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Interaction between pheromone and its receptor of the fission yeast Schizosaccharomyces pombe examined by a force spectroscopy study.

Sasuga S, Abe R, Nikaido O, Kiyosaki S, Sekiguchi H, Ikai A, Osada T - J. Biomed. Biotechnol. (2012)

Bottom Line: An AFM tip was modified with P-factor derivatives to perform force curve measurements.When the AFM tip was modified with truncated P-factor derivative lacking C-terminal Leu, the specific interaction between the tip and the cell surface was not observed.These results were also confirmed with an assay system using a green fluorescent protein (GFP) reporter gene to monitor the activation level of signal transduction following the interaction of Mam2 with P-factor.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Science, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Kanagawa, Yokohama, Japan.

ABSTRACT
Interaction between P-factor, a peptide pheromone composed of 23 amino acid residues, and its pheromone receptor, Mam2, on the cell surface of the fission yeast Schizosaccharomyces pombe was examined by an atomic force microscope (AFM). An AFM tip was modified with P-factor derivatives to perform force curve measurements. The specific interaction force between P-factor and Mam2 was calculated to be around 120 pN at a probe speed of 1.74 μm/s. When the AFM tip was modified with truncated P-factor derivative lacking C-terminal Leu, the specific interaction between the tip and the cell surface was not observed. These results were also confirmed with an assay system using a green fluorescent protein (GFP) reporter gene to monitor the activation level of signal transduction following the interaction of Mam2 with P-factor.

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Related in: MedlinePlus

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.
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fig1: 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.

Mentions: Coupling of peptides to AFM Si3N4 tips (OMCL-TR400PSA, Olympus, Tokyo, Japan; nominal value 0.02 N/m) was done using a heterobifunctional polyethylene glycol (PEG) linker as shown in Figure 1(a) [23, 24]. The AFM tips were cleaned in a UV ozone cleaner (UV/Ozone ProCleaner, Bioforce Nanosciences Inc., IA, USA) under ultraviolet light and exposed for 2 h to APTES (3-aminopropyl triethoxysilane) vapors in a 2-liter desiccator filled with argon and containing 30 μL of APTES and 10 μL of N,N-diisopropylethylamine (Sigma-Aldrich, Tokyo, Japan). The tips were then kept for up to 3 days in an argon-filled atmosphere until use. Amino-group bearing tips were incubated for 60 min with 1 mg/mL of N-hydroxy-succinimide ester-PEG-maleimide (NHS-PEG-MAL, 3400 Da, Nektar Therapeutics, Huntsville, AL) in PBS (phosphate-buffered saline). They were then washed several times with PBS to remove unanchored linker molecules. The final binding step was achieved by a reaction between the linker maleimide end and cysteine residues of peptides. The tips were incubated with each peptide (final concentration of 1 μM) in PBS for 30 min and then were abundantly washed with PBS to remove unbound peptides.


Interaction between pheromone and its receptor of the fission yeast Schizosaccharomyces pombe examined by a force spectroscopy study.

Sasuga S, Abe R, Nikaido O, Kiyosaki S, Sekiguchi H, Ikai A, Osada T - J. Biomed. Biotechnol. (2012)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3303674&req=5

fig1: 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.
Mentions: Coupling of peptides to AFM Si3N4 tips (OMCL-TR400PSA, Olympus, Tokyo, Japan; nominal value 0.02 N/m) was done using a heterobifunctional polyethylene glycol (PEG) linker as shown in Figure 1(a) [23, 24]. The AFM tips were cleaned in a UV ozone cleaner (UV/Ozone ProCleaner, Bioforce Nanosciences Inc., IA, USA) under ultraviolet light and exposed for 2 h to APTES (3-aminopropyl triethoxysilane) vapors in a 2-liter desiccator filled with argon and containing 30 μL of APTES and 10 μL of N,N-diisopropylethylamine (Sigma-Aldrich, Tokyo, Japan). The tips were then kept for up to 3 days in an argon-filled atmosphere until use. Amino-group bearing tips were incubated for 60 min with 1 mg/mL of N-hydroxy-succinimide ester-PEG-maleimide (NHS-PEG-MAL, 3400 Da, Nektar Therapeutics, Huntsville, AL) in PBS (phosphate-buffered saline). They were then washed several times with PBS to remove unanchored linker molecules. The final binding step was achieved by a reaction between the linker maleimide end and cysteine residues of peptides. The tips were incubated with each peptide (final concentration of 1 μM) in PBS for 30 min and then were abundantly washed with PBS to remove unbound peptides.

Bottom Line: An AFM tip was modified with P-factor derivatives to perform force curve measurements.When the AFM tip was modified with truncated P-factor derivative lacking C-terminal Leu, the specific interaction between the tip and the cell surface was not observed.These results were also confirmed with an assay system using a green fluorescent protein (GFP) reporter gene to monitor the activation level of signal transduction following the interaction of Mam2 with P-factor.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Science, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Kanagawa, Yokohama, Japan.

ABSTRACT
Interaction between P-factor, a peptide pheromone composed of 23 amino acid residues, and its pheromone receptor, Mam2, on the cell surface of the fission yeast Schizosaccharomyces pombe was examined by an atomic force microscope (AFM). An AFM tip was modified with P-factor derivatives to perform force curve measurements. The specific interaction force between P-factor and Mam2 was calculated to be around 120 pN at a probe speed of 1.74 μm/s. When the AFM tip was modified with truncated P-factor derivative lacking C-terminal Leu, the specific interaction between the tip and the cell surface was not observed. These results were also confirmed with an assay system using a green fluorescent protein (GFP) reporter gene to monitor the activation level of signal transduction following the interaction of Mam2 with P-factor.

Show MeSH
Related in: MedlinePlus