Volume measurement and biophysical characterization of mounds in epithelial monolayers after intracellular bacterial infection

A Stiff Extracellular Matrix Favors the Mechanical Cell Competition that Leads to Extrusion of Bacterially-Infected Epithelial Cells

“…Moreover, especially on glass coverslips, surrounding uninfected cells appeared much more polarized as compared to the other two conditions ( Figure 7A ). When we quantified the volume of extruded cell domains relative to that which infected cells exhibit on a soft 3 kPa matrix using alpha shapes ( Bastounis et al, 2021a ), we found it to be 2-fold increased for cells residing on stiff 35 kPa matrices and 1.78-fold for cells residing on glass. Therefore, we can conclude that both computationally and experimentally a stiffer ECM promotes infection mounding and also that above a certain stiffness threshold an asymptotic behavior is reached and further increase in stiffness does not lead to further increase in mounding.…”

“…Infection of MDCK cells with L.m. was performed as described previously ( Bastounis et al, 2021a ) using L.m. strain JAT607 (Species: L.m.…”

“…Volumes of infected extruded cell domains were calculated using custom MATLAB scripts as previously explained in detail ( Bastounis et al, 2021a ). Briefly, images of Hoechst-stained MDCK nuclei and of the bacterial fluorescence in and around a given infection focus were taken using a z-spacing of 0.2 μm.…”

“…Polyacrylamide hydrogels were prepared on glass bottom 24-well plates (MatTek, P24G-1.5-13-F) as previously described in ( Bastounis et al, 2021a ). Briefly, glass coverslips were pretreated with 0.5 M NaOH for 30 min at RT, rinsed with water and then incubated for 5 min at RT with 2% APTS ((3-Aminopropyl)triethoxysilane, Sigma, A3648-100ML) in 95% EtOH.…”

“…TFM was performed as previously described ( Lamason et al, 2016 ; Bastounis et al, 2021a ). More specifically, polyacrylamide hydrogels were equilibrated for 30 min at 37°C with cell media prior to cell seeding.…”