Measurement of the mechanical behavior of yeast membrane sensors using single-molecule atomic force microscopy

Autor(en): Heinisch, Juergen J.
Dupres, Vincent
Alsteens, David
Dufrene, Yves F.
Stichwörter: Biochemical Research Methods; Biochemistry & Molecular Biology; CELLS; DOMAINS; INTEGRITY; MID2; TRANSFORMATION; WSC1
Erscheinungsdatum: 2010
Herausgeber: NATURE PUBLISHING GROUP
Journal: NATURE PROTOCOLS
Volumen: 5
Ausgabe: 4
Startseite: 670
Seitenende: 677
Zusammenfassung: 
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 Ni2+-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 similar to 3 weeks.
ISSN: 17542189
DOI: 10.1038/nprot.2010.19

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