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First published online 20 June 2006
doi: 10.1242/jcs.03035

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Ligands on the string: single-molecule AFM studies on the interaction of antibodies and substrates with the Na⁺-glucose co-transporter SGLT1 in living cells

¹ Institute for Biophysics, Johannes Kepler University of Linz, Altenbergerstr. 69, Linz, A-4040, Austria
² Department of Epithelial Cell Physiology, Max Planck Institute of Molecular Physiology, Otto-Hahn-Str. 11, Dortmund 44227, Germany

View larger version (19K): [in a new window]   Fig. 1. Representative experiment of [14C]AMG uptake by nontransfected CHO cells and SGLT1 stably transfected G6D3 cells. Uptake was measured by incubation with 0.1 mM [14C]AMG for 15 minutes at 37°C in the presence of Na+ (KRH-NaCl, black bar), or in the absence of Na+ (KRH-NMG, white bar). Values are mean ± s.e.m., n=9; ***P<0.005 compared with levels in Na+-treated G6D3 cells.

View larger version (97K): [in a new window]   Fig. 2. Cell images of living G6D3 cells. (a) Topographical image and (b) deflection image of living cells imaged at a scan size of 70 µm. The bright regions of the image correspond to the nuclei region. The dominant features observed in both images are the cytoskeleton. (c) Crosssection analysis along the flattened cell showing the height of 832 nm. These images did not change during the course of the AFM experiments.

View larger version (31K): [in a new window]   Fig. 3. Recognition of SGLT1 on the surface of intact cells by an AFM tip carrying an epitopespecific antibody (PAN3-2). (a) Schematic representation of a force-distance cycle. The tip was moved toward the cell surface (dotted line, 12) and subsequently retracted (solid line) at a constant lateral position. During tip approach, the antibody forms a complex with an antigen that leads to a force signal with a distinct shape (3) during tip retraction. The force increases until dissociation occurs (4) at an unbinding force (fu). (b) Force curve showing specific interaction between the antibody and SGLT1 upon tipsurface retraction. The specific interaction is blocked by injecting free PAN3-2 antibodies in the solution (inset). (c) Quantitative comparison of binding probabilities of PAN3-2-coated tips on G6D3 (gray) and CHO cells (black) in the absence or presence of free PAN 3-2 in the medium. Values are mean ± s.e.m., n=2000-4000; P<0.005 compared with levels in the control group (unblocked). (d) Probability density function (pdf) giving the distribution of the unbinding force (fu) of PAN3-2 to SGLT1 (n=1000).

View larger version (27K): [in a new window]   Fig. 4. Effect of Na+ and phlorizin on the recognition probability of SGLT1 by PAN 3-2. (a) The binding probabilities of PAN3-2 tip on G6D3 cells in the presence of Na+ (KRH-NaCl buffer, gray) and (b) in the absence of Na+ (KRH-NMG buffer, blue) in the absence (dark columns) or in the presence (light columns) of phlorizin, a specific inhibitor of SGLT1. Values are mean ± s.e.m., n=2000-4000; and P<0.005 compared with levels in the control group (unblocked).

View larger version (22K): [in a new window]   Fig. 5. Recognition of SGLT1 on the surface of intact cells by a D-glucose primed AFM tip. (a) The effect of phlorizin and the antibody PAN32 in the presence of Na+ (KRH-NaCl) on the binding probabilities of D-glucose tip on G6D3 (gray) and CHO cells (black). (b) Probability density function (pdf) giving the distribution of the unbinding force fu required to disrupt single glucose-receptor interaction (n=1000). (c) Binding probabilities of D-glucose tip on G6D3 cells in the presence and in the absence of Na+ (KRH-NaCl) (gray) and KRH-NMG (sodium replaced by Nglucosamine) (blue), respectively. Values are mean ± s.e.m., n=2000-4000. *P<0.05 and ***P<0.005 compared with levels in the control group (unblocked).

View larger version (28K): [in a new window]   Fig. 6. Inhibition of D-glucose binding with the stereospecific monosaccharide. Binding probabilities of D-glucose tip and the effect of D-glucose (a, green), L-glucose (b, blue), and Dgalactose (c, brown). Values are mean ± s.e.m., n=2000-4000. D-glc, L-glc and D-gal are D-glucose, L-glucose and Dgalactose, respectively. Phlz, Phlorizin. *P<0.05, **P<0.01 and ***P<0.005 compared with levels in the relevant controls (absence of carbohydrates in solution).