Physiological function of Flo11p domains and the particular role of amyloid core sequences of this adhesin in Saccharomyces cerevisiae
Curation statements for this article:-
Curated by eLife
Evaluation Summary:
This work serves as an independent confirmation and expansion of previous work, suggesting that certain domains in fungal adhesins can form amyloid-like structures that are essential for cell-cell aggregation, but less so for cell-surface adhesion or invasive growth. Specifically, the expression of different adhesin constructs offers insight into the functional role of the different adhesin domains. Together, the results provide further insight into the molecular mechanisms underlying fungal adhesion, which does only further our understanding of basic fungal physiology, but also offers insight into fungal pathogenesis.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
This article has been Reviewed by the following groups
Listed in
- Evaluated articles (eLife)
- Microbiology and Infectious Disease (eLife)
Abstract
Flocculins are a family of glycosylated proteins that provide yeast cells with several properties such as biofilm formation, flocculation, invasive growth or formation of velum. These proteins are similarly organised with a N-terminal (adhesion) domain, a stalk-like central B-domain with several repeats and a C-terminal sequence carrying a cell wall anchor site. They also contain amyloid β-aggregation-prone sequences whose functional role is still unclear. In this work, we show that Flo11p differs from other flocculins by the presence of unique amyloid-forming sequences, whose the number is critical in the formation of adhesion nanodomains under a physical shear force. Using a genome editing approach to identify the function of domains in Flo11p phenotypes, we show that the formation of cellular aggregates whose density increases with the number of amyloid sequences cannot be attributed to a specific domain of Flo11p. The same is true for plastic adhesion and surface hydrophobicity the intensity of which depends mainly on the abundance of Flo11p on the cell surface. In contrast, the N and C domains of Flo11p are essential for invasive growth in agar, whereas a reduction in the number of repeats of the B domain weakens this phenotype. However, expression of FLO11 alone is not sufficient to trigger this invasion phenotype. Finally, we show that this flocculin contributes to the integrity of the cell wall.
Article activity feed
-
Author Response:
Reviewer #1:
This manuscript describes extensive phenotypic analyses of Flo11, a multifunctional adhesin in S. cerevisiae. Flo11 mediates a variety of adhesin-related activities including flocculation and adhesion to surfaces, and these activities can lead to biofilm and pellicle (floating biofilm) formation, as well as ability to mediate agar invasion. Flo11 activities are activated after shear force, which facilitates surface amyloid formation. Flo11 is highly diverse, with different yeast strains expressing alleles with sequence and repeat number differences. These differences contribute to differences in adhesion behaviors. The alleles have conserved regions including a secretion signal, a globular N-terminal region, and a C-terminal GPI anchor that mediates cell surface attachment. In between these regions are …
Author Response:
Reviewer #1:
This manuscript describes extensive phenotypic analyses of Flo11, a multifunctional adhesin in S. cerevisiae. Flo11 mediates a variety of adhesin-related activities including flocculation and adhesion to surfaces, and these activities can lead to biofilm and pellicle (floating biofilm) formation, as well as ability to mediate agar invasion. Flo11 activities are activated after shear force, which facilitates surface amyloid formation. Flo11 is highly diverse, with different yeast strains expressing alleles with sequence and repeat number differences. These differences contribute to differences in adhesion behaviors. The alleles have conserved regions including a secretion signal, a globular N-terminal region, and a C-terminal GPI anchor that mediates cell surface attachment. In between these regions are ~1000-1500 amino acids with variable sequence and repeat numbers and a variable number of potential amyloid-core sequences. The manuscript includes extensive sequence analysis of alleles from 4 strains, and analysis of the consequences of expression of different regions of the protein from various alleles. This major study includes detailed AFM analyses showing that a Flo11 allele from a wine-fermenting strain expression leads to presence of cell surface patches of Flo11 adhesin 10-100 nm diameter 10-20 nm elevation from the cell surface. SiN AFM probing determines both adhesive strength and stiffness of the adhesion patches. The regions containing the amyloid-core sequences are essential for most of the Flo11 activities, because the activities are inhibited by deletion of amyloid-forming core regions or by treatment of intact cells with a general amyloid inhibitor or a sequence-specific anti-amyloid peptide.
We thank the reviewer for these encouraging and stimulating comments.
The work is extensive and well-documented. Qualitative data on the different constructs shows how activity correlates to specific sequences in the protein. However, the analysis is compromised by the lack of data on the levels of surface expression of the different alleles and constructs. mRNA levels are reported, but for fungal adhesins these values often do not correlate with surface expression levels. The problem is especially confounding for the C-terminal deletion construct, because the deleted region includes the GPI addition signal: its deletion leads to secretion of free adhesin into the medium and decreased surface anchorage (Douglas et al. 1996 Eukaryot. Cell6:2214-2221).
This critical comment also raised by the Editor has been answered above. The main finding was to show that a Flo11 variant lacking the C-ter still show localization at the cell periphery as witnessed by immunofluorescence, suggesting that this variant can be retained at the cell wall by other structural components. This result appears to be in contradiction with the data from Douglas et (EC, 2007) except that as written in line 485-490 in the revised version), FLO11 was expressed on a 2μ plasmid under PGK1 promoter, leading to huge expression of Flo11p, which may exceed the capacity of cell wall to retain it.
The quantitative biophysical conclusions would be strengthened if the data from all tested cells were aggregated and presented, not just the data from the individual cell shown in each figure.
We have already answered to this question above, as it was one of the issues raised by the Editor.
If these critiques are addressed, the manuscript would greatly benefit from a summary figure and paragraph describing the activities attributed to each region of the sequence in L69 and other alleles. It would also benefit from reference to recent work demonstrating that cell-cell adhesion can be mediated by formation of amyloid-like bonds between cells.
This is indeed a good suggestion but the paper already contains 9 figures and 2 tables and we would like to extend this comparative analysis with all the FLO-encoded flocculins in S. cerevisiae, which will be the purpose of an ongoing short review on the subject to be submitted soon.
Reviewer #2:
The authors argue that the Flo1 protein of yeast can mediate cell-cell aggregation through amyloid formation. They identified a wine-making yeast strain that is particularly strong in this phenotype and this correlates with the presence of aggregation prone repeat regions in the protein. Their frequency seems to be important for the effect.
The paper makes an extensive case, although I have reservations on experimental specifics. They show AFM imaging of the cell surface (nanodomain formation) and extensive genome editing to alter the suspected regions, which largely (but not perfectly produces effects in line with the hypothesis). Also, they use an inhibitory peptide (not so well validated) and a widely used amyloid dye to disturb these amyloid formation. Taken together these data point toward a role for amyloid formation, similar to what was previously shown by Lipke for candida. However, I remain with some concerns regarding data, controls, interpretation that preclude firmly backing the story.
We thank this reviewer for his/her critical comments. We have done our best to answer to your relevant questions, comments and remarks.
Major Concerns:
There is no direct evidence of amyloid formation in situ.
The paper is not about to show amyloid fibers formation of Flo11p as this was already shown in a previous paper that Flo11p forms amyloids fibers in vitro. Moreover synthetic peptides with high β- aggregate potential sequences taken from these adhesins formed amyloids in vitro as well (see reference Ramsook et al., Eucaryotic cell, 9: 393-404 2010, and that we referenced in our paper).
The hydrophobic cluster analysis in figure 1 is not very clear - it is almost impossible to make anything out in those graphs
The HCA (hydrophobic cluster analysis) representation as shown in this figure has been used previously by others (see Chan et al., mSphere, e00128, 2016). This representation has the advantage for non-expert in the field to readily visualize what is common and different in term of sequences structure between the different Flo proteins. According to your request, we remove this figure and onoyt kep data on TRs in a supplementary File 1.
The term beta-aggregation potential is used throughout and not defined. I presume this refers to the TANGO score? You should call it TANGO score then. Where was the arbitrary cutoff of 70 for TANGO aggregation propensity determined? In itself it is only a prediction, not a demonstration of amyloid potential.
It is indeed a valuable suggestion although β-aggregation term is currently used in many other publications on this subject. TANGO is a statistical algorithm designed to predict β- aggregate potential sequences in protein. It does not predict whether or not these sequences will lead to amyloid formation, although correlation between β-aggregation sequences and amyloid formation has been discussed in (Fernandez-Escamilla AM, Rousseau F, Schymkowitz J, Serrano L. Prediction of sequence-dependent and mutational effects on the aggregation of peptides and proteins. Nat Biotechnol. 2004 Oct;22(10):1302-6. doi: 10.1038/nbt1012. Epub 2004 Sep 12. PMID: 15361882.).
The cutoff used for β-aggregation propensity was set arbitrarily set a 30% (see line 634 in the M&M section and in Table 1 and Supplementary File 2a) to discriminate which sequence in the protein have a high propensity to beta- aggregate. As shown in Table 1 and Supplementary File 2a, Flo11 protein exhibits several beta-aggregation prone sequences that are largely over this cut-off.
To do that you need to make the peptide and show it makes amyloid (and that this can be suppressed by thioS and your breaker peptide, see below)
These experiments have been carried out in a previous paper (See Ramsonk et al, Eukaryotic cell, 9, 393-404, 2010)
line 124: how many cells were analysed, is this pattern present in the population? analysing a single cell would seem insufficient to me - this comment applies to several analyses below.
As indicated in M&M, all AFM experiments were done on 3 biological replicates and for each replicate around 6 to 12 cells have been analyzed. Data reported in the figure are from a representative cell. However, as stated above and in accordance with the Editor request, we reported the aggregation of all data from 24 cells, which showed the bimodal behavior of the adhesion forces and stiffness, which are reported as box plots (see Figure 1G, H);
line 124: "this apparent roughness can be account for by proteins" - there is no data to support this, right? could be anything at this stage, it is just patch. I my mind you would need AFM-IR to ensure that you are actually observing a protein structure. This would immediately show you if its in the amyloid state as well.
As this stage, we do not have any idea what could be those aggregates and therefore we modified as follows (lines 115-118 in the revised version): The high-resolution height image (Fig. 1B) revealed a multitude of small aggregates on the cell surface with an average diameter of 100 nm and a height above the cell surface in the range of 15-20 nm (Fig. 1C).
line 134: possibly due to my ignorance: how do you conclude from those forces that one is the hydrophobic interaction and the other is protein unfolding? comes as a complete deus ex machina to me.
The difference between hydrophobic interactions and protein unfolding is obtained from the shape of the retraction force-distance curves as shown in Fig 1E. A hydrophobic interaction is a physicochemical interactions due to the surface tension which is exerted on the surface of materials. It is particularly related to the organization of the solvent (usually water) at the interface between the solid and the liquid. These forces are exerted at the immediate surface of the material and are characterized by a null distance between the AFM tip and the surface at the moment of rupture.
On the contrary, a protein unfolding, which as its name indicates, consists in unfolding a complex organic molecule (a protein) and which is characterized by a distance between the AFM tip and the surface, at the moment of rupture, equal to the length of the unfolded protein. If the protein unfolds in several steps, which is often the case for these molecules with complex folding, this results to sawtooth force curves profiles characteristic of the unfolding of proteins containing repeated domains.
line 151 (also mentioned later): the 'anti-amyloid peptide' is taken with a lot of faith: how do we know it works by disrupting amyloid? At least show it works on the peptide level (Tht curves) (again the AFM-IR measurement would solve this in one single effort). This experiment at least needs a control to show the cell is otherwise intact and other structures are still present. Designing potent amyloid disrupting peptide is not trivial - see various papers on the topic by David Eisenberg. I find it hard to believe these subtle mutations achieve all that they are claimed to achieve.
The use of a mutated peptide of the amyloid core sequences or amyloid perturbants are well established to investigate amyloid-like nature of nanodomains formation or in cell-cell aggregation. Therefore, it may be a question of semantic because in this case, we are working with a protein that forms cellular aggregates due to the presence of amyloid-core sequence. When these proteins unfold, they expose these sequences that were previously buried inside compact protein, leading to interaction between molecules of the same type, and hence this creates clusters of thousands of molecules that can eventually organize into nanodomain. Such interaction can be inhibited by a mutated peptide of the amyloid core sequence or by anti-amyloid dyes. This event is different from a protein whose amyloid formation is triggered by intermolecular associations leading to a refolded protein that often loses it function and this leads to condition of protein misfolding diseases neither on amyloid prions whose flocculin or adhesion in yeast does not belong to.
line 157: Maybe I am confused, but why would thioflavin S destabilise amyloid? it is widely used as an amyloid-specific dye, and I would expect its binding energy to stabilise the amyloid state. Later, around line 310, the compound is called a drug, but really, it is just a rotor dye with an affinity for amyloid. Do other amyloid dyes show similar effect (oligothiophenes, congo red, curcumin)?
It is indeed known that amyloid-dependent aggregation of yeast cells can be monitored by increased thioflavin T fluorescence as a result to its binding to β-amyloids structures. However, other amyloid binding dyes such as Congo red and in particular Thiflavin S had potent anti-aggregation effects (see for instance Ramsook et al. EC, 9, 393-404, 2010). Taking into account that these dyes bind to amyloids, they are thus employed to evaluate whether the cell-cell aggregation mediated by Flo11 (and the same for Als1 and Als5 in C. albicans) is due the presence of amyloid forming sequence in these proteins.
line 250: are we really comparing single cell instances of each construct?
Suggestions:
If the hypothesis of the authors is true, it should be possible to replace the amyloid domains with synthetic ones (STVIIE eg, Serrano and co-workers), or from another protein. Once you have sufficient of these, you should see the cell-interactions etc.
Also, introducing structure breaking residues in the repeats, like proline should stop the effect and would give strong support for the amyloid nature of the interactions over general hydrophobic patches.
Each of these strains expresses only one specific variant validated by genome sequencing. Our AFM analysis showed that nanodomains are no longer observed when Flo11p is lacking RR2 that contains the additional amyloid sequence motifs. Thus, we can conclude that amyloid formation sequences are indispensable for their formation. The question of disrupting the cell-cell interactions or preventing the formation of the nanodomains is presented earlier in the paper (see line152 -158 in the revised version).
Reviewer #3:
The authors observed nanodomains in FLO11 expressed S. cerevisiae strains when imaging the cell wall of the cell with a bare AFM tip. The adhesion to the tip as well as the stiffness of these domains were characterized using AFM. However, there was no direct proof/confirmation that these nanodomains were actually composed of Flo11 proteins.
We disagree with this statement because we clearly show that (i) the formation of nanodomains in response to AFM tip does not occur anymore in a mutant defective in FLO11, and (ii) the expression of the FLO11 gene from L69 strain in the lab strain BY leads to the formation of nanodomains at the cell surface of this strain, which is not seen upon overexpression of its endogenous FLO11. Thus, these data taken together demonstrate that a) Flo11 is responsible for nanodomains formation and b) this formation requires a peculiar Flo11 protein. We show actually that this peculiarity lies in the necessity to harbor sufficient amyloid-forming motifs in the protein sequence.
Previous research showed that Flo11p trans interacts and this trans interaction between cells could be (hypothesis) preceded by a cis interaction between the Flo proteins via beta-aggregation-prone amyloid-forming sequences. It seems that the obtained results could explain this trans-interaction (i.e. the "nanodomains"). However, in this model the main interaction is based on the Flo11 A-domain interaction that is responsible for the trans-interaction. This could not be confirmed in the present work where the cell-cell interaction results were only based on (qualitative) microscopic observation of cell aggregates. Additional, it has been previously shown that homotypic Flo11 A-domain interaction is pH dependent since only at low pH these domains hydrophobically interact. It seems that the current experiments were performed at a too high pH.
The suggestion that Flo11 dependent trans-interaction that characterizes cell-cell interaction may be preceded by a cis-interaction or homophobic interaction (ie interaction between Flo11p molecules leading to cluster) is still hypothetical. However, recent data from Lipke’ group (se Ho et al, mBio. 2019 Oct 8;10(5):e01766-19; Dehullu et al, Nanolett,19, 3846, 2019) showed that amyloid-forming sequences are implicated in these two types of interaction. Thus, while cis-interaction of Flo11p definitively requires amyloids motifs, our data are in line with this finding that the amyloid-forming sequence contribute to trans-interaction, although they are not essential to it. Altogether, we revised part of the Result section (see lines 312 -315 in the revised version) as well as the Discussion considering both model and our data (see line 484 to 514).
Finally, we have carried out the cell-cell adhesion experiments shown in Figure 6 at pH 5 (as for AFM experiments). This is now clearly stated in Mat & Meth.
-
Evaluation Summary:
This work serves as an independent confirmation and expansion of previous work, suggesting that certain domains in fungal adhesins can form amyloid-like structures that are essential for cell-cell aggregation, but less so for cell-surface adhesion or invasive growth. Specifically, the expression of different adhesin constructs offers insight into the functional role of the different adhesin domains. Together, the results provide further insight into the molecular mechanisms underlying fungal adhesion, which does only further our understanding of basic fungal physiology, but also offers insight into fungal pathogenesis.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed …
Evaluation Summary:
This work serves as an independent confirmation and expansion of previous work, suggesting that certain domains in fungal adhesins can form amyloid-like structures that are essential for cell-cell aggregation, but less so for cell-surface adhesion or invasive growth. Specifically, the expression of different adhesin constructs offers insight into the functional role of the different adhesin domains. Together, the results provide further insight into the molecular mechanisms underlying fungal adhesion, which does only further our understanding of basic fungal physiology, but also offers insight into fungal pathogenesis.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
-
Reviewer #1 (Public Review):
This manuscript describes extensive phenotypic analyses of Flo11, a multifunctional adhesin in S. cerevisiae. Flo11 mediates a variety of adhesin-related activities including flocculation and adhesion to surfaces, and these activities can lead to biofilm and pellicle (floating biofilm) formation, as well as ability to mediate agar invasion. Flo11 activities are activated after shear force, which facilitates surface amyloid formation. Flo11 is highly diverse, with different yeast strains expressing alleles with sequence and repeat number differences. These differences contribute to differences in adhesion behaviors. The alleles have conserved regions including a secretion signal, a globular N-terminal region, and a C-terminal GPI anchor that mediates cell surface attachment. In between these regions are …
Reviewer #1 (Public Review):
This manuscript describes extensive phenotypic analyses of Flo11, a multifunctional adhesin in S. cerevisiae. Flo11 mediates a variety of adhesin-related activities including flocculation and adhesion to surfaces, and these activities can lead to biofilm and pellicle (floating biofilm) formation, as well as ability to mediate agar invasion. Flo11 activities are activated after shear force, which facilitates surface amyloid formation. Flo11 is highly diverse, with different yeast strains expressing alleles with sequence and repeat number differences. These differences contribute to differences in adhesion behaviors. The alleles have conserved regions including a secretion signal, a globular N-terminal region, and a C-terminal GPI anchor that mediates cell surface attachment. In between these regions are ~1000-1500 amino acids with variable sequence and repeat numbers and a variable number of potential amyloid-core sequences. The manuscript includes extensive sequence analysis of alleles from 4 strains, and analysis of the consequences of expression of different regions of the protein from various alleles. This major study includes detailed AFM analyses showing that a Flo11 allele from a wine-fermenting strain expression leads to presence of cell surface patches of Flo11 adhesin 10-100 nm diameter 10-20 nm elevation from the cell surface. SiN AFM probing determines both adhesive strength and stiffness of the adhesion patches. The regions containing the amyloid-core sequences are essential for most of the Flo11 activities, because the activities are inhibited by deletion of amyloid-forming core regions or by treatment of intact cells with a general amyloid inhibitor or a sequence-specific anti-amyloid peptide.
The work is extensive and well-documented. Qualitative data on the different constructs shows how activity correlates to specific sequences in the protein. However, the analysis is compromised by the lack of data on the levels of surface expression of the different alleles and constructs. mRNA levels are reported, but for fungal adhesins these values often do not correlate with surface expression levels. The problem is especially confounding for the C-terminal deletion construct, because the deleted region includes the GPI addition signal: its deletion leads to secretion of free adhesin into the medium and decreased surface anchorage (Douglas et al. 1996 Eukaryot. Cell6:2214-2221).
The quantitative biophysical conclusions would be strengthened if the data from all tested cells were aggregated and presented, not just the data from the individual cell shown in each figure.
If these critiques are addressed, the manuscript would greatly benefit from a summary figure and paragraph describing the activities attributed to each region of the sequence in L69 and other alleles. It would also benefit from reference to recent work demonstrating that cell-cell adhesion can be mediated by formation of amyloid-like bonds between cells.
-
Reviewer #2 (Public Review):
The authors argue that the Flo1 protein of yeast can mediate cell-cell aggregation through amyloid formation. They identified a wine-making yeast strain that is particularly strong in this phenotype and this correlates with the presence of aggregation prone repeat regions in the protein. Their frequency seems to be important for the effect.
The paper makes an extensive case, although I have reservations on experimental specifics. They show AFM imaging of the cell surface (nanodomain formation) and extensive genome editing to alter the suspected regions, which largely (but not perfectly produces effects in line with the hypothesis). Also, they use an inhibitory peptide (not so well validated) and a widely used amyloid dye to disturb these amyloid formation. Taken together these data point toward a role for …
Reviewer #2 (Public Review):
The authors argue that the Flo1 protein of yeast can mediate cell-cell aggregation through amyloid formation. They identified a wine-making yeast strain that is particularly strong in this phenotype and this correlates with the presence of aggregation prone repeat regions in the protein. Their frequency seems to be important for the effect.
The paper makes an extensive case, although I have reservations on experimental specifics. They show AFM imaging of the cell surface (nanodomain formation) and extensive genome editing to alter the suspected regions, which largely (but not perfectly produces effects in line with the hypothesis). Also, they use an inhibitory peptide (not so well validated) and a widely used amyloid dye to disturb these amyloid formation. Taken together these data point toward a role for amyloid formation, similar to what was previously shown by Lipke for candida. However, I remain with some concerns regarding data, controls, interpretation that preclude firmly backing the story.
Major Concerns:
There is no direct evidence of amyloid formation in situ.
The hydrophobic cluster analysis in figure 1 is not very clear - it is almost impossible to make anything out in those graphs
The term beta-aggregation potential is used throughout and not defined. I presume this refers to the TANGO score? You should call it TANGO score then.
Where was the arbitrary cutoff of 70 for TANGO aggregation propensity determined? In itself it is only a prediction, not a demonstration of amyloid potential. To do that you need to make the peptide and show it makes amyloid (and that this can be suppressed by thioS and your breaker peptide, see below)line 124: how many cells were analysed, is this pattern present in the population? analysing a single cell would seem insufficient to me - this comment applies to several analyses below.
line 124: "this apparent roughness can be account for by proteins" - there is no data to support this, right? could be anything at this stage, it is just patch. I my mind you would need AFM-IR to ensure that you are actually observing a protein structure. This would immediately show you if its in the amyloid state as well.line 134: possibly due to my ignorance: how do you conclude from those forces that one is the hydrophobic interaction and the other is protein unfolding? comes as a complete deus ex machina to me.
line 151 (also mentioned later): the 'anti-amyloid peptide' is taken with a lot of faith: how do we know it works by disrupting amyloid? At least show it works on the peptide level (Tht curves)
(again the AFM-IR measurement would solve this in one single effort)
This experiment at least needs a control to show the cell is otherwise intact and other structures are still present.
Designing potent amyloid disrupting peptide is not trivial - see various papers on the topic by David Eisenberg. I find it hard to believe these subtle mutations achieve all that they are claimed to achieve.line 157: Maybe I am confused, but why would thioflavin S destabilise amyloid? it is widely used as an amyloid-specific dye, and I would expect its binding energy to stabilise the amyloid state. Later, around line 310, the compound is called a drug, but really, it is just a rotor dye with an affinity for amyloid. Do other amyloid dyes show similar effect (oligothiophenes, congo red, curcumin)?
line 250: are we really comparing single cell instances of each construct?
Suggestions:
If the hypothesis of the authors is true, it should be possible to replace the amyloid domains with synthetic ones (STVIIE eg, Serrano and co-workers), or from another protein. Once you have sufficient of these, you should see the cell-interactions etc.
Also, introducing structure breaking residues in the repeats, like proline should stop the effect and would give strong support for the amyloid nature of the interactions over general hydrophobic patches.
-
Reviewer #3 (Public Review):
The authors observed nanodomains in FLO11 expressed S. cerevisiae strains when imaging the cell wall of the cell with a bare AFM tip. The adhesion to the tip as well as the stiffness of these domains were characterized using AFM. However, there was no direct proof/confirmation that these nanodomains were actually composed of Flo11 proteins.
Previous research showed that Flo11p trans interacts and this trans interaction between cells could be (hypothesis) preceded by a cis interaction between the Flo proteins via beta-aggregation-prone amyloid-forming sequences. It seems that the obtained results could explain this trans-interaction (i.e. the "nanodomains"). However, in this model the main interaction is based on the Flo11 A-domain interaction that is responsible for the trans-interaction. This could not be …
Reviewer #3 (Public Review):
The authors observed nanodomains in FLO11 expressed S. cerevisiae strains when imaging the cell wall of the cell with a bare AFM tip. The adhesion to the tip as well as the stiffness of these domains were characterized using AFM. However, there was no direct proof/confirmation that these nanodomains were actually composed of Flo11 proteins.
Previous research showed that Flo11p trans interacts and this trans interaction between cells could be (hypothesis) preceded by a cis interaction between the Flo proteins via beta-aggregation-prone amyloid-forming sequences. It seems that the obtained results could explain this trans-interaction (i.e. the "nanodomains"). However, in this model the main interaction is based on the Flo11 A-domain interaction that is responsible for the trans-interaction. This could not be confirmed in the present work where the cell-cell interaction results were only based on (qualitative) microscopic observation of cell aggregates. Additional, it has been previously shown that homotypic Flo11 A-domain interaction is pH dependent since only at low pH these domains hydrophobically interact. It seems that the current experiments were performed at a too high pH.
-