Developmental appearance of phosphorylated intermediate filaments in nuclei of glioma and neuroblastoma cells
Date
2002-05
Authors
Weigum, Shannon E.
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Abstract
The structure and function of the proteinaceous skeleton in the nucleus of eukaryotic cells, termed the nucleoskeleton, is only vaguely known. Investigations into the role of intermediate filaments (IF) in the nucleus have primarily focused upon lamins, however some studies have suggested that there may be a role for non-lamin intermediate filaments in the nucleoskeleton. Recent studies of glioma and neuroblastoma cell lines have suggested that phosphorylated derivatives of IFs normally found in the cytoplasm can be demonstrated within nuclei. Additional reports indicate that IF phosphorylation may play an important role in regulating the dynamic organization of the intermediate filament network. In this study, the possibility that cytoplasmic phosphorylated IFs, recognized by Jl-31 and SMI-31 mABs, can be found in the nuclei of F98 glioma and SH-SY5Y neuroblastoma cells, respectively, under conditions of cell growth or differentiation was investigated. I identified the growth stages of cells grown in culture by determining growth rates as a function of cell density, and I identified actively proliferating cells by tracking DNA synthesis using uptake of 5'-bromo 2'-deoxyuridine. Using immunocytochemistry, Jl-31 and SMI-31 mAB nuclear labeling was exhibited primarily in proliferating cells with diminished nuclear labeling in quiescent cells found at high densities. SDS-PAGE and western blot analysis on whole cell lysates of F98 and SH-SY5Y cells were used to identify Jl-31 and SMI-31 antigens. The Jl-31 mAB appears to recognize a high molecular weight form of GFAP that contains phosphorylated serine residues in F98 glioma cells. SMI-31 mAB recognizes NF-H and NF-M peptide along with additional proteins in SH-SY5Y cells, but it was unclear if these proteins contained phosphorylated serine residues.
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Keywords
eukaryotic cells, cell nuclei, cytoplasmic filaments, phosphorylation
Citation
Weigum, S. E. (2002). Developmental appearance of phosphorylated intermediate filaments in nuclei of glioma and neuroblastoma cells (Unpublished thesis). Southwest Texas State University, San Marcos, Texas.