Homophilic and heterophilic cadherin bond rupture forces in homo- or hetero-cellular systems measured by AFM-based single-cell force spectroscopy

Abstract

Cadherins enable intercellular adherens junctions to withstand tensile forces in tissues, e.g. generated by intracellular actomyosin contraction. In-vitro single molecule force spectroscopy experiments can reveal cadherin–cadherin extracellular region binding dynamics such as bond formation and strength. However, characterization of cadherin-presenting cell homophilic and heterophilic binding in the proteins’ native conformational and functional states in living cells has rarely been done. Here, we used atomic force microscopy (AFM) based single-cell force spectroscopy (SCFS) to measure rupture forces of homophilic and heterophilic bond formation of N- (neural), OB- (osteoblast) and E- (epithelial) cadherins in living fibroblast and epithelial cells in homo- and hetero-cellular arrangements, i.e., between cells and cadherins of the same and different types. In addition, we used indirect immunofluorescence labelling to study and correlate the expression of these cadherins in intercellular adherens junctions. We showed that N/N and E/E-cadherin homophilic binding events are stronger than N/OB heterophilic binding events. Disassembly of intracellular actin filaments affects the cadherin bond rupture forces suggesting a contribution of actin filaments in cadherin extracellular binding. Inactivation of myosin did not affect the cadherin rupture force in both homo- and hetero-cellular arrangements, but particularly strengthened the N/OB heterophilic bond and reinforced the other cadherins’ homophilic bonds.

Community-building and promotion of technological excellence in molecular biophysics: the ARBRE–MOBIEU network

“…3 Geographical extent of the ARBRE-MOBIEU network membership in 2020, at the end of its funding by COST publications with multiple network members listed as authors. In addition, this special issue of the European Biophysics Journal includes a diversity of original research articles, describing for instance the secondary structure analysis of CueR (Balogh et al 2021), the characterisation of the complex formed between Fep1 and DNA (Miele et al 2021) the analysis of the Dps-DNA interaction in Marinobacter hydrocarbonoclasticus (Jacinto et al 2021), the characterisation of melanoma cell markers (Sobiepanek et al 2021), the single-molecule study of cadherin bond rupture forces (VijiBabu et al 2021), the study of a sulphur iron protein (Almeida et al 2021) and that of a manganese superoxide dismutase variant (Bonetta et al 2021). These are good examples of how the network is helping research groups enhance their understanding of biological systems through an improved use of biophysical approaches.…”

HAV‐Peptides Attached to Colloidal Probes Faithfully Detect E‐Cadherins Displayed on Living Cells

“…[15,17,18] Fichtner et al [18] found that the basic energy landscape of the homotypic E1-5 bond is governed by a wide potential (> 1 nm) and low off-rates (�10 À 4 s À 1 ). Recently, Radmacher and coworkers [19] investigated the rupture forces of homophilic and heterophilic bonds of N-(neural), OB-(osteoblast) and E-(epithelial) cadherins in living fibroblasts and epithelial cells using single-cell force spectroscopy (SCFS). They found that homotypic bonds are stronger than heterotypic ones.…”

Native extracellular matrix probes to target patient- and tissue-specific cell–microenvironment interactions by force spectroscopy

“…14–18 In the context of biophysical investigations, many AFM-based force spectroscopy (FS) configurations can be found: 19 a single-cell probe approaching a substrate coated with proteins; 20–25 an ECM-mimicking probe approaching an adherent cell; 11,26–30 a single-cell probe approaching an adherent cell. 16,31,32 Both cell–cell and cell–ECM interactions can be investigated, through the direct measurement of weak (10–100 pN) interaction forces, which are mostly cadherin or integrin related, respectively. 33–37…”

“…[14][15][16][17][18] In the context of biophysical investigations, many AFM-based force spectroscopy (FS) configurations can be found: 19 a single-cell probe approaching a substrate coated with proteins; [20][21][22][23][24][25] an ECM-mimicking probe approaching an adherent cell; 11,[26][27][28][29][30] a single-cell probe approaching an adherent cell. 16,31,32 Both cell-cell and cell-ECM interactions can be investigated, through the direct measurement of weak (10-100 pN) interaction forces, which are mostly cadherin or integrin related, respectively. [33][34][35][36][37] The main advantage of FS techniques using tips functionalised with single proteins 29,30,38 is the simplification of the complex cell-microenvironment interface, allowing us to study one specific molecular interaction at a time.…”