Satellite Cells in Skeletal Muscle of the Hibernating Dormouse, a Natural Model of Quiescence and Re-Activation: Focus on the Cell Nucleus

Satellite cells (SCs) participate in skeletal muscle plasticity/regeneration. Activation of SCs implies that nuclear changes underpin a new functional status. In hibernating mammals, periods of reduced metabolic activity alternate with arousals and resumption of bodily functions, thereby leading to repeated cell deactivation and reactivation. In hibernation, muscle fibers are preserved despite long periods of immobilization. The structural and functional characteristics of SC nuclei during hibernation have not been investigated yet. Using ultrastructural and immunocytochemical analysis, we found that the SCs of the hibernating edible dormouse, Glis glis, did not show apoptosis or necrosis. Moreover, their nuclei were typical of quiescent cells, showing similar amounts and distributions of heterochromatin, pre-mRNA transcription and processing factors, as well as paired box protein 7 (Pax7) and the myogenic differentiation transcription factor D (MyoD), as in euthermia. However, the finding of accumulated perichromatin granules (i.e., sites of storage/transport of spliced pre-mRNA) in SC nuclei of hibernating dormice suggested slowing down of the nucleus-to-cytoplasm transport. We conclude that during hibernation, SC nuclei maintain similar transcription and splicing activity as in euthermia, indicating an unmodified status during immobilization and hypometabolism. Skeletal muscle preservation during hibernation is presumably not due to SC activation, but rather to the maintenance of some functional activity in myofibers that is able to counteract muscle wasting.

Structure and dynamics of hnRNP-labelled nuclear bodies induced by stress treatments

We have previously described HAP, a novel hnRNP protein that is identical both to SAF-B, a component of the nuclear scaffold, and to HET, a transcriptional regulator of the gene for heat shock protein 27. After heat shock, HAP is recruited to a few nuclear bodies. Here we report the characterisation of these bodies, which are distinct from other nuclear components such as coiled bodies and speckles. The formation of HAP bodies is part of a general cell response to stress agents, such as heat shock and cadmium sulfate, which also affect the distribution of hnRNP protein M. Electron microscopy demonstrates that in untreated cells, similar to other hnRNP proteins, HAP is associated to perichromatin fibrils. Instead, in heat shocked cells the protein is preferentially associated to clusters of perichromatin granules, which correspond to the HAP bodies observed in confocal microscopy. Inside such clusters, perichromatin granules eventually merge into a highly packaged ‘core’. HAP and hnRNP M mark different districts of these structures. HAP is associated to perichromatin granules surrounding the core, while hnRNP M is mostly detected within the core. BrU incorporation experiments demonstrate that no transcription occurs within the stress-induced clusters of perichromatin granules, which are depots for RNAs synthesised both before and after heat shock.

Nuclear RNA Is Extruded from Apoptotic Cells

During spontaneous apoptosis of thymocytes there is extrusion of ribonucleoproteins (RNPs) from the cell. The aim of this investigation was to elucidate whether the RNP aggregates in apoptotic cells and bodies still contain RNA in an appreciable amount. We demonstrated by specific cytochemical techniques that the aggregates of nuclear RNPs extruded in the cytoplasm of spontaneously apoptotic thymocytes contain RNA in a sufficient amount to be detected cytochemically. These heterogeneous ectopic RNP-derived structures (HERDS) are formed by perichromatin fibrils, interchromatin granules, perichromatin granules, and nucleolar material. The RNA detected inside these clusters should therefore correspond to both mRNA and snRNA as well as to rRNA. We never observed DNA-contaning aggregates in the cytoplasm of apoptotic thymocytes. The presence of RNA in the HERDS that may be released from apoptotic cells suggests that the decrease in the amount of total RNA during apoptosis may be mostly linked to cellular extrusion rather than to degradation of RNA by RNase activities. Another interesting aspect of these results lies in the hypothesis of apoptosis as a possible cause for the presence of autoantibodies in the serum of patients with systemic autoimmune diseases.

Fine Structural Specific Visualization of RNA on Ultrathin Sections

We describe a new technique that allows specific visualization of RNA at the electron microscopic level by means of terbium citrate. Under the conditions presented here, terbium binds selectively to RNA and stains nucleoli, interchromatin granules, perichromatin fibrils, perichromatin granules, and coiled bodies in the cell nucleus, whereas ribosomes are the only contrasted structures in the cytoplasm. All the cell components contrasted by terbium are known to contain RNA. When ultrathin sections are pretreated with RNase A or nuclease S1 (specific for single-stranded nucleic acids), staining does not occur. Neither DNase nor pronase influences the reaction. We conclude that terbium staining is selective for RNA and especially for single-stranded RNA. The staining can be performed on thin sections of material embedded both in epoxy and in acrylic resins. The technique is not influenced by the aldehyde fixative used and can also be utilized after immunolabeling. The endproduct is very fine and, although weak in contrast, is suitable for high-resolution observations.

Use of human autoantibodies and immunoelectron microscopy to study structure and function of the nucleolus and nuclear bodies

By conventional electron microscopy, the formed elements of the nuclear interior include the nucleolus, chromatin, interchromatin granules, perichromatin granules, perichromatin fibrils, and various types of nuclear bodies (Figs. 1a-c). Of these structures, all have been reasonably well characterized structurally and functionally except for nuclear bodies. The most common types of nuclear bodies are simple nuclear bodies and coiled bodies (Figs. 1a,c). Since nuclear bodies are small in size (0.2-1.0 μm in diameter) and infrequent in number, they are often overlooked or simply not observed in any random thin section. The rat liver hepatocyte in Fig. 1b is a case in point. Historically, nuclear bodies are more prominent in hyperactive cells, they often occur in proximity to nucleoli (Fig. 1c), and sometimes they are observed to “bud off” from the nucleolar surface.