Date of Degree

10-2014

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biochemistry

Advisor(s)

Peter lipke

Subject Categories

Biochemistry

Abstract

Candida albicans adhesins have amyloid-forming sequences (Ramsook et al. 2010, Otoo et al. 2008). Similarly, Tango and Waltz predicted that amyloid-forming sequences are also present in Saccharomyces cerevisiae flocculins, Flo1p and Flo11p. The cell surface of Flo1p- and Flo11p-expressing cells have ordered domains that are thioflavin T fluorescent and Congo red birefringent, two hallmarks of amyloids. Flo1p and Flo11p amyloids were important for activities of the flocculins including cell-to-cell adhesion, cell-to-substrate adhesion, and agar invasion. In addition, amyloid-perturbing dyes thioflavin S and Congo red inhibited aggregation, biofilm formation and agar invasion.

Force-induced formation and propagation of adhesion nanodomains in Als5p-expressing cells were demonstrated with single-molecule atomic force microscopy (Alsteens et al. 2010). Because amyloid formation can be triggered by force, we investigated whether laminar flow and mechanical stress could activate amyloid formation and therefore increase adhesion. Shearing S. cerevisiae cells expressing Als5p or C. albicans at 0.8 dyne/cm2 increased quantity and strength of cell-to-surface and cell-to-cell binding, compared to 0.02 dyne/cm2. Mechanical stress from vortex-mixing at 2500 rpm also increased the aggregation of S. cerevisiae cells expressing Als5p or C. albicans. Similarly, cells expressing Flo1p and Flo11p also showed shear-and mechanical stress-dependent binding, and biofilm formation.

I report here for the first time that catch bonding behavior in yeast cells was mediated by amyloid formation. Amyloids mediate both sensing and response in the presence of force. Adhesin-expressing cells binding to surfaces under shear stress were less likely to be washed off from the substrate than cells that were not stressed. This is characteristic of catch bonding. Catch bonding behavior was accompanied by the formation of amyloid nanodomains through the clustering at homotypic amyloid sequences. Thus, these nanodomains increased binding avidity of the adhesin-expressing cells to other cells (flocculation and aggregation assays) and to substrate surfaces.

Furthermore we have devised ways of quantifying forces needed to activate aggregation, cell adhesion, and amyloids on the surface of yeast cells. Two different types of force, mechanical stress from vortex-mixing and shear stress from laminar flow increased adhesion and biofilm formation. Additionally, we quantified amyloid formation in live whole cell yeast suspensions in response to force. Fluorescent confocal microscopy and flow cytometry were used to quantify surface amyloids. Force-activated cells had punctate nanodomains with increased thioflavin T staining. Collectively, the assays can now be used to quantify amyloids in other fungal adhesins.

These results demonstrate that 1. there are functional amyloids present in fungal adhesins Flo1p and Flo11p from S. cerevisiae, 2. amyloid formation mediates adhesion, agar invasion and biofilm, 3. amyloid nanodomains mediate force-sensitive catch-bonding, and 4. force-sensitive amyloid formation on the yeast cell wall surface can be quantified.

Included in

Biochemistry Commons

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