Heinisch JJ, et al. (2010) Measurement of the mechanical behavior of yeast membrane sensors using single-molecule atomic force microscopy. Nat Protoc 5(4):670-7
Abstract: In Saccharomyces cerevisiae, surface stresses acting on the cell wall or plasma membrane are detected by a group of five membrane sensors: Wsc1, Wsc2, Wsc3, Mid2 and Mtl2. Here we present protocols to measure the mechanical properties of Wsc1 sensors in their native cellular environment, using the combination of genetic manipulations with single-molecule atomic-force microscopy (AFM). We describe procedures (i) for obtaining genetically modified sensors that are fully functional and suitable for AFM analysis, i.e., elongated Wsc1 derivatives terminated with a His-tag, and (ii) for detecting and stretching single Wsc1 sensors on the surface of living S. cerevisiae cells, using AFM tips functionalized with Ni(2+)-NTA groups. These procedures are multidisciplinary to implement and need competent researchers from at least two disciplines: molecular biology and nanotechnology. For experienced researchers in biological AFM, the entire protocol can be completed in approximately 3 weeks.
|Status: Published||Type: Journal Article||PubMed ID: 20360762|
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