Research ArticleBIOTECHNOLOGY

Kinetics of small molecule interactions with membrane proteins in single cells measured with mechanical amplification

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Science Advances  23 Oct 2015:
Vol. 1, no. 9, e1500633
DOI: 10.1126/sciadv.1500633
  • Fig. 1 Detection of molecular interactions with membrane proteins in cells through mechanical amplification.

    (A) Schematic illustration of the experimental setup based on an inverted phase-contrast microscope with a 40× phase 2 objective. (B) Differential optical detection for accurate tracking of cell edge changes induced by analyte-receptor interaction. (C) Schematic of a typical binding curve as determined from the cell edge movement. (D) The root mean square of the fixed cell edge change is 0.46 nm. (E) Illustration of cell edge changes over time during the binding process, where i, ii, and iii correspond to the stages marked in (C). Blue and red rectangles in (B) and (E) are the ROIs for differential detection.

  • Fig. 2 Large molecule interactions: WGA interaction with glycoproteins.

    (A and B) Phase-contrast images of fixed CP-D cells with binding of WGA [0.56 μΜ (20 μg/ml) (A) and 0.14 μM (5 μg/ml) (B)]. The white arrows mark the cells under analysis. (C and D) Averaged cell edge movement over the whole cell (black dots) and global fitting (red curves) for 0.56 μΜ (20 μg/ml) (C) and 0.14 μM (5 μg/ml) (D) WGA, respectively. Scale bars, 10 μm.

  • Fig. 3 Small-molecule interactions: Acetylcholine interaction with nAChRs in cells.

    (A) Phase-contrast image of fixed hα4β2-transfected SH-EP1 cells, where the white arrow marks the cell under analysis. Scale bar, 20 μm. (B) Averaged cell edge movement over the whole cell (black dots) and global fitting results (red curves) during the binding process for acetylcholine of different concentrations (from bottom to top: 1, 5, 20, and 100 nM). (C) Cell edge movement at equilibrium versus acetylcholine concentration. The equilibrium constant (KD) was determined to be ~26 nM by fitting the data with the Langmuir isotherm.

  • Fig. 4 Heterogeneity of small-molecule interactions with cell membrane receptors.

    (A and C) Phase-contrast images of fixed hα4β2-transfected SH-EP1 cells, where the numbers mark the cells under analysis. Scale bars, 20 μm. (B) Binding kinetics of cells 1 and 2 (100 nM acetylcholine). (D) Binding kinetics of cells 3 and 4 (100 nM acetylcholine). (E) Binding kinetics at different locations of the cell in (F) (100 nM acetylcholine). (F) Phase-contrast image of a fixed hα4β2-transfected SH-EP1 cell. The color represents the amount of cell membrane edge deformation. Scale bar, 20 μm.

  • Table 1 Association rate constants (kon), dissociation rate constants (koff), and equilibrium constants (KD) for four cells as shown in Fig. 4, A to D.
    kon (M−1 s−1)koff (s−1)KD (nM)
    Cell 17.3 ± 0.2 × 1051.61 ± 0.02 × 10−222.0 ± 0.6
    Cell 28.3 ± 0.3 × 1052.17 ± 0.03 × 10−226.1 ± 0.7
    Cell 33.4 ± 0.2 × 1052.98 ± 0.03 × 10−288.0 ± 4.0
    Cell 45.8 ± 0. 9 × 1051.72 ± 0.01 × 10−229.9 ± 0.5
    Mean6.2 × 1052.12 × 10−241.5
    SD2.1 × 1050.62 × 10−231.2

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/1/9/e1500633/DC1

    WGA and glycoprotein interactions in a live cell

    Fig. S1. WGA and glycoprotein interactions in a live cell.

    Anti-EGFR antibody interaction with EGFR in fixed A431 cells

    Fig. S2. Anti-EGFR antibody interaction with EGFR in fixed A431 cells.

    Negative control for nAChR and acetylcholine interaction

    Fig. S3. Negative control for nAChR and acetylcholine interaction.

    nAChR immunofluorescence

    Fig. S4. Phase-contrast and immunofluorescence images of nAChR-positive and nAChR-negative cells.

    Noise power spectrum of cell edge movement of a fixed CP-D cell

    Fig. S5. Noise power spectrum of cell edge movement of a fixed CP-D cell.

    Molecular binding–induced membrane deformation along cell edges

    Fig. S6. Molecular binding–induced membrane deformation along cell edges.

    Calibration of the differential imaging intensity and cell edge movement

    Fig. S7. Calibration of the differential detection of cellular edge movement.

    Differential detection method

    Fig. S8. Diagram illustrating the procedure of the differential detection method.

    Statistic analysis of WGA and glycoprotein interactions on fixed CP-D cells

    Table S1. Binding kinetics between WGA and glycoprotein on different fixed CP-D cells.

    Statistic analysis of WGA and glycoprotein interactions on live CP-D cells

    Table S2. Binding kinetics between WGA and glycoprotein on different live CP-D cells.

    Statistic analysis of acetylcholine and nAChR interaction on fixed SH-EP1-hα4β2 cells.

    Table S3. Binding kinetics between acetylcholine and nAChRs on different fixed SH-EP1-hα4β2 cells.

    Reference (43)

  • Supplementary Materials

    This PDF file includes:

    • WGA and glycoprotein interactions in a live cell
    • Fig. S1. WGA and glycoprotein interactions in a live cell.
    • Anti-EGFR antibody interaction with EGFR in fixed A431 cells
    • Fig. S2. Anti-EGFR antibody interaction with EGFR in fixed A431 cells.
    • Negative control for nAChR and acetylcholine interaction
    • Fig. S3. Negative control for nAChR and acetylcholine interaction.
    • nAChR immunofluorescence
    • Fig. S4. Phase-contrast and immunofluorescence images of nAChR-positive and
    • nAChR-negative cells.
    • Noise power spectrum of cell edge movement of a fixed CP-D cell
    • Fig. S5. Noise power spectrum of cell edge movement of a fixed CP-D cell.
    • Molecular binding–induced membrane deformation along cell edges
    • Fig. S6. Molecular binding–induced membrane deformation along cell edges.
    • Calibration of the differential imaging intensity and cell edge movement
    • Fig. S7. Calibration of the differential detection of cellular edge movement.
    • Differential detection method
    • Fig. S8. Diagram illustrating the procedure of the differential detection method.
    • Statistic analysis of WGA and glycoprotein interactions on fixed CP-D cells
    • Table S1. Binding kinetics between WGA and glycoprotein on different fixed CPD cells.
    • Statistic analysis of WGA and glycoprotein interactions on live CP-D cells
    • Table S2. Binding kinetics between WGA and glycoprotein on different live CPD cells.
    • Statistic analysis of acetylcholine and nAChR interaction on fixed SH-EP1-hα4β2 cells.
    • Table S3. Binding kinetics between acetylcholine and nAChRs on different fixed SH-EP1-hα4β2 cells.
    • Reference (43)

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