There is a growing interest in the adhesive interactions occurring between cell pairs that allow for mating to occur ^[1][2][3][1,2,3]. In prokaryotic cells and yeasts, the role played by surface adhesion proteins (adhesins) in selective interactions has been the subject of intense research for many years. These interactions play key roles in colonization, biofilm formation, and, in pathogens, direct interactions with host tissues and cells that lead to infection [4]. Moreover, the role of force in these interactions is gaining recognition ^[5][6][7][5,6,7]. While measures of affinity under equilibrium conditions are very useful in the characterization of receptor–ligand interactions, adhesive interactions often occur under out-of-equilibrium conditions, that is to say that binding needs to occur and be strong under physical stress. The study of adhesive receptor–ligand interactions, thus, requires appropriately adapted approaches. Accordingly, great progress has been made in atomic force microscopy (AFM) force spectroscopy approaches [8].

2. Characteristics of SCFS

Single-cell force spectroscopy (SCFS) has been instrumental in improving our understanding of cell–substrate adhesion, cell–cell adhesion, and cell–host adhesion down to the molecular level (for recent reviews, herwein direct the reader toward ^[6][8][6,8]). Briefly, the principle of SCFS consists of attaching a single cell to a soft, force-sensitive AFM cantilever (Figure 12a). This cell probe can then be used to measure interactions with other surfaces, including those of other single cells. By bringing the cell probe in contact with the surface of another cell and subsequently retracting it away, while recording the forces between the two cells, force–distance (FD) curves are generated, from which the strength of adhesion between the two cells can be determined accurately. Importantly, the velocity at which the cell probe is approached and pulled away from the sampled cell can also be controlled precisely. For most measurements, an arbitrary constant approach/retract velocity of ~1–10 µm·s⁻¹ is used to roughly match physiological conditions ^[9][37]. However, varying the retraction velocity allows assessing the effect of different force loading rates on the tensile strength of molecular complexes formed between adhesive molecules on the surfaces of the interacting cells. Performing such dynamic force spectroscopy experiments can provide information on the energy landscape underlying forced unbinding of receptor–ligand complexes ^[10][38]. AFM can also be utilized to investigate the binding dynamics between single cells and polypeptide ligands. ThWe authors previously used this technique (called single-molecule force spectroscopy, AFM-SMFS) to detect and analyze the unfolding of C. albicans Als5p. In those experiments, a single yeast cell expressing a V5 epitope-tagged Als5p was immobilized onto a polymer membrane. Als5p-V5 was detected, and the adhesion forces were measured using AFM tips coupled to anti-V5 antibodies ^[11][39]. Notably, recent AFM-SCFS experiments have revealed binding dynamics associated between the SARS-CoV-2 spike protein and human ACE2 receptor. In those experiments, the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein was attached to the AFM tip, and the purified ACE2 receptor or ACE2-expressing cells were immobilized onto their respective surface ^[12][13][40,41]. A new, exciting AFM technology that has improved the ease, robustness, and speed of SCFS experiments is fluidic force microscopy (FluidFM), which relies on AFM cantilevers containing a microchannel that is connected to a pump ^[14][42]). These microfluidic probes are capable of extracting contents from or injecting foreign material into eukaryotic cells through precise pressure control at the aperture ^[15][16][43,44]. Alternatively, FluidFM allows capturing single cells in a fast, noninvasive manner that does not require (bio)chemical adhesives that may interfere with cellular function. Next, the captured cell is used to probe another surface such as a different cell or model substrate (Figure 12b) ^[15][16][17][43,44,45]. The reversibility of the adhesion between cell and probe makes it possible to pick up and release a single cell in a controlled manner, and this technique has been applied to isolate single bacteria expressing a desired phenotype from a mixed microbial population ^[18][46]. Regarding force spectroscopy, FluidFM has been applied to uncover the biophysical dynamics governing bacterial adhesion to hydrophobic surfaces ^[19][47] and to quantify forces in the adhesion to abiotic surfaces of yeast and mammalian cells ^[11][39].

2.1. SCFS Application to Studying Yeasts

2.1.1. Intercellular Adhesion

While earlier studies of Als interactions relied on the use of purified binding partners (e.g., antibodies) attached to AFM tips, recent FluidFM-based SCFS analyses unraveled the forces in Als homophilic binding occurring between single pairs of yeast cells ^[20][21][51,54]. FluidFM-based SCFS allows monitoring of the interaction between two single living yeast cells as they are brought into contact and subsequently separated, while recording adhesin-dependent attractive forces existing between the cell pair as a function of distance or time (Figure 2a3a). Force curves may exhibit specific profiles characteristic of the unfolding patterns of adhesins under study (Figure 2a3a, right), while specificity may be further supported using appropriate negative controls, such as mutant cells not expressing the adhesin (Figure 2b3b).

Figure 23.

SCFS demonstration of the cellular role of a gene product in cell interaction. FluidFM-based SCFS allows for many different strains to be used as cell probes successively. Each cell probe can be brought into contact with many different cells in a petri dish. (

a

,

left

) A yeast cell probe attached to the cantilever interacts with a cell in the petri dish on the surface of the microscope stage through multiple cycles of contact and retraction of the probe. In the case of a mating experiment, the two cells are of opposite mating types. (

middle

) Details of cell surface showing cell membrane and cell wall, including blue glucan fibers and adhesins covalently crosslinked to the glucan through modified C-terminal GPI-anchors. (

right

) Histogram of rupture forces on successive adhesion events as the cells are brought into contact, and then separated; the inset shows force–distance curves for three representative contacts. (

b) The situation when the cell on the microscope stage is a mutant cell, in this case, lacking a cell surface adhesin. This phenotype would result from mutation in the adhesin gene, in a gene required for cellular localization of the adhesin, or in a gene that regulates expression of these genes. In this case, the experiment yields infrequent cell–cell adhesions; thus, 97% of the F–D curves show no force needed for separation. The F–D plots are intended as illustrations only and are reprinted from Dehullu et al. ^[22]

) The situation when the cell on the microscope stage is a mutant cell, in this case, lacking a cell surface adhesin. This phenotype would result from mutation in the adhesin gene, in a gene required for cellular localization of the adhesin, or in a gene that regulates expression of these genes. In this case, the experiment yields infrequent cell–cell adhesions; thus, 97% of the F–D curves show no force needed for separation. The F–D plots are intended as illustrations only and are reprinted from Dehullu et al. [11].

2.1.2. Intercellular Adhesion during Mating

As such, FluidFM-based SCFS was recently applied to study adhesive interactions in yeast mating by bringing a pair of MATα and MATa cells in contact while meticulously controlling contact force and time ^[23][36]. Subsequent pulling revealed the forced extension and unbinding behavior of single interacting α-agglutinin/a-agglutinin partners ^[23][36]. Under minimal contact time (0.1 s) and contact force (0.25 nN) conditions, a relatively weak single bond strength of approximately 100 pN was observed for pheromone-induced cells. Strikingly, increasing either the contact time (1 s) or the contact force (1 nN) led to sharply increased magnitudes of rupture forces, as well as the frequencies of binding events occurring. This supports the notion of high-avidity α-agglutinin/a-agglutinin interactions playing an important role in strengthening adhesion during MATα–MATa mating. A question was whether contact-facilitated agglutinin expression over longer timescales may result in enhanced adhesion between cell pairs. Meticulous control of cell–cell contact is one of the unique capabilities of SCFS. Therefore, this question could be addressed by taking force measurements before and after an extended cell–cell contact. In essence, the adhesion force was measured between a mating pair, and then the cells were brought into contact for 30 min, after which a set of adhesion force measurements were again performed. This approach revealed that initial MATα–MATa cell contact promotes surface expression of agglutinins, resulting in increased strength of the adhesive contact between a mating cell pair. AFM is uniquely suited to answer another question: how does mechanical stress (for example, caused by fluid shear flow encountered during the normal lifestyles of most microbes) influence the bond strength of the MATα–MATa cell pair? By increasing the pulling speed of the cell probe, the effect that high force loading rates have on forced unbinding of molecular complexes can be investigated. Remarkably, it was observed that high loading rates correlated with increased bond strength. This result indicates that mechanical stress may enhance agglutinin-dependent intercellular adhesion required for mating. This study proved the concept of using SCFS as a powerful tool to study cell–cell mating interactions, a major scientific breakthrough that can now be applied to various mating systems.

2.2. SCFS Application to Studying Bacteria

2.2.1. Bacterial Adhesion

Recent SCFS-based analyses in bacteria demonstrated remarkable mechanostability of the single-molecular complexes formed by a number of MSCRAMM (microbial surface component-recognizing adhesive matrix molecules) adhesins and their ligands. The prototypical example is that of Staphylococcus epidermidis SdrG binding to the human plasma protein fibrinogen (Fg). X-ray crystallographic studies unraveled an exotic binding mechanism called “dock, lock, and latch” wherein a terminal peptide of Fg is tightly confined within a groove located at the center of the adhesin’s immunoglobulin-like head domain ^[24][55]. This observation fueled speculation that the complex may resist high tensile loads relevant to the in vivo context of an arterial infection. Direct evidence for this was brought by SCFS studies that quantified the mechanical strength of single SdrG–Fg interactions occurring on single living S. epidermidis cells, showing that tensile loads exceeding 2 nN could be sustained before rupture occurred ^[25][26][56,57]. To put it into perspective, this is more than ten times stronger than the streptavidin–biotin complex and practically as strong as a covalent bond ^[27][58]. Follow-up SCFS and SMFS studies found similar mechanostabilities for several related Staphylococcal MSCRAMM adhesins (reviewed in ^[28][59]). Another discovery that was made for several of these MSCRAMM adhesins is the enhancement of their binding to their ligands by mechanical stress; that is, the faster the complexes were pulled apart, the greater the tensile load that they could sustain ^[29][30][60,61]. These results exemplified the property of catch bonding, i.e., bonds whose stability (lifetime) is enhanced by tensile load ^[31][32][62,63].

2.2.2. Bacterial Adhesion in the Context of Mating

Bacillus thuringiensis is the organism used in Bt insecticides and is, thus, relatively well-studied. The subspecies israelensis harbors a large plasmid (pXO16) that confers the ability to transfer itself to many other strains ^[33][34][67,68]. MPS is a result of the Agr⁺ adhesin encoded on the plasmid. Neither its structure nor its ligand is known. Here too, SCFS studies have brought novel insights into intercellular binding occurring during mating. An SCFS approach was devised where a single recipient cell was attached to a special bioadhesive-coated colloidal probe, which could be used to force probe a donor cell attached to a surface (Figure 4a). It was found that that the bonds induced between single donor and recipient cells are remarkably strong (~2 nN) and depended on the presence of a 27 kb fragment of the pX016 plasmid encoding two giant proteins containing collagen-binding B-type domains, as well as the peptidoglycan-anchoring LPXTG motif ^[35][69]. An exciting possibility brought by the technique was to mechanically induce the transfer of the plasmid between cell partners and to measure the time of plasmid transfer (Figure 4b).

Figure 4. SCFS to probe and control bacterial conjugation. (a) Measuring forces in the adhesion of single pXO16wt or pXO16Δagr donor cells to single recipient cells. Top: Schematic of the approach. The pXO16 plasmid is represented by a red and green (pXO16wt) or green (pXO16Δagr) circle. The red part indicates the Agr region encoding large surface proteins (also indicated as a red outline around cells) that bind to recipient cells not expressing such proteins. Bottom: Histogram plots of the adhesion forces between recipient and donor cells carrying pXO16wt (left) pXO16Δagr (right). (b) Controlling plasmid transfer. Top: Schematic showing the approach consisting of (i) contact between recipient and donor cells for 15 min allowing DNA transfer, (ii) followed by retraction and rest for 45 min allowing maturation, i.e., proper expression and surface localization of agr-encoded proteins, and (iii) eventual measurements of cell–cell adhesion forces to evaluate changes in cell surface properties caused by plasmid transfer. Bottom: Histogram plots of the adhesion forces showing a loss of strong adhesion as a result of the presence of agr-encoded proteins on both cells. Using the resulting pXO16wt-cell probe to probe a naïve recipient cell restored strong adhesion as shown in the bottom left panel of (a). Figure adapted with permission from [69].

3. Summary and Conclusions

A limited number of adhesive factors important for the process of mating to occur have now been discovered and characterized in both eukaryotic and prokaryotic organisms. Conventional biochemical approaches to studying receptor–ligand interactions have pointed to high avidity in these interactions where parallel bonding, sometimes involving clustering of binding partners on the surfaces of the cells, maximizes the strength of intercellular adhesion. As adhesive interactions often need to combat shear forces that pull interacting partners apart, their characterization requires techniques that allow quantifying the tensile forces that they can resist before they rupture. As we have been detailed here, AFM SCFS approaches are suited to this task.

In the field of pathogen–host adhesion, specifically, SCFS approaches have been critical to several recent key discoveries. These include the discovery of the strongest non-covalent single-molecular interaction in nature existing between a bacterial adhesin and its ligand ^[25][26][56,57], that the binding strength of certain adhesins is enhanced by mechanical stress ^[29][30][60,61], and that some adhesins form unusual catch bonds, whose bond lifetime is enhanced by tensile load ^[36][37][64,70]. These studies serve as models for the cell–cell adhesions during mating.

In the field of mating, SCFS has now been used in a bacterial and a fungal mating system. Both studies have revealed strong interactions (nN per cell pair) due to multiple binding interactions between specific gene products. In the yeast system, herwein can compare the strength of cell–cell binding to thermodynamic and kinetic characteristics, as well as known cell surface concentrations. In yeasts, the cell–cell interactions are actually strengthened under tensile force. This technique can now assay enough cell pairs to allow phenotypic characterization of the interactions between cells with specific mutations or gene deletions in known or putative adhesins ^[23][35][36,69] or differences in surface expression levels ^[23][36]. What else lies ahead? Other possibilities offered by AFM force spectroscopy remain to be tapped into; for example, dynamic force spectroscopy can be applied to quantify out-of-equilibrium thermodynamic and kinetic parameters of single-molecular interactions as has been demonstrated for single virus–cell interactions by the Alsteens group ^{[38][39][40][41][42][43][44][45]}[71,72,73,74,75,76,77,78]. This methodology can serve as an excellent platform to investigate the effect of small-molecule inhibitors of adhesive interactions under physiologically relevant conditions. Force clamp AFM allows measuring the stabilities of receptor–ligand complexes under sustained discrete tensile loads ^[36][64]. A question that this methodology could help address is how adhesive interactions during mating respond to mechanical stresses and whether they are enhanced by them. AFM SCFS protocols exist for the study of interactions between single living mammalian cell pairs ^[8][15][46][8,43,79]; therefore, it can also be applied to investigate the forces in the adhesive interactions between gametes.

AFM has already proven itself as a singularly powerful tool to investigate single-molecular interactions that govern cellular adhesion. ItWe isare confident that the SCFS modality will play a key role in future breakthroughs on cell–cell mating.