ). In another study, melt coextruded aligned poly(ε-caprolactone) (PCL) fi bers were modifi ed with photochemical gradient of IKVAV peptide, which provided directional cues for neuronal outgrowth of PC-12 cells (Kim et al 2015 ).…”

Smart polymeric gels: Redefining the limits of biomedical devices

“…The integrin-binding activity of natural adhesion proteins can be partially mimicked by oligopepetide sequences [95,96], such as the ubiquitous RGD (i.e., arginineglycine-aspartic acid) [97][98][99][100], RGD and PHSRN (i.e., proline-histidine-serine-arginineasparagine) (in synergy) [101,102], IKVAV (i.e., isoleucine-lysine-valine-alanine-valine) [103][104][105], YIGSR (tyrosine-isoleucine-glycine-serine-arginine) [106,107], PHSRN , and so on. Harden et al went a step further from the triblock architecture initially proposed by Tirrell et al by conjugating RGD to the central block of the modular triblock protein architecture.…”