ASSEMBLY OF MICROFILAMENTS AND MICROTUBULES FROM AXONALLY TRANSPORTEDACTIN AND TUBULIN AFTER AXOTOMY

The slow component (SC) of axonal transport conveys structural protein s, regulatory proteins, and glycolytic enzymes toward the axon tip at 1-6 mm/day, Following axon interruption (axotomy), the rate of outgrow th 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 charact erize the acceleration of SCb, we measured the equilibrium between sub units and polymers for both actin and tubulin, We radiolabeled newly s ynthesized proteins in rat motor neurons by microinjecting [S-35]methi onine into the spinal cord 7 days after crushing the sciatic nerve (85 mm from the spinal cord), Nerves were removed 7 days later for homoge nization in polymer-stabilizing buffer (PSB) and centrifugation, follo wed by SDS-PAGE of supernatants (S) and pellets (P), We removed beta-t ubulin, actin, and the medium-weight neurofilament protein (NF-M) from each gel by using the fluorogram as a template, After solubilizing ge l segments for liquid scintillation spectrometry, we expressed counts as a polymerization ratio: P/[S + P]. In the nerve segments that conta ined 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 e xperiment, we added 12 mu M 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 did increase the ratio in axotomized nerves, from 0.44 to 0.63 (P < 0.05); polymerization rat ios for SCb actin were unaffected, We conclude that the assembly of mi crofilaments and microtubules increases to provide cytoskeletal elemen ts for axon sprouts, The resulting loss of actin and tubulin subunits may play a role in the acceleration of SCb. (C) 1996 Wiley-Liss, Inc.