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

Abstract

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 β‐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 β‐tubulin. In a separate experiment, we added 12 μM taxol to PSB to stabilize newly assembled microtubules. Adding taxol did not alter the polymerization ratio for SCb β‐tubulin in sham‐axotomized nerves but did 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. © 1996 Wiley‐Liss, Inc.

The nerve conditioning lesion: A strategy to enhance nerve regeneration

“…In contrast, the fast transport pathway is not affected by conditioning . CLs augment the amount of tubulin and actin available to the developing growth cone and accelerate the SCb pathway by 20 to 25% . Axotomy accelerates the polymerization of actin and tubulin by 50% and 43%, respectively.…”

“…Axotomy accelerates the polymerization of actin and tubulin by 50% and 43%, respectively. This shift in the monomer‐to‐polymer ratio decreases the total number of molecules being transported, accelerating the SCb pathway and promoting intra‐axonal microtubule assembly at the axonal tip . Tyrosination of tubulin is similarly induced by conditioning, suggesting an overall dynamic shift of microtubule formation .…”

The Basis for Diminished Functional Recovery after Delayed Peripheral Nerve Repair

“…The molecular response of axotomized neurons with upregulation of actin, tubulin, GAP-43, and CAP-23, the expression of the neonatal isoform, ␣-tubulin, and the downregulation of neurofilament protein (Tetzlaff et al, 1988;Miller et al, 1989;Jacob and McQuarrie, 1996;Fu and Gordon, 1997;Bomze et al, 2001;Woolf, 2001;Mason et al, 2002) has been interpreted as evidence of a molecular switch from a transmitting to a growth mode, the genes being referred to as growth-associated genes (GAGs) (Gordon, 1983;Miller et al, 1989;Fu and Gordon, 1997). GAG expression is relatively short-lived, however, declining from a peak at 7 d to baseline levels within 6 months (You et al, 1997;McPhail et al, 2004).…”

Retrograde Axonal Degeneration (“Dieback”) in the Corticospinal Tract after Transection Injury of the Rat Spinal Cord: A Confocal Microscopy Study

“…One of the more prominent theories involves the increase in axoplasmic flow in axons following trauma (Jacob and McQuarrie, 1996;Kao et al, 1977;Kreutzberg, 1995). The constant pushing force produced by this flow is thought to be one of the main contributors to the tissue swelling and subsequent rupture that is characteristic of axon tips as they undergo dieback.…”