Nonlamin components of the lamina: a paucity of proteins.

Current models of nuclear organization propose that nuclear functions are modulated in part by reversible tethering of chromatin loops to structural elements of the nucleoplasm and the nuclear envelope. Lamins are the best-characterized proteins of the lamina portion of the nuclear envelope and are involved in binding chromatin to the inner nuclear membrane. However, they are not a universal feature of eukaryotic nuclei and do not account fully for the putative functions of the lamina in all organisms.

Splenic T lymphocytes die preferentially during heat-induced apoptosis: NuMA reorganization as a marker.

We are investigating nuclear events during apoptosis in mouse splenic lymphocytes cultured immediately after isolation (controls) or after heat treatment (42 degreesC, 30 minutes), and have found that hyperthermia increased the level of apoptosis to double that of spontaneous apoptosis in controls within 6 hours. Immunolabelling for Nuclear Mitotic Apparatus Protein (NuMA) suggested that splenocytes were responding heterogeneously to the heat treatment. Whereas all nuclei in controls and about half of nuclei in heat-treated samples showed the usual diffuse nucleoplasmic labelling, 40-60% of nuclei in heated samples also contained numerous bright spots.

Unique behaviour of NuMA during heat-induced apoptosis of lymphocytes.

Nuclear collapse is a key feature of apoptosis, reflecting the DNA and protein fragmentation observed biochemically. We have compared nuclear events during spontaneous and heat-induced (42 degrees C for 30 min) apoptosis at the level of individual cells, monitoring overall chromatin organization by staining with 4,6'-diamidino-2-phenylindole (DAPI), DNA cleavage by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling (TUNEL), and the nuclear antigens NuMA, PI2, and lamin B by immunofluorescence microscopy. The data were correlated with analyses at the population level by flow cytometry and immunoblotting.

Is DNA topoisomerase II beta a nucleolar protein?

We have carried out immunofluorescence labelling of two human cell types, HeLa cells and peripheral blood lymphocytes, prepared by several different fixation/permeabilization protocols using a variety of antibodies against DNA Topoisomerase II (Topo II). We have found that the distribution of Topo II alpha was overall similar doing interphase and mitosis to that previously reported, regardless of antibody and of sample preparation. On the other land, the interphase distribution of Topo II beta was quite variable, depending both on the antibody and on the method used to prepare the sample.

Organization of DNA topoisomerase II isotypes during the cell cycle of human lymphocytes and HeLa cells.

We have monitored the organization of DNA topoisomerase II (Topo II) in relation to chromatin disaggregation during mitogen stimulation of lymphocytes and to the mitotic chromosome condensation cycle by immunofluorescence microscopy with isozyme-specific antibodies. Labelling for both Topo II alpha and Topo II beta was diffusely nucleoplasmic and non-nucleolar in resting lymphocytes and the pattern changed little during stimulation. Topo II alpha labelling intensity increased in parallel with the extent of cell stimulation, but a fraction of fully stimulated cells was labelled very brightly.