Kin discrimination in social yeast is mediated by cell surface receptors of the Flo11 adhesin family
- Department of Genetics, Philipps-Universität Marburg, Germany
- Department of Biosystems Science and Engineering, ETH Zürich, Switzerland
- Department of Biochemistry, Philipps-Universität Marburg, Germany
- Max Planck Institute for Terrestrial Microbiology, Germany
- LOEWE Center for Synthetic Microbiology, Philipps-Universität Marburg, Germany
Figures
Figure 1 with 2 supplements
Functional model and phylogeny of fungal Flo11A domains.
(A) Model showing self adhesion of two S. cerevisiae Flo11 molecules (ScFlo11) conferred by homotypic binding of ScFlo11A adhesion domains (green sphere), which are covalently anchored via ScFlo11BC …
Figure 1—figure supplement 1
Multiple sequence alignment of Flo11A-type domains from different Saccharomycetales species.
Sequences are described in Supplementary file 1 and were alignment by using the Clustal Omega web tool (McWilliam et al., 2013). Numbers after species names indicate percentage identity to Flo11A …
Figure 1—figure supplement 2
Functional analysis of Flo11A-type domains from different Saccharomycetales species.
Biofilm formation of flo11∆ yeast strain RH2662 expressing Flo11A domains from different yeast species (Supplementary file 3) was measured on agar surfaces after 5 d of growth as previously …
Figure 2 with 4 supplements
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 …
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Figure 2—source data 1
Single cell-cell adhesion forces determined by SCFS and presented in Figure 2.
- https://cdn.elifesciences.org/articles/55587/elife-55587-fig2-data1-v2.xlsx
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 …
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 …
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 …
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 …
Figure 3 with 1 supplement
Functional mapping of the Flo11A protein surface at single cell and population level.
(A) Structural model of ScFlo11A (PDB code 4UYR). Functionally relevant aromatic surface residues of aromatic bands I and II are depicted in orange (as shown in Figure 3—figure supplement 1). …
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Figure 3—source data 1
Single cell-cell adhesion forces determined by SCFS and presented in Figure 3.
- https://cdn.elifesciences.org/articles/55587/elife-55587-fig3-data1-v2.xlsx
Figure 3—figure supplement 1
Structure-based functional analysis of ScFlo11A.
(A) Structural model of Flo11A from S. cerevisiae strain S288c (PDB code 4UYR). Functionally relevant aromatic surface residues of bands I and II are shown in orange. Surface-exposed aromatic …
Figure 4 with 4 supplements
Structural and functional properties of Flo11A from Komagataella pastoris at single cell and population level.
(A) Crystal structure of KpFlo11A at 1.40 Å resolution (PDB code 5FV5). The structural model shows solvent-exposed aromatic residues (Tyr, Trp) in green. Disulfide bonds are shown in yellow. (B) …
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Figure 4—source data 1
Single cell-cell adhesion forces determined by SCFS and presented in Figure 4.
- https://cdn.elifesciences.org/articles/55587/elife-55587-fig4-data1-v2.xlsx
Figure 4—figure supplement 1
Overall structure of the KpFlo11A domain from Komagataella pastoris.
(A) Crystal structure of KpFlo11A with one molecule per asymmetric unit (asu). The fibronectin type III domain is shown in brown, the neck subdomain in orange and the apical region in yellow (PDB …
Figure 4—figure supplement 2
Structural comparison between ScFlo11A and KpFlo11A.
(A) Structural superposition between ScFlo11A (green) and KpFlo11A (brown). The root-mean-square deviation is 1.048 Å for 685 Cα atoms. Tyr and Trp residues are highlighted as sticks. Disulfide …
Figure 4—figure supplement 3
Rigidity of aromatic surface patches in ScFlo11A and KpFlo11A and overall conservation.
(A) The crystal structures of ScFlo11A and KpFlo11A with highlighted Tyr and Trp residues colored by temperature factors (B-factors). High flexibility is indicated by red and low flexibility by blue …
Figure 4—figure supplement 4
SPR binding analysis of KpFlo11A-KpFlo11A and KpFlo11A-ScFlo11A interactions.
Sensorgrams of KpFlo11A-KpFlo11A interactions (A) and KpFlo11A-ScFlo11A interactions (B) with concentrations from 5 to 350 µM KpFlo11A or ScFlo11A, respectively, in solution. KpFlo11A was …
Figure 5 with 1 supplement
Flo11A-mediated separation of competitive yeast strains.
(A) Competitive biofilm assays. Yeast strains expressing the indicated Flo11 variants were mixed in a 1:1 ratio and were grown to a mat biofilm for three weeks. To distinguish between competing …
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Figure 5—source data 1
Quantification of RFP and GFP signals in mixed biofilms presented in Figure 5.
- https://cdn.elifesciences.org/articles/55587/elife-55587-fig5-data1-v2.xlsx
Figure 5—figure supplement 1
Quantitative analysis of biofilms upon competitive assays.
Yeast strains expressing the indicated Flo11 variants were mixed in a 1:1 ratio (OD600 of cells) and grown to a mat biofilm for three weeks. Competing yeast strains were tagged by expression of …
Figure 6 with 1 supplement
Structural and functional properties of ScΣFlo11A from S. cerevisiae strain Σ1278b at single cell and population level.
(A) A structural model of ScΣFlo11A as obtained as snap shot by molecular dynamics analysis. The ScΣFlo11A-specific insert differing from ScFlo11A is shown in magenta. Aromatic surface residues …
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Figure 6—source data 1
Single cell-cell adhesion forces determined by SCFS and presented in Figure 6.
- https://cdn.elifesciences.org/articles/55587/elife-55587-fig6-data1-v2.xlsx
Figure 6—figure supplement 1
Functional analysis of ScΣFlo11A.
Biofilm formation of yeast strains expressing ScFlo11A, ScΣFlo11A or ScFlo11AΣins were measured on agar surfaces after 3 d of growth as previously described (Roberts and Fink, 1994). Images of …
Figure 7
Correlation between SCFS and QCAM data.
Correlation between maximum adhesion forces obtained by SCFS analysis and total aggregate areas obtained by QCAM analysis was calculated by Spearman's rank correlation test. Dots show SCFS and QCAM …
Figure 8
Model for kin discrimination by Flo11.
(A) Structural elements for Flo11-directed discrimination. Shown are ScFlo11A, ScΣFlo11A and KpFlo11A variants that differ either by the topology of the conserved aromatic bands shown as orange or …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Saccharomycetales) | Flo11-type A domain sequences | various | See Supplementary file 1 | |
| Genetic reagent Saccharomyces cerevisiae | Flo11-type A domain variants carrying yeast strains | See Supplementary file 2 | ||
| Antibody | Rabbit polyclonal anti-ScFlo11A | (Kraushaar et al., 2015) | Previously produced in our lab (1:10.000) | |
| Antibody | Mouse monoclonal anti-STREP II Chromeo 488 | IBA Lifesciences, Göttingen, Germany | Cat# 2-1544-050 | (1:1.000) |
| Recombinant DNA reagent | Flo11-type A domain variants carrying plasmids | various | See Supplementary file 3 | |
| Sequence-based reagent | Primer Ampl_FLO11_wt_fw | This study | PCR primers | CTC AAA AAT CCA TAT ACG CAC ACT |
| Sequence-based reagent | Primer Ampl_FLO11_wt_BC_rev | This study | PCR primers | GTA GAG CTG GAT GGA GTT GG |
| Peptide, recombinant protein | ScFlo11A | (Kraushaar et al., 2015) | Previously produced in our lab | |
| Peptide, recombinant protein | KpFlo11A | This study | ||
| Commercial assay or kit | Amine coupling kit | GE Healthcare, Solingen, Germany | ||
| Software, algorithm | Matlab | MathWorks | RRID:SCR_001622 | See section ‘QCAM’ and ‘Competitive biofilm assay’ |
| Software, algorithm | GraphPad Prism, v7.0 | GraphPad Software, La Jolla, CA, USA | RRID:SCR_002798 | |
| Software, algorithm | XDS | (Kabsch, 2010) | RRID:SCR_015652 | |
| Software, algorithm | CCP4 | Collaborative Computational Project, Number 4, 1994 | RRID:SCR_007255 | |
| Software, algorithm | Phenix suite | (Adams et al., 2002) | RRID:SCR_014224 | |
| Software, algorithm | Coot | (Emsley et al., 2010) | RRID:SCR_014222 | |
| Software, algorithm | Refmac5 | (Murshudov et al., 2011) | RRID:SCR_014225 | |
| Software, algorithm | PyMOL v1.8.0.1 | (Delano, 2002) | RRID:SCR_000305 | |
| Software, algorithm | Biacore T100 evaluation software, v1.1 | GE Healthcare, Solingen, Germany | ||
| Software, algorithm | Clustal Omega web tool | (McWilliam et al., 2013) | RRID:SCR_001591 | |
| Software, algorithm | PRANK web tool | (Löytynoja and Goldman, 2010) | RRID:SCR_017228 | |
| Software, algorithm | FigTree v1.4.2 | http://tree.bio.ed.ac.uk | RRID:SCR_008515 | |
| Software, algorithm | STRIDE web server | (Heinig and Frishman, 2004) | ||
| Software, algorithm | Modeller 9v7 | (Sali and Blundell, 1993) | RRID:SCR_008395 |
Additional files
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Supplementary file 1
Flo11A domain sequences.
- https://cdn.elifesciences.org/articles/55587/elife-55587-supp1-v2.docx
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Supplementary file 2
Yeast strains.
- https://cdn.elifesciences.org/articles/55587/elife-55587-supp2-v2.docx
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Supplementary file 3
Plasmids.
- https://cdn.elifesciences.org/articles/55587/elife-55587-supp3-v2.docx
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Supplementary file 4
Crystal structure data collection, processing and refinement.
- https://cdn.elifesciences.org/articles/55587/elife-55587-supp4-v2.docx
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Supplementary file 5
Structural analysis of KpFlo11A.
- https://cdn.elifesciences.org/articles/55587/elife-55587-supp5-v2.docx
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Supplementary file 6
Quantification of Flo11A protein amounts.
- https://cdn.elifesciences.org/articles/55587/elife-55587-supp6-v2.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/55587/elife-55587-transrepform-v2.pdf
