THE NUCLEUS OF HELA-CELL CONTAINS TUBULAR STRUCTURES FOR CA2+ SIGNALING

It has long been assumed that Ca2+ are translocated from the cytosol t o the cell nucleus by a long distance to activate transcription machin ery buried deep in the nucleoplasm. However, this model has been recen tly challenged. When HeLa cells were loaded with fluo-3, highly fluore scent spots of similar to 2 mu m in diameter were observed in the cell nucleus while the fluo-3 signals were low in their neighbouring nucle oplasm as determined by confocal microscopy. These fluorescent spots w ere devoid of but usually associated with chromatin on their boundary. When cells were stimulated by ionomycin (1 mu M), the fluo-3 fluoresc ence in these spots increased faster than that in their neighbouring n ucleoplasm. In another experiment, optical sections with hot spot(s) w ere used to construct 3-D images to study the morphology of the hot sp ots. Views of reconstruction from different angles indicated that the hot spots formed a tubular structure with a connection to the nucleocy toplasmic interface. Moreover, injection of calcium green-dextran (70 kDa), a Ca2+-sensitive indicator conjugated with an inert molecule of large molecular size, into the cytosol leads to a formation of signals also in a tubular shape inside the nucleoplasm. This suggests that th e (channels' are real inside the nucleus and they are derived from an invagination of the double-membraned nuclear envelope. Taken together, our results indicate (1) tubular structures are found inside the cell nucleus; (2) they are extended horn the cytosol into the nucleus thro ugh the invagination of the double membraned nuclear envelope; (3) mol ecules of molecular size up to 70 kDa could penetrate into these 'tunn els'; (4) Ca2+ can be released or transported into the cell nucleus th rough these tubular structures after ionomycin stimulation; and (5) th e structures are usually associated with chromatin. (C) 1998 Academic Press.