Spatiotemporal characterization of endothelial cell motility and physical forces during exposure to Borrelia burgdorferi.

Cell motility and biomechanics are critical in various (patho)physiological processes, including the regulation of vascular barrier integrity, which can be subverted by bacterial pathogens. Here, we present a protocol on how to expose endothelial cells (ECs) to vector-borne Borrelia burgdorferi (Bb) and characterize EC kinematics and dynamics during exposure to live or heat-inactivated Bb through traction force and monolayer stress microscopy. Modifications to this protocol may be necessary for studying how different cell types interact with Bb or other microorganisms. For complete details on the use and execution of this protocol, please refer to Yuste et al. (2022).1.

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

Cell competition refers to the mechanism whereby less fit cells ("losers") are sensed and eliminated by more fit neighboring cells ("winners") and arises during many processes including intracellular bacterial infection. Extracellular matrix (ECM) stiffness can regulate important cellular functions, such as motility, by modulating the physical forces that cells transduce and could thus modulate the output of cellular competitions. Herein, we employ a computational model to investigate the previously overlooked role of ECM stiffness in modulating the forceful extrusion of infected "loser" cells by uninfected "winner" cells. We find that increasing ECM stiffness promotes the collective squeezing and subsequent extrusion of infected cells due to differential cell displacements and cellular force generation. Moreover, we discover that an increase in the ratio of uninfected to infected cell stiffness as well as a smaller infection focus size, independently promote squeezing of infected cells, and this phenomenon is more prominent on stiffer compared to softer matrices. Our experimental findings validate the computational predictions by demonstrating increased collective cell extrusion on stiff matrices and glass as opposed to softer matrices, which is associated with decreased bacterial spread in the basal cell monolayer in vitro. Collectively, our results suggest that ECM stiffness plays a major role in modulating the competition between infected and uninfected cells, with stiffer matrices promoting this battle through differential modulation of cell mechanics between the two cell populations.