ABSTRACT
The precise localization of transcribed DNA and resulting RNA is an important aspect of the functional architecture of the nucleus. To this end we have developed a novel in situ hybridization approach in combination with immunoelectron microscopy, using sense and anti-sense RNA probes that are derived from total cellular or cytoplasmic poly(A+) RNA. This new technology is much more gentle than classical in situ hybridization using DNA probes and shows excellent preservation of nuclear structure. Carried out on ultrathin sections of fixed and resin-embedded COS-7 cells, it revealed at high resolution the localization of the genes that code for the cellular mRNAs. Quantitative analysis shows that most transcribed DNA is concentrated in the perichromatin region, i.e. the interface between subchromosomal compact chromatin domains and the interchromatin space essentially devoid of DNA. The RNA that is produced is found mainly in the perichromatin region and the interchromatin space. These results imply that in the mammalian nucleus the chromatin fiber is folded so that active genes are predominantly present in the perichromatin region, which is the most prominent site of transcription.
Subject(s)
Cell Nucleus/chemistry , Chromatin/chemistry , DNA/analysis , Transcription, Genetic , Animals , COS Cells , Chlorocebus aethiops , In Situ Hybridization/methods , RNA/biosynthesis , RNA ProbesABSTRACT
Growth hormone (GH) is a signaling molecule regulating cell proliferation, differentiation and metabolism via activation of specific cell surface receptors and subsequent triggering of signal transduction pathways. This is associated with GH/GH receptor internalization and accumulation of GH in several subcellular compartments, including mitochondria. To assess the functional relevance of such mitochondrial accumulation, we first confirmed the occurrence of mitochondrial GH uptake ex vivo as early as 10 min after (125)I-GH injection to the rats. We next showed that intact (125)I-GH accumulates in mitochondrial fractions in vitro in a specific, rapid and saturable manner with an apparent affinity (K(d)) of 1.44 nM. At the electron-microscopic level, immunoreactive GH density within mitochondria increased after in vitro hormone incubation, without any modification of the sub-mitochondrial distribution pattern. The presence of GH in the inter-membrane space and at the inner membrane seen by electron microscopy was confirmed by SDS-PAGE and autoradiography after mitochondrial fractioning thus suggesting the involvement of GH in the respiration control. To test this hypothesis further, we performed polarographic and spectrophotometric assays on isolated mitochondria. These assays pointed to a direct, selective and dose-dependent effect of GH on the inhibition of succinate dehydrogenase and cytochrome C oxidase activities. The latter inhibition was in contrast with indirect, GH receptor-initiated stimulation of cytochrome C oxidase activity observed in GH-treated whole BRL cells transfected to express this receptor. Altogether, these data show that GH is specifically imported in mitochondria, where it operates a direct metabolic effect, independently of cell surface receptors and signal transduction.
Subject(s)
Electron Transport/physiology , Growth Hormone/metabolism , Mitochondria, Liver/metabolism , Animals , Cell Line , Electron Transport Complex IV/metabolism , Iodine Radioisotopes , Liver/metabolism , Liver/ultrastructure , Male , Mitochondria, Liver/ultrastructure , Mitochondrial Membranes/metabolism , Mitochondrial Membranes/ultrastructure , Rats , Rats, Wistar , Receptors, Somatotropin/metabolism , Succinate Dehydrogenase/metabolism , Time FactorsABSTRACT
Growth hormone (GH) binding to its receptor (GHR) initiates GH-dependent signal transduction and internalization pathways to generate the biological effects. The precise role and way of action of GH on mitochondrial function are not yet fully understood. We show here that GH can stimulate cellular oxygen consumption in CHO cells transfected with cDNA coding for the full-length GHR. By using different GHR cDNA constructs, we succeeded in determining the different parts of the GHR implicated in the mitochondrial response to GH. Polarography and two-photon excitation fluorescence microscopy analysis showed that the Box 1 of the GHR intracellular domain was required for an activation of the mitochondrial respiration in response to a GH exposure. However, confocal laser scanning microscopy demonstrated that cells lacking the GHR Box 1 could efficiently internalize the hormone. We demonstrated that internalization mediated either by clathrin-coated pits or by caveolae was able to regulate GH mitochondrial effect: these two pathways are both essential to obtain the GH stimulatory action on mitochondrial function. Moreover, electron microscopic and biochemical approaches allowed us to identify the caveolar pathway as essential for targeting GH and GHR to mitochondria.