Figure 2. Quantification of Flo11-mediated cell adhesion forces.

(A) Outline of a single-cell force spectroscopy (SCFS) experiment (upper scheme). (i) The probe cell is immobilized on an AFM cantilever and brought into contact at a defined speed with a target cell adhering to a glass substrate until a preset contact force is reached. After a defined contact time (ii), the cantilever is retracted until the probe cell is fully separated from the target cell (iii and iv). During approach and retraction, the cantilever deflection and thus, the force acting on the probe cell, is detected by using a laser and a photodiode and is recorded in a force-distance curve (lower scheme) that allows calculation of the maximum adhesion force (F_max). (B) Single yeast probe cells immobilized on an AFM cantilever. The image was obtained by differential interference contrast imaging. Scale bar corresponds to 15 µm. (C) Confocal laser scan microscopy after staining with FUN-1 dye. Fungal cells internalize FUN-1 and the dye is seen as diffuse green cytosolic fluorescence. FUN-1 is then transported to the vacuole in metabolically active cells and subsequently compacted into fluorescent, red cylindrical intravacuolar structures (CIVS), here pseudo-colored in purple to indicate healthy cells (Millard et al., 1997). Scale bar corresponds to 15 µm (D) Adhesion forces mediated by ScFlo11A at single cell level were determined for homotypic or heterotypic interaction of yeast cells presenting no Flo11A (ΔA) or ScFlo11A by SCFS (Figure 2—source data 1). Average F_Max values measured after 20 s of contact time are shown as red bars and were calculated from at least 15 independent individual measurements (grey dots). The average adhesion forces mediated by cells lacking ScFlo11A or with ScFlo11A are shown by dotted lines, and corresponding SD areas are shown by grey and green bands. (E) Cell-cell aggregation strength mediated by ScFlo11A in homogeneous populations was determined using yeast strains presenting no Flo11A (ΔA) or ScFlo11A by QCAM (Figure 2—figure supplement 4). Total area covered (mm²) by all cell aggregates of a given size category (I - IV) per image area (1 cm²) is shown as a quantitative measure for cell-cell aggregation. Error bars indicate standard deviation obtained by at least three independent measurements. The average total aggregate areas obtained with cells lacking Flo11A or with regular ScFlo11A are shown by dotted lines, and corresponding SD areas are shown by grey and orange bands. Significance was calculated applying an unpaired t-test (D) or a Wilcoxon rank sum test (E) with p>0.05 (n.s), 0.05 ≥ P > 0.01 (*), 0.01 ≥ P > 0.001 (**), p≤0.001 (***).

Figure 2—figure supplement 1. Preparation of single yeast cells suitable for SCFS analysis.

(A) Outline of the single-cell force spectroscopy (SCFS) experiment. Yeast cells were grown and harvested in SC-4 medium and collected to be centrifuged and resuspended in YEPD medium. Resuspended cells were then plated, dissociated and vortexed and replated on a new dish for SCFS. (B) Confocal laser scanning microscopy after staining the ScFlo11A domain with fluorescently-labelled antibodies (green) and yeast nuclei with DAPI (red) from harvested cells after growing in SC-4 media. Scale bar, 5 µm. (C) SEM of pipettes used to separate cells. (D) Example of an isolated cell attached to an AFM cantilever for adhesion studies. The ScFlo11A domain was stained with fluorescently-labelled antibodies (green) and yeast nuclei with DAPI (red) to monitor surface expression of adhesins. Scale bar, 15 µm. (E–F) Correlation between cell surface presence of ScFlo11A and magnitude of cell-cell adhesion forces. Amount and localization of ScFlo11A on probe and target cells was visualized by staining ScFlo11A (green) and nuclei (red) followed by confocal laser scan microscopy. Scale bar, 5 µm. Cells used for SCFS analysis were stained after determination of adhesion forces and imaged mounted on the AFM cantilever (probe cell) or a glass surface (target cell). Z-stacks of images recorded at 0.4 µm distance are shown for a ScFlo11A-presenting probe cell in contact with a non-presenting target cell (ScFlo11A⁺ vs ScFlo11A^-) and for a ScFlo11A-presenting probe and target cell configuration (ScFlo11A⁺ vs ScFlo11A⁺). Average maximum adhesion forces (F_max) were obtained after 20 s by contact time of ScFlo11A-presenting probe cells with non-presenting target cells (Figure 2—source data 1). Forces of individual cell-cell pairs were measured by SCFS (dots) and average values calculated (red line).

Figure 2—figure supplement 2. Controls for cell adhesion on cantilever.

(A) Cells were tested for adhesion experiments by functionalizing them to the cantilever using covalent chemistry and measured against a PEG-passivated surface. (B) Cell viability of covalently attached cells on cantilever was only 35% according to FUN1 staining. (C) Viable cells were selected and measured on PEG coated glass surfaces and showed almost no adhesion (Figure 2—source data 1). (D) Selected adhesion curves from the three different Flo11A constructs from experiment described in panel C). (E) Cells were tested for adhesion experiments by attaching them to a cantilever which were passively coated with ConA and measured against a PEG-passivated surface. (F) Cell viability of ConA attached cells on cantilever was 95% according to FUN1 staining. (G) Viable cells were selected and measured on ConA coated glass surfaces and showed almost similar adhesive values for all Flo11A variants (Figure 2—source data 1). (H) Selected adhesion curves from the three different Flo11A constructs described in panel G).

Figure 2—figure supplement 3. Time course of average force values obtained by single-cell force spectroscopy (SCFS).

(A–F), Shown are average maximum adhesion forces obtained by SCFS using yeast cells expressing different Flo11A domains in homotypic or heterotypic configurations as indicated. Average maximum adhesion force values of force time traces were measured at time intervals of 0.5, 1, 5, 10, 20 s. Individual measurements can be found in Figure 2—source data 1, Figure 3—source data 1, Figure 4—source data 1 and Figure 4—source data 1.

Figure 2—figure supplement 4. Quantitative cell aggregation microscopy (QCAM).

To obtain a quantitative measure for cell-cell aggregation, the QCAM method was developed as described in the Materials and Methods section. (A) Cells are grown on a solid agar surface to build a biofilm, before a defined volume of cells is harvested and transferred to a defined volume of liquid medium. (B,C) Cell aggregates are subsequently separated by vortexing, and a defined volume is spread on solid medium for microscopy. (D) Microscopic 2D images of a defined area are further analyzed by automated cell aggregate size analysis. Scale bar, 1 mm. (E,F) Data obtained from the image analysis is further sorted and quantified by determination of the total area covered by cell aggregates (in units of mm²) of a given size category (I - IV) in relation to the total image area (1 cm²). Aggregates with a size smaller than 10⁻⁴ mm² are below the threshold for detection.