Characterization of intermediate filaments in PC12 cells

Abstract

A 57 kDa protein is the major polypeptide in intermediate filament (IF)-enriched cytoskeletal preparations obtained from the neuronal cell line PC12 (rat pheochromocytoma). Under the conditions used to assemble IF in vitro from other cultured cell lines, 10 nm filaments are formed after 2 cycles of disassembly-assembly from PC12 IF-enriched cytoskeletal preparations; the 57 kDa protein is the major component of the final IF pellet. The 57 kDa protein is immunologically related to the BHK-21 fibroblast 55 kDa protein (vimentin), but a comparison of the peptide maps of PC12 57 kDa and BHK 55 kDa indicates that they are different proteins. With the use of a polyclonal antiserum to the PC12 57 kDa protein, immunofluorescence observations of PC12 cells not treated with NGF reveal a juxtanuclear "knot"-like structure. After NGF treatment, the "knots" are less prominent and many IF arrays are seen coursing through the cytoplasm and extending into the neurites. These immunofluorescence observations of the distribution of IF are corroborated by fine-structural analyses. SDS-PAGE analyses indicate that IF-enriched cytoskeletons isolated from NGF-treated cells have a polypeptide composition similar to that of untreated cells, that is, the 57 kDa protein remains the major polypeptide. SDS-PAGE and immunoblotting analyses show that untreated and NGF-treated PC12 cells also contain relatively minor amounts of the 68, 150, and 200 kDa neurofilament triplet (NFT) proteins. Under immunofluorescence, only 5% of untreated PC12 cells are found to contain a juxtanuclear "knot" labeled with NFT antibodies, but with time following NGF treatment, the number of fluorescent cells increases. After about 2 weeks of NGF treatment, all of the PC12 cells appear to contain NFT antibody-positive filamentous structures. As assessed by immunofluorescence, the NFT polypeptides appear to codistribute with the 57 kDa protein in both untreated and NGF-treated PC12 cells. These data indicate that PC12 cells contain IF composed of a complex set of polypeptides, including a previously unidentified 57 kDa IF protein. While NGF may induce production of NFT polypeptides, there does not appear to be a "switch" from known mesenchymal IF polypeptide expression to NFT polypeptide expression upon stimulation of PC12 cells with NGF.