Dokument

Summary:
Wiring of the brain is established by axons, which elongate from a neuron and are guided through the brain to their target neurons. Growth cones are actin-rich structures located at the tips of axons and are responsible for sensing environmental cues as well as controlling directed axonal outgrowth. Motility and function of growth cones are mediated by underlying actin dynamics, which in turn are regulated by actin-binding proteins (ABPs). Among these proteins is the family of cyclase-associated proteins (CAPs), which comprises two members (CAP1, CAP2), which are both expressed in the brain. Despite recent progress in uncovering their molecular function in actin dynamics, their physiological role during brain development remains largely unknown. Therefore, we used knockout (KO) mouse models for both CAPs to investigate their function in brain development. We found both proteins expressed in the embryonic brain as well as in cultured neurons and being localized within growth cones. CAP1-KO brains displayed impaired fiber track formation, but had no alterations in neuron migration or precursor proliferation. In addition, CAP1-KO neurons were delayed in development and exhibited shorter and thicker neurites. This was accompanied by enlarged growth cones, which had fewer filopodia, reduced motility, impaired actin dynamics and consequently disturbed responses to guidance cues. Instead, the loss of CAP2 did not cause any changes in brain morphology or neuron differentiation. Alterations in differentiation and morphology in CAP1-KO neurons as well as growth cone size could be rescued by overexpression of either CAP1 or CAP2, suggesting functional redundancy of both proteins. Further analysis exploiting CAP1 mutants revealed that the helical fold domain and therefore the interaction with the actin regulator cofilin1 is important in mediating growth cone function. Establishing a neuron replating protocol to study early neuron differentiation and growth cone function upon knockout of either CAP1, cofilin1 or both ABPs allowed a more detailed analysis on the functional interaction of CAP1 and cofilin1 in the growth cone. This approach revealed that both proteins synergistically regulate F-actin dynamics within the growth cone and that they are functionally dependent on each other. Taken together, this study showed that CAP1 and CAP2 are redundant in regulating growth cone function in vitro, but that CAP1 is the dominant family member in neuron differentiation and brain development. Furthermore, this study provides a new protocol for studying protein function during early aspects of neuron differentiation and showed that CAP1 and cofilin1 functionally interact in the growth cone and regulate its dynamics, thereby providing new insights into the physiological role of CAP1-cofilin1 interaction.

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