Fig. 6. Analysis of cells expressing GFP-tagged modified SFAs. (A) Sequence of the C-terminal domain of SFA. Underlined residues are truncated in C18; the deletion in C6 is double underlined. Lower rows show the point mutations used to modify the penultimate serine. (B) Phase contrast and fluorescent image of strain C18-11 cells fixed with -20°C methanol. The GFP-tagged protein was concentrated near the basal bodies. Bar, 10 µm. (C) Western blot analysis of strains expressing truncated or mutated SFA molecules using anti-SFA. Upper panel: distribution of SFA, and GFP-tagged SFA or SFAC18 in control (C), GFP8 and C18-11 cells. Note the reduced amounts of SFA in GFP8 and 18-11. In the latter, most of the GFP-tagged protein remained in the supernatant, whereas full-length SFA-GFP was mostly insoluble. The amounts loaded in each lane corresponded to 3.6x10⁵ cells (whole cell), 1.8x10⁶ cells (pellets) and 3.3x10⁵ cells (supernatants). Lower panel: comparison of pellets and supernatants from GFP8, SE12 and SA11. Note that most of the SFA-GFP-SE was soluble, whereas SFA-GFP-SA was highly insoluble. Similar amounts of pellets and supernatants were loaded. (D) Fluorescence images of strain SA11 cultivated at 15°C before and immediately after a heat shock of 60 minutes. Note the star-like arranged SFA fibers and the dotted signals after heat shock. Bar, 10 µm.