Journal: mBio

Article Title: Differential stability of Gcn4p controls its cell-specific activity in differentiated yeast colonies

doi: 10.1128/mbio.00689-24

Figure Lengend Snippet: Localization of constitutively overproduced Gcn4p-GFP in colonies. ( A ) Growth and U/L cell differentiation of colonies of p TEF -Gcn4p strains. Left: Giant colonies of wt-Gcn4p-GFP, wt-p TEF -Gcn4p-GFP, and wt-p TEF -Gcn4p-HA strains in acidic and alkaline phases of development grown on GMA-BCP plates. Right: Vertical cross-section of a 4-day-old microcolony in the alkaline phase of the wt-p TEF -Gcn4p-GFP strain and wt strain BY4742 (as control). Shown are the correct U/L cell differentiation and the morphology of U and L cells visualized by DIC. Green bar, U cells; red bar, L cells. ( B and C ) Cellular localization of Gcn4p-GFP in colonies of the wt-p TEF -Gcn4p-GFP and gcn2 -p TEF -Gcn4p-GFP strain in acidic (left panels, orange bar) and alkaline (right panels, purple bar) phases of their development. The areas of colonies in the acidic phase (orange rectangles; 1, upper and 2, lower cells) and the alkaline phase (purple rectangle; 1, U and 2, L cells) are shown at higher magnification. Yellow bar, 10 μm; turquoise arrows show examples of nuclear localization. Large vacuoles without GFP fluorescence are visible in L cells (examples indicated by yellow arrows). Representative results of three independent biological experiments are shown. A comparison of Gcn4p-GFP fluorescence with background fluorescence is shown in Fig. S4. ( D ) Nuclear and cytosolic localization of Gcn4p-GFP in U and L cells from 4-day-old microcolonies of the wt-p TEF -Gcn4p-GFP strain. Cells with Gcn4p-GFP were stained with DAPI to visualize the position of the nucleus. The DIC image shows the morphology of the U and L cells. White bar, 10 μm.

Article Snippet: Cells were observed using a Carl Zeiss Axio Observer.Z1 fluorescence microscope with DAPI filter (335–383 nm for excitation and 420–470 nm for emission), GFP filter (450–490 nm for excitation and 500–550 nm for emission) and DIC (to distinguish U and L cell types) equipped with an Axiocam 506 and an Apochromat 63×/1.20W, using ZEN 2012 software (blue edition).

Techniques: Cell Differentiation, Fluorescence, Comparison, Staining