Single Molecule Interaction in Living Cells: A Case Study

The section presents the AFM-based studies on properties of single molecular complexes composed of an individual N- cadherin molecule and monoclonal antibody against N-cadherins (referred here as Ncadh-GC4 complex). The force spectroscopy measurement were carried out directly on a surface of living human bladder cells (HCV29—non-malignant cancer cell of ureter and T24 bladder cells from transitional cell carcinoma). The single-molecule measurements were performed with the aim to answer the question whether an individual molecule or its part displays different binding properties in normal and cancerous cells [54]. In these experiments, the monoclonal antibody against N-cadherin was directly immobilized on a surface of the AFM probe. The results presented in this section illustrates the potential of single molecule unbinding experiments in characterization of the given interaction type.

Properties of N-Cadherin in Bladder Cancer Studied by AFM

Cadherins (see Chapter 2) are transmembrane proteins having both extracellular and cytoplasmic domains (Fig. 5.25). The extracellular domain consists of five cadherins repeats (called ectodomains), each of about 110 amino acids residues. Between two repeats, calcium ions are bound, participating in the formation of calcium-dependent, homophilic bonds. The cytosolic domain of the cadherin is directly associated with b-catenin or/and plakoglobin ( g-catenin) [54, 55]. However, there is one exception: Plakoglobin can associate with both classical cadherins (e.g., E- or N-cadherin) and desmosomal cadherins while g-catenin associates only with the members of the classical cadherin family. Both g-catenin and plakoglobin bind to g-catenin, which links the cadherin/catenin complex to the actin cytoskeleton [56, 57].

Illustration of the cadherin-catenin complex embedded in cell membrane. Reprinted with permission from [2]

Figure 5.25 Illustration of the cadherin-catenin complex embedded in cell membrane. Reprinted with permission from [2].

In order to detect N-cadherin on a surface of living cells, the monoclonal antibody against the cadherin extracellular domain was applied (GC4 antibody) [58]. The used antibody inhibits adherens junction formation and disrupts existing junctions in cultured cells. The binding site for the antibody is located between the two first, outermost ectodomains ([59], denoted usually as EC1 and EC2, Fig. 5.25).

 
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