Redistribution of GFAP and alphaB-crystallin after thermal stress in C6 glioma cell line.
Some intermediate filament (IF) proteins expressed in the development of glia include nestin, vimentin, and glial fibrillary acidic protein (GFAP). However, GFAP is the major intermediate filament protein of mature astrocytes. To determine the organization of GFAP in glial cells, rat GFAP cDNA tagged with enhanced green fluorescent protein (EGFP) was transfected into the rat C6 glioma cell line. After selection, two stable C6-EGFP-GFAP cell lines were established. Stable C6-EGFP-GFAP cell lines with or without heat shock treatment were analyzed by immunocytochemistry, electron microscopy, and Western blot analysis. In the transient transfection study, EGFP-GFAP transiently expressed in C6 cells formed punctate aggregations in the cytoplasm right after transfection, but gradually a filamentous structure of EGFP-GFAP was observed. The protein level of nestin in the C6-EGFP-GFAP stable clone was similar to that in the pEGFP-C1 transfected C6 stable clones and non-transfected C6 cells, whereas the level of vimentin was reduced in Western blotting. Interestingly, the expression level of small heat shock protein alphaB-crystallin in C6-EGFP-GFAP cells was also enhanced after transfection. Immunostaining patterns of C6-EGFP-GFAP cells showed that GFAP was dispersed as a fine filamentous structure. However, after heat shock treatment, GFAP formed IF bundles in C6-EGFP-GFAP cells. In the meantime, alphaB-crystallin also colocalized with IF bundles of GFAP in C6-EGFP-GFAP cells. The heat-induced GFAP reorganization we found suggested that small heat shock protein alphaB-crystallin may play a functional role regulating the cytoarchitecture of GFAP.
Tseng
Wei-Chia
WC
Department of Anatomy and Cell Biology, College of Medicine, National Taiwan University, No. 1, Section 1, Jen-Ai Road, Taipei, 100, Taiwan.
Lu
Kuo-Shyan
KS
Lee
Wen-Ching
WC
Chien
Chung-Liang
CL
D000818:Animals; D018929:Cell Culture Techniques; D045744:Cell Line, Tumor; D015870:Gene Expression; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D050888:Heat-Shock Proteins, Small / Q000378:metabolism*; D006358:Hot Temperature / Q000009:adverse effects; D007150:Immunohistochemistry; D007382:Intermediate Filaments / Q000378:metabolism*; D009457:Neuroglia / Q000378:metabolism* / Q000648:ultrastructure; D051381:Rats; D038203:alpha-Crystallin B Chain / Q000378:metabolism*
D005904:Glial Fibrillary Acidic Protein; D050888:Heat-Shock Proteins, Small; D038203:alpha-Crystallin B Chain; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins