Stable and metastable cytoskeletal polymers carried by slow axonal transport

Abstract

The proteins carried by the slow axonal transport in the rat sciatic motor axons were radiolabeled by injecting 35S-methionine into the spinal cord, and the distribution of their solubility through the 2 main components of slow transport (SCa and SCb) was considered. For this purpose, a cytoskeleton-stabilizing buffer was designed in which a pellet enriched in macromolecular and polymeric structures was separated from the solubilized proteins. The monomer/polymer ratios for tubulin were quantified in the 2 rate components. Our results indicate that 90% of the total tubulin was carried with SCa. Of this, 75% was in a polymeric state, versus only 50% of the tubulin carried with SCb. The monomeric tubulin recovered in the soluble fraction was concomitantly transported with the polymerized microtubules, suggesting that it might represent metastable regions of these microtubules. The insoluble and soluble fractions of the transported actin were measured. Actin was mostly (70%) transported with SCb. Of this, more than 80% was recovered in the soluble fraction, but we cannot say whether it was in a monomeric or polymeric state, nor if it was transported free or bound to a structure solubilized during fractionation. The other 30% of the actin, most of it transported with SCa, was recovered in the polymer-enriched fraction, probably bound to a stabilized polymer, such as the microtubules.(ABSTRACT TRUNCATED AT 250 WORDS)

Defective axonal transport of neurofilament proteins in neurons overexpressing peripherin

“…However, the mechanism by which the IF aggregates occur in these animals is not clear. Previous investigations have shown that peripherin is mainly transported by SCa in the sciatic nerve (Filliatreau et al . 1988; Chadan et al .…”

“…The slow component a (SCa) carries NFs and microtubules at a rate of 0.1–1 mm/day. α‐Internexin and peripherin are co‐transported with NF triplets in axons (Filliatreau et al . 1988; Kaplan et al .…”

Aberrant neurofilament phosphorylation in sensory neurons of rats with diabetic neuropathy.

“…Measurement of protein levels using Western blotting. The L 4 and L 5 d o rsal root ganglia (DRG) (dorsal roots 1 cm adjacent to ganglion) and 2 cm distal segments of peroneal and sural nerve were taken and frozen in liquid nitrogen and homogenized (32). Briefly, samples were homogenized using a Polytron (Kinematica, Luzerne, Switzerland) in 0.1 mmol/l PIPES (pH 6.9), 5.0 mmol/l MgCl 2 , 5.0 mmol/l EGTA, 0.5% Triton X-100, 20% glycerol, 1.0 mmol/l phenylmethylsulfonyl fluoride, and a mixture of protease inhibitors (1.0 µg/ml pepstatin A, 1.0 µg/ml leupeptin, 10 µg/ml benzoyl-L-arginine methyl ester, 10 µg/ml pt o s y l -L-arginine methyl ester, 10 µg/ml soybean trypsin inhibitor, 10 µg/ml L-1 -t o s ylamide-2 phenylethylchloromethyl ketone, and 7 µg/ml aprotinin).…”

Enhancing intrinsic growth capacity promotes adult CNS regeneration

“…Soluble proteins such as unassembled actin, clathrin, tubulin, and enzymes of glycolysis are transported in SCb at a net rate of 3-6 mm/day in these axons [18,19]. The conditioning lesion changes the velocity of SCb and increases the unassembled tubulin transporting in SCb.…”