Understanding how cell adhesion proteins form adhesion domains is a key challenge in cell biology. Here, we use single-molecule atomic force microscopy (AFM) to demonstrate the force-induced formation and propagation of adhesion nanodomains in living fungal cells, focusing on the covalently anchored cell-wall protein Als5p from Candida albicans. We show that pulling on single adhesins with AFM tips terminated with specific antibodies triggers the formation of adhesion domains of 100-500 nm and that the forceinduced nanodomains propagate over the entire cell surface. Control experiments (with cells lacking Als5p, single-site mutation in the protein, bare tips, and tips modified with irrelevant antibodies) demonstrate that Als5p nanodomains result from protein redistribution triggered by force-induced conformational changes in the initially probed proteins, rather than from nonspecific cell-wall perturbations. Als5p remodeling is independent of cellular metabolic activity because heat-killed cells show the same behavior as live cells. Using AFM and fluorescence microscopy, we also find that nanodomains are formed within ∼30 min and migrate at a speed of ∼20 nm·min −1 , indicating that domain formation and propagation are slow, time-dependent processes. These results demonstrate that mechanical stimuli can trigger adhesion nanodomains in fungal cells and suggest that the force-induced clustering of adhesins may be a mechanism for activating cell adhesion.single-molecule techniques | microbial adhesion | glycoproteins | fungi C ell adhesion is a ubiquitous feature of living cells and plays essential roles in a variety of cellular processes, including neuronal interactions, cellular communication, inflammation, and microbial infection (1-5). A growing body of evidence indicates that, in mammalian cells, the early stage of adhesion involves the formation of adhesion domains (i.e., focal adhesion complexes) composed of aggregated proteins (6, 7). A remarkable trait of adhesion domains is their ability to grow and strengthen under force (8-10). Whether such force-dependent behavior also occurs with microbial adhesins is unknown.Adhesins in the Candida albicans Als gene family bind the pathogen to host tissues and initiate biofilm formation (11,12). Als proteins are covalently attached to the cell wall and consist of an N-terminal Ig-like region, which initiates cell adhesion, followed by a threonine-rich region with an amyloid-forming sequence (T), a tandem repeat (TR) region that participates in cellcell aggregation, and a stalk region projecting the molecule away from the cell surface (Fig. 1A) (13-15). Previous studies in C. albicans and in a Saccharomyces cerevisiae surface display model showed that Als5p-mediated adhesion involves a rapid cellto-ligand adhesion step, followed by slower cell-to-cell aggregation mediated by the T and TR regions (13, 15). These kinetics suggest that, following adhesion to ligands, Als5p may undergo conformational changes that mediate cellular aggregation. Consistent with this, Rauceo ...

Cell adhesion molecules in Saccharomyces cerevisiae and Candida albicans contain amyloid-forming sequences that are highly conserved. We have now used site-specific mutagenesis and specific peptide perturbants to explore amyloid-dependent activity in the Candida albicans adhesin Als5p. A V326N substitution in the amyloid-forming region conserved secondary structure and ligand binding, but abrogated formation of amyloid fibrils in soluble Als5p and reduced cell surface thioflavin T fluorescence. When displayed on the cell surface, Als5p with this substitution prevented formation of adhesion nanodomains and formation of large cellular aggregates and model biofilms. In addition, amyloid nanodomains were regulated by exogenous peptides. An amyloid-forming homologous peptide rescued aggregation and biofilm activity of Als5pV326N cells, and V326N substitution peptide inhibited aggregation and biofilm activity in Als5pWT cells. Therefore, specific site mutation, inhibition by anti-amyloid peturbants, and sequence-specificity of pro-amyloid and anti-amyloid peptides showed that amyloid formation is essential for nanodomain formation and activation.