Cytotypic differences in the protein composition of the axonally transported cytoskeleton in mammalian neurons

Abstract

Many of the structural and functional differences between axons are thought to reflect underlying differences in the biochemical composition and dynamic aspects of the axonal cytoskeleton and cytomatrix. In this study we investigated how the composition of the 2 slow components of axonal transport, SCa and SCb, which convey the cytoskeleton and cytomatrix, differs in axons that are structurally and functionally distinct. For this comparison we analyzed axons of retinal ganglion cells in the optic nerve (ON), axons of dorsal root ganglion (DRG) cells, and axons of ventral motor neurons (VMN) in adult rats. 35S-Methionine-labeled proteins transported with the peak of SCa and SCb were analyzed using high-resolution 2-dimensional polyacrylamide gels (2D-PAGE) and fluorography, and the amounts of major SCa and SCb proteins were quantified. The polypeptide composition of both SCa and SCb was found to be largely similar in DRG and VMN axons, but major qualitative as well as quantitative differences between these axons and ON axons were found. Notable among these were higher ratios of neurofilament protein to tubulin in SCa in DRG and VMN axons compared to ON axons, and significantly larger amounts of 2 microtubule-associated proteins relative to tubulin in SCa of ON axons than in both VMN and DRG axons. Tubulin was the major SCb protein in VMN and DRG axons, but it was not present in SCb in ON axons. Additionally, relatively larger amounts of 2 metabolic enzymes, creatine phosphokinase and nerve-specific enolase, were present in SCb in ON axons than in DRG or VMN axons. The results indicate that significant biochemical heterogeneity among different types of axons can be identified by examining the slow components of axonal transport.

Fast axonal transport of kinesin in the rat visual system: functionality of kinesin heavy chain isoforms.

“…These values were added together and defined as the total amount of radiolabeled kinesin present in the homogenate. McQuarrie et al, 1986;Oblinger et al, 1987) The absolute mass of kinesin in the optic nerve of several animals was determined to insure that differences in the amount of radiolabeled kinesin at different time points post-labeling were due to differences in specific activity, not to differences in the amount of kinesin present in the axons of different animals. Optic nerves from two non-radiolabeled rats were harvested and homogenized in lysis buffer.…”

“…Rates of movement for different subclasses of membrane bounded organelles and cytoskeletal structures through the axon are known (Willard et al, 1974;McQuarrie et al, 1986;Oblinger et al, 1987). If rate(s) of movement for kinesin in the axon can be determined, then roles for this mechanochemical enzyme in the movement of specific types of axonal structures can be assessed.…”

Production of native creatine kinase B in insect cells using a baculovirus expression vector

“…The peptide immunogen BW-17 (Ser-Asn-Ser-His-Asn-AlaLeu-Lys-Leu-Arg-Phe-Pro-Ala-Glu-Asp-Glu-Phe) corre sponding to a unique stretch o f amino acids (residues [4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] from the N-terminal end of the human B-CK was produced by standard solid phase synthesis [24], The purity of the peptide was assessed by determination of its amino acid com position and by routine HPLC analysis. Next, activated BW-17 peptide was coupled to BSA using MHS as described (25), yielding a ratio o f 16 moles of peptide per mole of BSA.…”

“…The B-CK subunit forms an especially interest ing study object in this respect. Not only are transcriptional and translational principles involved in its expression, also the modulation of enzymatic activity by protein modifica tion [phosphorylation; 4] and non-covalent protein and membrane associations [5,6] do play a role. B-CK studies may therefore form a paradigm for the study of other CKisoforms or enzymes with a similar role in the ATP regula tory networks.…”

Partial sequence of the rat heavy neurofilament polypeptide (NF‐H) Identification of putative phosphorylation sites

“…70 kDa (NF-L), 160 kDa (NF-M) and 200 kDa (NF-H) and are involved in the maintenance of neuronal caliber [1]. Phosphorylation probably plays a major role in the functioning of the two larger neurofilament polypeptides (NF-M and NF-H), the levels of phosphorylation being altered developmentally and coincident with a change in neurofilament function [2,3]. Both NF-M and NF-H contain a heavily phosphorylated carboxy region which may influence the formation of filament-filament axonal cross bridges [4,5].…”