Abstract
Coordinated control of the growth cone cytoskeleton underlies axon extension and guidance. Members of the collapsin response mediator protein (CRMP) family of cytosolic phosphoproteins regulate the microtubule and actin cytoskeleton, but their roles in regulating growth cone dynamics remain largely unexplored. Here, we examine how CRMP4 regulates the growth cone cytoskeleton. Hippocampal neurons from CRMP4-/- mice exhibited a selective decrease in axon extension and reduced growth cone area, whereas overexpression of CRMP4 enhanced the formation and length of growth cone filopodia. Biochemically, CRMP4 can impact both microtubule assembly and F-actin bundling in vitro. Through a structure function analysis of CRMP4, we found that the effects of CRMP4 on axon growth and growth cone morphology were dependent on microtubule assembly, whereas filopodial extension relied on actin bundling. Intriguingly, anterograde movement of EB3 comets, which track microtubule protrusion, slowed significantly in neurons derived from CRMP4-/- mice, and rescue of microtubule dynamics required CRMP4 activity toward both the actin and microtubule cytoskeleton. Together, this study identified a dual role for CRMP4 in regulating the actin and microtubule growth cone cytoskeleton.
Highlights
Intricate regulation of the growth cone cytoskeleton controls growth cone dynamics
Through a structure function analysis of CRMP4, we found that the effects of CRMP4 on axon growth and growth cone morphology were dependent on microtubule assembly, whereas filopodial extension relied on actin bundling
CRMP4 activity toward microtubules regulates growth cone size and neurite outgrowth, whereas CRMP4 activity toward actin bundling is important for filopodial extension and may be more important for acute growth cone rearrangements in response to guidance cues
Summary
Intricate regulation of the growth cone cytoskeleton controls growth cone dynamics. Results: Loss of CRMP4 disrupts growth cone cytoskeletal dynamics, growth cone expansion, and axon growth. Members of the dihydropyrimidinase-like or collapsin response mediator protein (CRMP) family of cytosolic phosphoproteins are excellent candidates to dually regulate actin and microtubule cytoskeletal rearrangements underlying growth cone dynamics and neurite extension. CRMP family members (CRMP1–5) play important roles in neuronal differentiation, axonal growth, guidance, axon/dendrite specification, and microtubule organization including establishing the microtubule asymmetry that underlies protein sorting axons and dendrites (10 –20). We focus on elucidating the role of CRMP4 in regulating the actin and microtubule growth cone cytoskeleton in hippocampal neurons and the impact of its activity on growth cone dynamics and extension. We found that CRMP4 is responsible for organizing both the actin and microtubule structure within hippocampal growth cones and that this activity underlies growth cone morphology and axon extension
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