Calpain-Mediated Proteolysis of Talin and FAK Regulates Adhesion Dynamics Necessary for Axon Guidance

Abstract

Guidance of axons to their proper synaptic target sites requires spatially and temporally precise modulation of biochemical signals within growth cones. Ionic calcium (Ca²⁺) is an essential signal for axon guidance that mediates opposing effects on growth cone motility. The diverse effects of Ca²⁺ arise from the precise localization of Ca²⁺ signals into microdomains containing specific Ca²⁺ effectors. For example, differences in the mechanical and chemical composition of the underlying substrata elicit local Ca²⁺ signals within growth cone filopodia that regulate axon guidance through activation of the protease calpain. However, how calpain regulates growth cone motility remains unclear. Here, we identify the adhesion proteins talin and focal adhesion kinase (FAK) as proteolytic targets of calpain in <i>Xenopus laevis</i> spinal cord neurons both <i>in vivo</i> and <i>in vitro</i> Inhibition of calpain increases the localization of endogenous adhesion signaling to growth cone filopodia. Using live cell microscopy and specific calpain-resistant point-mutants of talin (L432G) and FAK (V744G), we find that calpain inhibits paxillin-based adhesion assembly through cleavage of talin and FAK, and adhesion disassembly through cleavage of FAK. Blocking calpain cleavage of talin and FAK inhibits repulsive turning from focal uncaging of Ca²⁺ within filopodia. In addition, blocking calpain cleavage of talin and FAK <i>in vivo</i> promotes Rohon-Beard peripheral axon extension into the skin. These data demonstrate that filopodial Ca²⁺ signals regulate axon outgrowth and guidance through calpain regulation of adhesion dynamics through specific cleavage of talin and FAK.<b>SIGNIFICANCE STATEMENT</b> The proper formation of neuronal networks requires accurate guidance of axons and dendrites during development by motile structures known as growth cones. Understanding the intracellular signaling mechanisms that govern growth cone motility will clarify how the nervous system develops and regenerates, and may identify areas of therapeutic intervention in disease or injury. One important signal that controls growth cones is that of local Ca²⁺ transients, which control the rate and direction of axon outgrowth. We demonstrate here that Ca²⁺-dependent inhibition axon outgrowth and guidance is mediated by calpain proteolysis of the adhesion proteins talin and focal adhesion kinase. Our findings provide mechanistic insight into Ca²⁺/calpain regulation of growth cone motility and axon guidance during neuronal development.