FIGURE 3.

Localization of CinY2 and CinW2 in T. pseudonana biosilica. A, promoter-dependent location of cingulin-GFP fusion proteins expressed in T. pseudonana. Shown are confocal fluorescence microscopy images of live cells (girdle band view) at different stages of the cell cycle expressing CinY2 and CinW2 under control of the nitrate reductase promoter/terminator cassette (Pnr) or their native promoters Pciny2 and Pcinw2, respectively. Green, location of the cingulin-GFP fusion proteins; red, chloroplast autofluorescence. For orientation, the middle column shows schematic images of cross-sections of T. pseudonana cells at the respective cell cycle stages: silica (black symbols), plasma membranes (blue lines), and cytosol (gray). For clarity, intracellular organelles were omitted in the schematic drawings. B, accessibility of organic microrings in T. pseudonana biosilica. Biosilica and insoluble organic matrices were isolated from transformants expressing CinY2-GFP or CinW2-GFP under control of their native promoters. The biosilica and the organic matrices were subjected to immunolabeling using anti-GFP as primary antibody and an Alexa647-labeled secondary antibody. GFP and Alexa647 fluorescence was quantified in individual biosilica and organic microring particles. The right column shows the overlays of the images from the other three columns. Scale bars, 2 μm.