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

Abstract

Atomic force microscopy (AFM) was used to probe topology, conformational changes and initial substratecarrier interactions of Na+-glucose co-transporter (SGLT1) in living cells on a single-molecule level. By scanning SGLT1-transfected Chinese hamster ovary (CHO) cells with AFM tips carrying an epitope-specific antibody directed against the extramembranous C-terminal loop 13, significant recognition events could be detected. Specificity was confirmed by the absence of events in nontransfected CHO cells and by the use of free antigen and free antibody superfusion. Thus, contrary to computer predictions on SGLT1 topology, loop 13 seems to be part of the extracellular surface of the transporter. Binding probability of the antibody decreased upon addition of phlorizin, a specific inhibitor of SGLT1, suggesting a considerable conformational change of loop 13 when the inhibitor occludes the sugar translocation pathway. Using an AFM tip carrying 1-thio-D-glucose, direct evidence could be obtained that in the presence of Na+ a sugarbinding site appears on the transporter surface. The binding site accepts the sugar residue of the glucoside phlorizin, free D-glucose, and D-galactose, but not free Lglucose and probably represents the first of several selectivity filters of the transporter. This work demonstrates the potential of AFM to study the presence and dynamics of plasma membrane transporters in intact cells on the single molecule level.

Imaging and measuring the rituximab-induced changes of mechanical properties in B-lymphoma cells using atomic force microscopy

“…For those adherent cells, the most commonly used method is to directly culture them on a glass support for about 1-2 days and these cells can adhere to the support tightly [24]. Sometimes in order to enhance the cell adherence, adhesion proteins such as poly-L-lysine are coated onto the substrate [25]. For those microbial cells which can not spread over solid substrates [26], one method is by coating the substrate with poly-L-lysine [27], and the other method is by mechanically trapping with the use of porous polymer membranes which is generally suited for the sphere-shaped microbial cells [28].…”

“…High-resolution of living cells are hampered by cell deformation, as the considerable deformation induced by AFM tip [29]. Owing to the high flexibility and softness of the cell membrane, it is hard to directly visualize the proteins on the living cell surface [25]. To visualize the single proteins on the cell membrane, one method is to adsorb the cell membrane segments onto a flat support (such as mica) [30].…”

Investigating the Molecular Specific Interactions on Cell Surface Using Atomic Force Microscopy

“…This phenomenon can be explained by Bell-Evans model [55], which characterizes the behavior of molecular unbinding pulled by an external force. SMFS is a mature single-molecule technique and many researchers have used SMFS to measure the molecular binding force on cells grown in vitro, such as receptor-drug [41], receptor-ligand [56], fibrinogen-erythrocyte [57], and aptamer-protein [58], showing that the binding force of receptor-ligand was in the range of 20-200 pN [59]. Here, we measured the CD20-rituximab binding force on patient cancer cells based on the fluorescence recognition of the specific cancer cell surface marker and the binding force was in the range of molecular binding force.…”

Localization of the ergtoxin-1 receptors on the voltage sensing domain of hERG K+ channel by AFM recognition imaging

“…Here, we introduce an alternative way to probe the surface topology of hERG channel by using AFM (dynamic recognition imaging and single molecule force spectroscopy). Due to continuous progress in the technical aspects of the AFM and tip functionalization procedures, the investigations of receptor-ligand interactions on living cells at the single-molecule level have become possible [32][33][34]. To localize receptor-ligand recognition in vitro systems and in living cells, fluorescence techniques (immunochemistry [35] or single molecule optical microscopy [36] are commonly used.…”