SUMMARY: Fungi are nonmotile eukaryotes that rely on their adhesins for selective interaction with the environment and with other fungal cells. Glycosylphosphatidylinositol (GPI)-cross-linked adhesins have essential roles in mating, colony morphology, host-pathogen interactions, and biofilm formation. We review the structure and binding properties of cell wall-bound adhesins of ascomycetous yeasts and relate them to their effects on cellular interactions, with particular emphasis on the agglutinins and flocculins of Saccharomyces and the Als proteins of Candida . These glycoproteins share common structural motifs tailored to surface activity and biological function. After being secreted to the outer face of the plasma membrane, they are covalently anchored in the wall through modified GPI anchors, with their binding domains elevated beyond the wall surface on highly glycosylated extended stalks. N-terminal globular domains bind peptide or sugar ligands, with between millimolar and nanomolar affinities. These affinities and the high density of adhesins and ligands at the cell surface determine microscopic and macroscopic characteristics of cell-cell associations. Central domains often include Thr-rich tandemly repeated sequences that are highly glycosylated. These domains potentiate cell-to-cell binding, but the molecular mechanism of such an association is not yet clear. These repeats also mediate recombination between repeats and between genes. The high levels of recombination and epigenetic regulation are sources of variation which enable the population to continually exploit new niches and resources.

Yeast cell wall proteins, including Cwp1p and α-agglutinin, could be released by treating the cell wall with either β-1,3- or β-1,6-glucanases, indicating that both polymers are involved in anchoring cell wall proteins. It was shown im-munologically that both β-1,3- and β-1,6-glucan were linked to yeast cell wall proteins, including Cwp1p and α-agglutinin. It was further shown that β-1,3-glucan was linked to the wall protein through a β-1,6-glucan moiety. The β-1,6-glucan moiety could be removed from Cwp1p and other cell wall proteins by cleaving phosphodiester bridges either enzymatically using phosphodiesterases or chemically using ice-cold aqueous hydrofluoric acid. These observations are consistent with the notion that cell wall proteins in Saccharomyces cerevisiae are linked to a β-1,3-/β-1,6-glucan heteropolymer through a phosphodiester linkage and that this polymer is responsible for anchoring cell wall proteins. It is proposed that this polymer is identical to the alkali-soluble β-1,3-/β-1,6-glucan heteropolymer characterized by Fleet and Manners (1976,1977).

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 force-induced 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.

The cell adhesion protein alpha-agglutinin is bound to the outer surface of the Saccharomyces cerevisiae cell wall and mediates cell-cell contact in mating. alpha-Agglutinin is modified by addition of a glycosyl phosphatidylinositol (GPI) anchor as it traverses the secretory pathway. The presence of a GPI anchor is essential for cross-linking into the wall, but the fatty acid and inositol components of the anchor are lost before cell wall association (Lu, C.-F., J. Kurjan, and P. N. Lipke, 1994. A pathway for cell wall anchorage of Saccharomyces cerevisiae alpha-agglutinin. Mol. Cell. Biol. 14:4825-4833). Cell wall association of alpha-agglutinin was accompanied by an increase in size and a gain in reactivity to antibodies directed against beta 1,6-glucan. Several kre mutants, which have defects in synthesis of cell wall beta 1,6-glucan, had reduced molecular size of cell wall alpha-agglutinin. These findings demonstrate that the cell wall form of alpha-agglutinin is covalently associated with beta 1,6-glucan. The alpha-agglutinin biosynthetic precursors did not react with antibody to beta 1,6-glucan, and the sizes of these forms were unaffected in kre mutants. A COOH-terminal truncated form of alpha-agglutinin, which is not GPI anchored and is secreted into the medium, did not react with the anti-beta 1,6-glucan. We propose that extracellular cross-linkage to beta 1,6-glucan mediates covalent association of alpha-agglutinin with the cell wall in a manner that is dependent on prior addition of a GPI anchor to alpha-agglutinin.