Regulation of filopodia by transmembrane agrin

Filopodial activity is implicated in control of growth and movement in many cell types. Neuronal filopodia are involved in control of axon guidance, neurite branching and in synapse formation. GTPase molecular switches and their effectors of actin assembly play a recognized role in filopodia formation but the upstream molecules that control filopodia formation and activity in response to extracellular cues are much less well characterized. Here we describe a novel function of the transmembrane isoform of the proteoglycan agrin (TM‐agrin), which is expressed predominantly in the CNS. Roles for agrin have been suggested in the regulation of neurite outgrowth, excitatory signaling and synapse formation. We found that over‐expression of TM‐agrin caused the formation of excess filopodia on neurites of hippocampal neurons cultured for up to a week. Conversely, suppression of agrin expression by transfection with a siRNA plasmid reduced the number of filopodia. Time‐lapse analysis suggested that endogenous TM‐agrin regulates filopodia number by increasing the stability of filopodia and increasing initiation. Over‐expression of TM‐agrin induced the formation of filopodia in several cell lines and branched retraction fibers in most cell lines tested. Analysis of filopodia induction by deletion mutants of TM‐agrin in COS‐7 cells and hippocampal neurons indicated that motifs in the extracellular portion of the N‐terminal half of TM‐agrin are essential. Cdc42 and Rac activation were strongly increased in COS‐7 cells transfected with TM‐agrin, suggesting a role for these GTPases downstream of agrin. By positively regulating the number and stability of filopodia in developing neurons, TM‐agrin may influence the pattern of neurite outgrowth and synapse formation. This work was funded by the NHLBI Intramural Research Program.