The influence of sub-micron inhibitory clusters on growth cone substratum attachments and CD44 expression

Proteoglycan expression patterns in the central nervous system guide neuronal pathfinding during development, but also disrupt regeneration after injuries. To deepen our understanding of the molecular level effects of proteoglycan spatial arrangements on neuronal pathfinding, we designed micropatterning stamps for the precise placement of near single molecule chondroitin sulfate proteoglycan (CSPG) clusters into regularly spaced arrays. Actin ultrastructural analysis in dorsal root ganglion neurons grown on laminin-coated substrata patterned with aggrecan cluster arrays revealed filopodial and lamellapodial edge contact avoidance of individual clusters, while growth cone lamellapodia and central domains were able to span multiple clusters over a range of cluster densities. Total internal reflection fluorescence microscopy interrogation of growth cone substratum morphology further revealed persistence of integrin mediated substratum adhesion and local out-of-plane membrane bending over clusters on the height scale of aggrecan glycosaminoglycan side chains. Direct imaging of cell adhesion molecule CD44 expression in growth cones revealed an aggrecan dose dependent upregulation in CD44 molecules. Evidence of CD44 clustering coinciding with underlying aggrecan molecules implies CSPG–CD44 interactions. The results reveal the limited local repulsive effect of CSPGs on neuronal structures and provide evidence that CD44 upregulation in neurons is affected by local CSPG expression.