Interaction between Pheromone and Its Receptor of the Fission Yeast Schizosaccharomyces pombe Examined by a Force Spectroscopy Study
Figure 1
Schematic overview of experiments. (a) AFM tip modification with peptides. Si3N4 AFM tips are aminosilanized by exposure to APTES vapors. A heterobifunctional PEG linker is anchored to amino-group bearing tips through its NHS end. Peptide is attached to the PEG linker free end via a maleimide-cysteine bond. (b) Force spectroscopy method. Peptide-modified AFM tips approached the cell surface on a 200 nm z-scan size at a speed of 1.74 μm/s and were retracted at the same speed. (c) The reporter assay. Pheromone binding to its receptors on the cell surface activates the intracellular signaling pathway that leads to the expression of GFP. The released Gpa1 (Galpha) with GTP from a heterotrimeric G protein activates the MAP kinase cascade of Byr2 (MAP3K), Byr1 (MAP2K), and Spk1 (MAPK). Activation of Byr2 also requires Ras1.
