Assembly of microfilaments and microtubules from axonally transported actin and tubulin after axotomy

The slow component (SC) of axonal transport conveys structural proteins, regulatory proteins, and glycolytic enzymes toward the axon tip at 1-6 mm/day. Following axon interruption (axotomy), the rate of outgrowth corresponds to the rate of SCb-the fastest subcomponent of SC. Both axonal outgrowth and SCb accelerate 20-25% after axotomy. Tubulin and actin are the major proteins being carried by SCb. To further characterize the acceleration of SCb, we measured the equilibrium between subunits and polymers for both actin and tubulin. We radiolabeled newly synthesized proteins in rat motor neurons by microinjecting [35S]methionine into the spinal cord 7 days after crushing the sciatic nerve (85 mm from the spinal cord). Nerves were removed 7 days later for homogenization in polymer-stabilizing buffer (PSB) and centrifugation, followed by SDS-PAGE of supernatants (S) and pellets (P). We removed beta-tubulin, actin, and the medium-weight neurofilament protein (NF-M) from each gel by using the fluorogram as a template. After solubilizing gel segments for liquid scintillation spectrometry, we expressed counts as a polymerization ratio: P/[S+P]. In the nerve segments that contained radiolabeled Scb proteins, located 24-36 mm from the spinal cord, axotomy increased the polymerization ratio of SCb actin from 0.23 to 0.36 (P < 0.05) but had no effect on SCb beta-tubulin. In a separate experiment, we added 12 microM taxol to PSB to stabilize newly assembled microtubules. Adding taxol did not alter the polymerization ratio for SCb beta-tubulin in sham-axotomized nerves but aid increase the ratio in axotomized nerves, from 0.44 to 0.63 (P < 0.05); polymerization ratios for SCb actin were unaffected. We conclude that the assembly of microfilaments and microtubules increases to provide cytoskeletal elements for axon sprouts. The resulting loss of actin and tubulin subunits may play a role in the acceleration of SCb.