Measurement of the mechanical behavior of yeast membrane sensors using single-molecule atomic force microscopy.
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.
Heinisch
Jürgen J
JJ
Fachbereich Biologie/Chemie, Universität Osnabrück, Osnabrück, Germany. Juergen.Heinisch@Biologie.Uni-Osnabrueck.DE
Dupres
Vincent
V
Alsteens
David
D
Dufrêne
Yves F
YF
D001696:Biomechanical Phenomena; D055592:Biophysical Phenomena; D002462:Cell Membrane / Q000502:physiology; D008565:Membrane Proteins / Q000235:genetics / Q000502:physiology; D018625:Microscopy, Atomic Force / Q000379:methods*; D036103:Nanotechnology; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000502:physiology; D015398:Signal Transduction
D008565:Membrane Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins