Associations between intermediate filament proteins expressed in cultured dorsal root ganglion neurons.
The developmental profile of the neurofilament (NF) triplet proteins, alpha-internexin and peripherin in cultured dorsal root ganglion neurons from gestation day 15 rat embryos was determined by Western blot analysis. At the outset (day 0 in culture), the neurons contained mostly alpha-internexin. A significant increase in peripherin levels was seen at days 1-2, in the midsized (NFM) and low molecular weight (NFL) NF subunits at days 2-3, and in the high molecular weight (NFH) NF subunit at days 5-6. Immunofluorescence microscopy showed that the five intermediate filament proteins were co-localized in all neuronal cell bodies and neurites. Analysis of Triton X-100 extracts from okadaic acid-treated dorsal root ganglion cultures revealed that peripherin and alpha-internexin followed the same fragmentation pattern observed with NFs. Interactions between the various neuronal intermediate filament proteins in these extracts were assessed by immunoprecipitation under native conditions using antibodies specific for the individual proteins. Co-immunoprecipitation of NFH with NFL, NFM with NFL, NFM with alpha-internexin, and alpha-internexin with peripherin demonstrated that the intermediate filament cytoskeleton in cultured sensory neurons is a highly integrated structure.
Athlan
E S
ES
Department of Biochemistry, McGill University, Montreal, Quebec, Canada.
Sacher
M G
MG
Mushynski
W E
WE
D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D015153:Blotting, Western; D002352:Carrier Proteins / Q000032:analysis / Q000276:immunology / Q000378:metabolism; D002478:Cells, Cultured / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005136:Eye Proteins / Q000032:analysis / Q000276:immunology / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D005727:Ganglia, Spinal / Q000166:cytology; D007381:Intermediate Filament Proteins / Q000032:analysis / Q000276:immunology / Q000378:metabolism*; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D016900:Neurofilament Proteins / Q000032:analysis / Q000276:immunology / Q000378:metabolism; D009474:Neurons / Q000737:chemistry / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D017830:Octoxynol; D019319:Okadaic Acid / Q000494:pharmacology; D064531:Peripherins; D011233:Precipitin Tests; D051381:Rats
D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D005136:Eye Proteins; D007381:Intermediate Filament Proteins; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D016900:Neurofilament Proteins; D064531:Peripherins; C046643:alpha-internexin; C044882:neurofilament protein L; C051774:neurofilament protein H; C061195:neurofilament protein M; D019319:Okadaic Acid; D017830:Octoxynol