Ultrastructural study of depolarization-induced translocation of NRF-2 transcription factor in cultured rat visual cortical neurons.
Nuclear respiratory factor (NRF)-2 or GA-binding protein is a potential transcriptional, bigenomic coordinator of mitochondrial and nuclear-encoded subunits of cytochrome oxidase genes. It is composed of an alpha subunit that binds DNA and a beta subunit that has the transactivating domain. Previously, we found that the level of NRF-2 paralleled that of cytochrome oxidase under normal and functionally altered states. The goal of our present study was to increase the resolution to the ultrastructural level and to quantify changes before and after depolarizing stimulation. We used a pre-embedding immunogold-silver method for the two subunits of NRF-2 in cultured rat visual cortical neurons. NRF-2alpha and beta were normally located in both the nucleus and the cytoplasm. In the nucleus, both subunits were associated primarily with euchromatin rather than heterochromatin, consistent with active involvement in transcription. In the cytoplasm, they were associated mainly with free ribosomes and occasionally with the Golgi apparatus and the outer membrane of the nuclear envelope. Labelling was not found in the mitochondria, confirming the specificity of the antibodies. Neuronal depolarization by KCl for 5 h induced a six- to seven-fold increase in the nuclear-to-cytoplasmic ratio of both subunits (P < 0.001) without increases in total labelling densities. These results strongly indicate that both NRF-2alpha and NRF-2beta respond to increased neuronal activity by translocating from the cytoplasm to the nucleus, where they engage in transcriptional activation of target genes. Our results also indicate that the cytoplasmic to nuclear movement of transcription factors is a dynamic process induced by neuronal activity.
Yang
Shou Jing
SJ
Department of Cell Biology, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA.
Liang
Huan Ling
HL
Ning
Gang
G
Wong-Riley
Margaret T T
MT
D021581:Active Transport, Cell Nucleus / Q000502:physiology; D000818:Animals; D000831:Animals, Newborn; D002467:Cell Nucleus / Q000737:chemistry / Q000187:drug effects / Q000648:ultrastructure; D002478:Cells, Cultured; D003593:Cytoplasm / Q000737:chemistry / Q000187:drug effects / Q000648:ultrastructure; D004268:DNA-Binding Proteins / Q000032:analysis / Q000378:metabolism* / Q000648:ultrastructure*; D051268:GA-Binding Protein Transcription Factor; D008564:Membrane Potentials / Q000502:physiology; D009474:Neurons / Q000737:chemistry / Q000187:drug effects / Q000648:ultrastructure*; D011189:Potassium Chloride / Q000494:pharmacology; D051381:Rats; D014157:Transcription Factors / Q000032:analysis / Q000378:metabolism* / Q000648:ultrastructure*; D014793:Visual Cortex / Q000737:chemistry / Q000187:drug effects / Q000648:ultrastructure*
D004268:DNA-Binding Proteins; D051268:GA-Binding Protein Transcription Factor; C495649:Gabpa protein, rat; C495653:Gabpb2 protein, rat; D014157:Transcription Factors; D011189:Potassium Chloride