Figure 4

Disruption of nuclear architecture by Rab24(D123I) inclusions. NIH 3T3 cells expressing mycRab24(D123I) were processed for immunofluorescence microscopy 24 h after transfection. (A) The inclusions detected by staining with the anti-myc monoclonal antibody appeared to occupy areas of the nucleus devoid of DAPI staining (arrows). In some cells (upper panels) the nucleus was well defined, and it was possible to distinguish between inclusions localized inside and outside the nucleus. In other cells (lower panels), the inclusions were more diffuse, the nucleus was distorted, and it was difficult to discern the boundaries between the nuclear and cytoplasmic compartments. (B) mycRab24(D123I) inclusions were localized with rabbit anti-myc polyclonal antibody followed by FITC-conjugated GAR IgG. The nuclear envelope was highlighted by co-staining with a monoclonal antibody against lamins A and B, followed by rhodamine-conjugated GAM IgG. In some cells with immunoreactive inclusions (upper panels) a well defined nuclear envelope was visible, whereas in others (bottom panels) the nuclear envelope appeared to be completely disrupted. Where both cytoplasmic and nuclear inclusions were present (top and middle panels), it appeared that the myc-reactive aggregates inside the nucleus contained lamins (arrows), while those outside the nucleus did not (arrowheads). For comparison, panel C shows the nuclear lamin staining typically observed in non-transfected cells. The scale bar represents 10 microns.