| dc.description.abstract | The cell surface of yeast cells are covered with a specific set of molecules that are involved in cellular communication, adhesion and microbial infection. In an effort to expand the knowledge of the relevant interactions involved in these processes, the interactions between the lectins Concanavalin A (ConA) and Wheat germ agglutinin (WGA) and the cell wall polysaccharides of the model organism Saccharomyces cerevisiae, were quantified using the ultrasensitive force probe optical tweezers. By including genetically modified strains with defects in the synthetic pathways of the main components of the yeast cell wall, information about the location of each component can be obtained. In addition to the wild type, the mutant strains ∆mnn9, ∆gas1 and ∆chs3 were studied, each having a defect in the synthesis of cell wall mannan, β-glucan and chitin, respectively.
The receptor for ConA was found in the cell surface of all the strains under study. Binding strengths from 3 pN to ∼70 pN were observed, at a loading rate intervall of 40-300 pN/s.
Using the Bell-Evans model to determine properties of the energy land- scape, single energy barriers were identified at xβ = 0.42 nm, xβ = 0.41 nm, xβ = 0.47 nm and xβ = 0.64 nm for wild type, ∆gas1, ∆chs3 and ∆mnn9, respectively. The interaction between ConA and ∆mnn9 most likely involves the extended binding site on ConA and the trimannoside of the exposed core oligosaccharide on cell wall proteins.
For the ConA - yeast interactions, dissociation rates in the absence of applied force were estimated to be k0 = 3.99 s−1, k0 = 4.53 s−1, k0 = 1.85 s−1 and k0 = 3.59 s−1, for wild type, ∆gas1, ∆chs3 and ∆mnn9, respectively. This corresponds to bond lifetimes of τ0 = 0.25 s, τ0 = 0.22 s, τ0 = 0.54 s and τ0 = 0.28 s, respectively.
The receptor for WGA was not found at the surface of the cell wall for wild type, ∆mnn9 or ∆chs3. However, interactions were observed frequently for ∆gas1. Binding strengths ranging from 4 pN to 55 pN were observed, at a loading rate interval of 45-300 pN/s.
The Bell-Evans model revealed two energy barriers in the energy landscape. An inner barrier is located at xβ = 0.14 nm and an outer barrier at xβ = 0.90 nm. For these energy barriers, dissociation rates of k0 = 3.5 s−1 and k0 = 2.42 s−1 were estimated, respectively. This corresponds to lifetimes of τ0 = 0.29 s and τ0 = 0.41 s, respectively.
Immobilization of mannan on amino polystyrene beads is possible using a two step procedure involving oxidation and subsequent formation of a Schiff base. Insufficient surface coverage for single molecule studies with optical tweezers is acheived when introducing ∼ 10 aldehyde groups per mannan
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molecule and allowing the immobilization reaction to take place in methanol. Immobilization of chitosan on carboxy polystyrene beads using EDCA as a crosslinker is dependent on the degree of acetylation, chain length and the pH of the reaction mixture, and is not possible for relatively short chitosan samples with FA = 0.49 at pH 5.8. | en |
