o cell wall polysaccharide. The overlapping binding specificities and variable expression of Als proteins make them difficult to study in C. albicans. Nevertheless, expression in Saccharomyces cerevisiae has shown that Als1p, Als3p and Als5p bind similarly to a variety of substrates and are involved in endothelial cell adhesion, fungal aggregation, and tissue invasion. Als-mediated adhesion to substrate is followed 50 min later by aggregation of Als-expressing yeast cells. This aggregation is accompanied by a cell surface conformational change in the Als proteins to mediate stronger adhesive interactions. This activation is independent of cellular metabolism, because it occurs in heat-killed cells. The T domains of Als proteins contain amyloid-forming sequences that are highly conserved. Amyloids are insoluble fibrillar protein aggregates whose cores consist of crystalline arrays of identical sequences in many molecules of the amyloid protein. In the presence of amyloids, 
Congo red absorbance is redshifted and increased, and the fluorescence emission of thioflavin T increases several fold. At high concentrations, these dyes can perturb amyloid structure. Some amyloidforming bacterial adhesins can elicit cell-cell and cell-substrate adhesion leading to the formation of biofilms. Other known roles for amyloid include amyloid-like stacking of residues in b-helices in viral spike proteins, curli in gram negative bacteria, sequestration of regulatory proteins in yeast March 2011 | Volume 6 | Issue 3 | e17632 Amyloids in Cell Aggregation and Biofilms 2 March 2011 | Volume 6 | Issue 3 | e17632 Amyloids in Cell Aggregation and Biofilms , packing of pro-hormones in secretory vesicles, and as template activity for melanin assembly. Previously, we showed that the amyloid sequence in the T region of Als proteins mediates amyloid formation, and that amyloid binding dyes can inhibit aggregation in the S. cerevisiae surface display model. However, there is no GSK2330672 direct data for the roles of Als amyloid sequences per se in vivo. We have therefore created a version of Als5p with its amyloid sequence disrupted by a single site substitution, and assayed its effects in the S. cerevisiae display model. We report the effects of this mutation on aggregation and biofilm formation in a model system. In addition, we have tested amyloid-forming and amyloid-inhibiting peptides for their effect in vivo in C. albicans. Finally, we provide direct evidence for the formation of amyloid adhesion nanodomains in C. albicans, using single-molecule atomic force microscopy . Results Cells expressing a mutation in the amyloid sequence of Als5p exhibit less efficient aggregation S. cerevisiae cells expressing Als5p form large aggregates, 21609844 similar to those seen in C. albicans. This cell aggregation can be inhibited by amyloid binding dyes. A V326N mutation in the Als5p sequence reduces TANGO b-aggregation potential of the amyloid region from 93% to 4%. To test the hypothesis that the amyloid forming sequence is critical for cell aggregation, we incorporated the V326N mutation into full length wild type Als5p, and expressed it in S. cerevisiae. Immunofluorescence confirmed that that both wild type Als5p and the substitution sequence were expressed on the yeast cell surface, with non-amyloid Als5pV326N expression slightly greater than Als5pWT. In agreement with previous results, cells expressing Als5pWT bound to heat denatured BSAcoated beads and formed large aggregates . Cells expre