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

Figures

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  Fig. 1. Schematic representation AFM-based SCFS experimental setup. a This cartoon represents the capturing of cell by a tipless cantilever in a stepwise manner. 1-The conA functionalized tipless cantilever and a cell with round morphology is chosen with the aid of optical microscopy. 2-The cantilever is approached towards the cell at certain velocity (5 µm/sec) and contact force (3.5 nN). 3-Given the contact time of 5 s, the cell attached cantilever is retracted with the same velocity (5 µm/sec). b Optical image shows the cell attached cantilever. c Force curve recorded during cell pick up was shown and retract curve (blue arrow) contains rupture events due to unspecific binding and tethers. 4-After a recovery time of 10 min, the interaction between cell attached to the cantilever and cell grown as monolayer was conducted. d Cell–cell interaction force curve shows rupture events that corresponded to the extracellular cadherin–cadherin bond breakage. Here, multiple rupture events (specific binding) were recorded

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  Fig. 2. Fibroblast intercellular cadherin expression and rupture force measurement. Histogram shows the rupture force (red bar) recorded for a NF–NF, b SF–SF and c DF–DF interactions. Cadherin involvement in the rupture events (Supplementary Fig. 1A) was controlled by EGTA (blue bar) addition to the cell–cell interaction setup. This leads to the respective loss of rupture events (Supplementary Fig. 1B). d Increasing the approach and retract velocity of the cantilever linearly increases the cadherin rupture force for NF–NF (black square), SF–SF (red upper triangle) and DF–DF (blue lower triangle). NF and SF displays large rupture forces than DF at all velocities. n represents the number of independent experiments performed for each category (see Materials and Methods section). e Dual immunofluorescence data shows N-cadherin expression (red fluorescence) in all fibroblasts adherens junctions. OB-cadherin expression (green fluorescence) was seen only in DF. The overlay (orange fluorescence) represents the heterophilic binding of N-cadherin and OB-cadherin which is encountered only in DF. Blue fluorescence indicates the nuclei. Scale bar 20 µm

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  Fig. 3. Heterocellular fibroblasts-MDCK interactions rupture force measurement and cadherin expression. Ruptures forces displayed as histograms recorded with (blue bar) and without EGTA (red bar) for a MDCK-MDCK, b NF-MDCK, c SF-MDCK and d DF-MDCK. e The cadherin rupture force shows linear relationship with cantilever approach and retract velocity for MDCK-MDCK (sandal circle), NF-MDCK (black filled square), SF-MDCK (red filled upper triangle), DF-MDCK (blue filled lower triangle). n represents the number of independent experiments performed for each category (see Materials and Methods section). f Immunofluorescence data shows predominant E-cadherin expression in MDCK-MDCK adherens junction. Subpopulations of MDCK express N-cadherin but not OB-cadherin. Dual immunofluorescence data shows N-cadherin (red fluorescence in G, I) homophilic binding and loss of E-cadherin (H) in NF-MDCK, SF-MDCK and DF-MDCK. Due to the similar excitation and emission wavelength of fluorescence tags (secondary antibody), dual immunostaining g for N-cadherin (red fluorescence) and E-cadherin (sandal fluorescence) is difficult to interpret. The E-cadherin loss seen in h confirms the N-cadherin homophilic binding…

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  Fig. 4. Effect of Cytochalasin D treatment on the rupture force of homocellular and heterocellular adherens junctions. The cityscape plot summarise the effect of cytochalasin D (5 µM) on homocellular- a NF–NF, b SF–SF, c DF–DF, d MDCK-MDCK and heterocellular- e NF-MDCK, f SF-MDCK, g DF-MDCK adherens junctions. Rupture events were recorded without the drug as control (black line), with DMSO (red line) and with cytochalasin D (blue line). n = 2 for each category; n represents the number of independent experiments performed for each category (see Materials and Methods section). The corresponding median values were listed in Table 2 and plotted in Supplementary Fig.5

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  Fig. 5. Effects of ML-7 treatment on the rupture force of homocellular and heterocellular adherens junctions. The cityscape plot summarise the effect of ML-7 (5 µM) on homocellular- a NF–NF, b SF–SF, c DF–DF, d MDCK-MDCK and heterocellular- e NF-MDCK, (F) SF-MDCK, g DF-MDCK adherens junctions. Rupture events were recorded without the drug as control (black line), with DMSO (red line) and with ML-7 (blue line). n = 2 for each category; n represents the number of independent experiments performed for each category (see Materials and Methods section). The corresponding median values were listed in Table 3 and plotted in Supplementary Fig. 8

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  Fig. 6. Acto-myosin contractility influences cadherin extracellular domain binding dynamics. Cartoon representations depict the effect of actin filaments disruption a and myosin inactivation (B) on cadherin homophilic and heterophilic adhesion pairs. a Actin filaments disassembly resulting of cytochalasin D treatment leads to weakening of homophilic and heterophilic cadherin adhesions. b Myosin inactivation by inhibiting myosin light chain kinase (MLCK) using ML-7 treatment leads to N/OB heterophilic adhesion reinforcement whereas dissimilar effects were seen in other cadherin homophilic and heterophilic adhesions. This pictures the stabilization and reinforcement of cadherin homophilic and heterophilic adhesion by actin filaments, with no cross linkers-myosin generating contractile forces and with myosin inactivation at the cadherin-catenin-actin complex