RESUMO
Nuclear tubules (NTs) were found in the nucleus of HeLa cells. Although no function has been ascribed to these structures, our previous data has shown that they are the sites of Ca(2+) release with mitochondria shuttled around. In the present study, we further characterized these NTs through different fluorescent dye-labeling and red fluorescent protein transfection experiments. We found that doxorubicin (Dox) is a good indicator to demonstrate the NTs since Dox is fluorescent and DNA is able to quench its fluorescence. By using confocal and electron microscopy, we show that the number and nature of the NTs in HeLa vary from cell to cell, ranging from tubular to intricately branched structures. Additionally, these NTs are double-membrane invaginations of the nuclear envelope and usually lie close to nucleolus. At rest, NTs appeared to be stable and their mouths are always closed. Upon Ca(2+) ionomycin stimulation, various forms of dynamism, including membrane protrusion to the nucleus, enlargement and shrinkage of the NTs, and distortion of the nuclear envelope and NTs were observed over a time scale of minutes. These observations suggest that the NT represents a specialized and dynamic compartment inside the nucleus under the control of Ca(2+).
Assuntos
Núcleo Celular/ultraestrutura , Células HeLa/ultraestrutura , Laranja de Acridina/farmacologia , Carbocianinas/farmacologia , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , DNA/efeitos dos fármacos , DNA/metabolismo , Doxorrubicina/farmacologia , Corantes Fluorescentes/farmacologia , Humanos , Ionomicina/farmacologia , Microscopia Confocal , Microscopia Eletrônica de TransmissãoRESUMO
Calcium is an important messenger that controls many nuclear functions such as gene expression in mammalian cells but the regulation of nuclear Ca(2+) remains unclear. It has long been thought that Ca(2+) is translocated from the cytosol by a long distance to the nucleus through the nuclear pore complexes to activate or suppress gene transcription. However, this model is at best an incomplete one. With an aid of confocal and transmission electron microscopy, we demonstrated here that tubules, in a vertical or horizontal orientation, extended deep inside the nucleus of HeLa cells. These nuclear tubules (NTs) are double-membraned invaginations of the nuclear envelope and are usually associated with nucleolus. Also, membrane bound vesicles are found inside and inositol 1,4,5 trisphosphate (IP(3)) receptors are enriched in some but not all of these tubular structures. Interestingly, shuttling of mitochondria was observed in the NT and cytoplasm of the HeLa cells loaded with dihydro-rhod-2/AM. After stimulation with histamine that increases cytosolic [Ca(2+)] through IP(3) production, a slow rise of dihydro-rhod-2 fluorescence for the measurement of intra-mitochondrial Ca(2+) was observed in the area of NT indicating that Ca(2+) was sequestered by mitochondria inside the tubular invagination. Our work therefore suggests that the NTs and mitochondrial activities represent a specialized compartment and dynamic process involved in the regulation of Ca(2+) inside the cell nucleus.