Resolving tractions across cell‐cell adhesion reveals the role of intercellular shear in plithotaxis

Background Endothelial cells form a selectively permeable layer by attaching to their neighbors through various adhesion molecules. Some of these molecules are interlinked through cytoskeletal filaments and exposed to cytoskeletal forces. Importantly, cytoskeletal forces are balanced continuously at the cell‐cell and cell‐substrate adhesion maintaining monolayer homeostasis and preventing cellular motion. Using Monolayer Stress Microscopy, we have previously quantified cytoskeletal forces across cellular monolayers. We identified that locations with orientation‐dependent (or anisotropic) tension exhibited motion along the orientation of highest tension (or equivalently, the orientation of minimal shear forces). This phenomenon, known as plithotaxis, would occur if the intercellular junctions are unable to support shear forces. Here we examine the validity of this structural basis for plithotaxis in cultured cell monolayers. Methods We seeded clusters of pulmonary endothelial cells on a collagen‐coated hydrogel and quantified the cell‐substrate forces by analyzing the motion of beads embedded within the gel. Using these forces, we analyzed mechanical equilibrium of the cell monolayer and recovered a two‐dimensional monolayer stress tensor (or cytoskeletal forces). By segmenting phase contrast image of the cell monolayer, we detected the location of the intercellular boundaries. The monolayer stress tensor at the intercellular boundaries was then resolved into Fn and Ft. Using these forces, we then computed a two‐dimensional moment tensor for each cell. This moment tensor is then used in assessing a novel definition of plithotaxis, not as a property of discrete points but instead, as a property of each cell within the monolayer. Results Fn and Ft exhibited unique characteristics for endothelial cells from distinct pulmonary vascular segments, including arteries (PAEC), microvessels (PMVEC) and veins (PVEC). The highest contrast was seen between PAECs and PMVECs. PMVECs exhibited the fewest intercellular segments with negative Fn, Ft was largest and most comparable in magnitude to Fn, and the cells appeared to move along the principal axis of the moment tensor, indicating that intercellular shear forces are not negligible and plithotaxis can be defined at the lengthscale of a cell. Moreover, plithotaxis was more pronounced for the cells that had higher median Ft/Fn, indicating that intercellular shear force and plithotaxis are in fact positively correlated. Conclusion Plithotaxis arise not from inability but instead from the strong ability of intercellular junctions to support shear forces. Our work opens new research avenues for systematic examination of the relationship between structure and physical function of the intercellular junctions. Support or Funding Information Abraham Mitchell Cancer Research Fund, Research and Scholarly Development Grant, P01 HL66299 (NIH/NHLBI), F32 HL144040‐01 (NHLBI), University of South Alabama Honors College Scholarship. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .