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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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Force histogram of unbinding events with (red columns) or without (blue columns) free 1 μM P-factor. (a) Unbinding events between AFM tip modified with P-factor and mam2Δ strain cells. (b) Unbinding events between AFM tip modified with P-factorΔLeu and mam2+ strain cells.
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fig3: Force histogram of unbinding events with (red columns) or without (blue columns) free 1 μM P-factor. (a) Unbinding events between AFM tip modified with P-factor and mam2Δ strain cells. (b) Unbinding events between AFM tip modified with P-factorΔLeu and mam2+ strain cells.

Mentions: A force-volume mode of AFM was carried out to examine specific interactions between pheromone and pheromone receptor. Using the AFM tip cross-linked with P-factor derivatives via a heterobifunctional PEG linker, 1024 AFM force curves were then obtained over different spots on mam2+ strain cells expressing pheromone receptors. Although most of retraction curves (around 90%) showed no interaction (Figure 2(a), upper curve), some retraction curves presented a downward deflection abruptly ending with a force jump (Figure 2(a), others). Since the PEG linker used to attach the peptide to the AFM tip has a total length of 30 nm, only events occurring after 30 nm extension were considered valid interaction events and were included into further analyses. The distribution of unbinding force is shown in Figure 2(b). To verify the specificity of the unbinding force, force curves were also obtained in the presence of 1 μM-free P-factor where specific interaction was expected to be inhibited. Ranging from 100 to 160 pN, 122 interaction peaks (11.9%) were detected without free P-factor while 33 unbinding events (3.2%) were detected with free P-factor. The number of events clearly decreased and the unbinding probability fell to 3.2 from 11.9%. Next, we carried out force curve measurements to examine interaction force between the AFM tip modified with P-factor and mam2Δ strain cells expressing no pheromone receptors, and between the AFM tip modified with P-factorΔLeu and mam2+ strain cells. When mam2Δ strain cells were used for force curve measurements with P-factor-modified tips, the unbinding probabilities were not affected with or without free P-factor, showing 2.7% without free P-factor and 2.9% with free P-factor (Figure 3(a)). When the AFM tip was modified with truncated P-factor derivative lacking C-terminal Leu, which was reported to have no P-factor function, the specific interaction was not observed, and the unbinding probabilities were almost the same with (3.5%) or without (3.1%) free P-factor (Figure 3(b)). From these three kinds of force curve measurements, the unbinding forces observed in the first force curve measurement were supposed to be caused by the specific interaction between P-factor and its receptor.


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)

Force histogram of unbinding events with (red columns) or without (blue columns) free 1 μM P-factor. (a) Unbinding events between AFM tip modified with P-factor and mam2Δ strain cells. (b) Unbinding events between AFM tip modified with P-factorΔLeu and mam2+ strain cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig3: Force histogram of unbinding events with (red columns) or without (blue columns) free 1 μM P-factor. (a) Unbinding events between AFM tip modified with P-factor and mam2Δ strain cells. (b) Unbinding events between AFM tip modified with P-factorΔLeu and mam2+ strain cells.
Mentions: A force-volume mode of AFM was carried out to examine specific interactions between pheromone and pheromone receptor. Using the AFM tip cross-linked with P-factor derivatives via a heterobifunctional PEG linker, 1024 AFM force curves were then obtained over different spots on mam2+ strain cells expressing pheromone receptors. Although most of retraction curves (around 90%) showed no interaction (Figure 2(a), upper curve), some retraction curves presented a downward deflection abruptly ending with a force jump (Figure 2(a), others). Since the PEG linker used to attach the peptide to the AFM tip has a total length of 30 nm, only events occurring after 30 nm extension were considered valid interaction events and were included into further analyses. The distribution of unbinding force is shown in Figure 2(b). To verify the specificity of the unbinding force, force curves were also obtained in the presence of 1 μM-free P-factor where specific interaction was expected to be inhibited. Ranging from 100 to 160 pN, 122 interaction peaks (11.9%) were detected without free P-factor while 33 unbinding events (3.2%) were detected with free P-factor. The number of events clearly decreased and the unbinding probability fell to 3.2 from 11.9%. Next, we carried out force curve measurements to examine interaction force between the AFM tip modified with P-factor and mam2Δ strain cells expressing no pheromone receptors, and between the AFM tip modified with P-factorΔLeu and mam2+ strain cells. When mam2Δ strain cells were used for force curve measurements with P-factor-modified tips, the unbinding probabilities were not affected with or without free P-factor, showing 2.7% without free P-factor and 2.9% with free P-factor (Figure 3(a)). When the AFM tip was modified with truncated P-factor derivative lacking C-terminal Leu, which was reported to have no P-factor function, the specific interaction was not observed, and the unbinding probabilities were almost the same with (3.5%) or without (3.1%) free P-factor (Figure 3(b)). From these three kinds of force curve measurements, the unbinding forces observed in the first force curve measurement were supposed to be caused by the specific interaction between P-factor and its receptor.

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