From an axon into a growth cone after axotomy: A model for cytoskeletal dynamics

Using live confocal microscope imaging of molecular probes, retrospective immunolabeling, classical electron microscopy, and cultured Aplysia neurons this chapter describes the experimental approaches to document the cascades of cytoskeleton remodeling that underlie the transformation of a cut axonal-end into a growth cone (GC). Rapture of the axon's plasma membrane is followed by massive influx of calcium through the cut end, leading to depolymerization of the microtubules (MTs) and the actin filaments. The elevated free intracellular calcium concentration ([Ca²⁺]_i) activates calpain which cleaves the submembrane spectrin skeleton. Repair of the ruptured membrane barrier is followed by the recovery of the (Ca²⁺)_i and the restructuring of the cytoskeleton in a way that totally differs from that of a differentiated axon. The typical unipolar orientation of axonal MTs is changed to form two distinct MT-based vesicle traps. One traps capture and concentrates anterogradely transported Golgi-derived vesicles while the other concentrates retrogradely transported vesicles. The trapped Golgi-derived vesicles fuse with the plasma membrane from which the submembrane spectrin skeleton was removed by calpain. Actin filaments repolymerize to form radially oriented bundles that generate the mechanical force underlying the extension of the GC's lamellipodium. Feedback interactions between the cytoskeletal elements and the transported cargo as well as interactions with membrane targets participate in the definition of cytoskeleton restructuring and the formation of competent GCs.