Figure 4.

H3::GFP localized to both nuclei in the transformed SvH3G strain. Nuclear localization of H3::GFP and comparative immunolocalization of the microtubule cytoskeleton in S. vortens trophozoites (DAPI, blue; H3::GFP, green; anti-α-tubulin, red). UPPER PANELS: Panel (a) shows the GFP fluorescence of a field of live S. vortens cells. Panels (b-d) show a different field of fixed S. vortens cells, with (b) marking the DAPI-stained nuclei of the cells, (c) marking the nuclear GFP localization of the cells, and (d) showing the merged image of panels (b) and (c). The upper panels further illustrate that the H3::GFP transgene is expressed in a high proportion of cells (b-d). LOWER PANELS: Panels (e-h) show the same fixed S. vortens cell, with (e) marking the DAPI-stained nuclei, (f) marking the GFP localization, (g) marking the microtubule cytoskeleton, and (h) showing a merged image of panels (e-g). In the lower panels, staining of a single cell with anti-α-tubulin immunostaining (g, h in red) labels the eight axonemes and the lateral ridges of the microtubule cytoskeleton. The H3::GFP expressed from the native promoter localizes to both nuclei in both live cells (a) and fixed cells (b-h), and the H3::GFP transgene co-localizes to the DAPI-stained nuclei (b-f), but not to the microtubule cytoskeleton as visualized by anti-α-tubulin immunostaining (g, h). Though H3::GFP co-localizes with DAPI, H3::GFP has a more punctate staining pattern. Images are representative of GFP localizations observed in over 250 cells per strain. Scale bar = 2 μm.