The ability of adherent cells to sense changes in the mechanical properties of their extracellular environments is critical to numerous aspects of their physiology. It has been well documented that cell attachment and spreading are sensitive to substrate stiffness. Here, we demonstrate that this behavior is actually biphasic, with a transition that occurs around a Young's modulus of ∼7 kPa. Furthermore, we demonstrate that, contrary to established assumptions, this property is independent of myosin II activity. Rather, we find that cell spreading on soft substrates is inhibited due to reduced myosin-II independent nascent adhesion formation within the lamellipodium. Cells on soft substrates display normal leading-edge protrusion activity, but these protrusions are not stabilized due to impaired adhesion assembly. Enhancing integrin-ECM affinity through addition of Mn recovers nascent adhesion assembly and cell spreading on soft substrates. Using a computational model to simulate nascent adhesion assembly, we find that biophysical properties of the integrin-ECM bond are optimized to stabilize interactions above a threshold matrix stiffness that is consistent with the experimental observations. Together, these results suggest that myosin II-independent forces in the lamellipodium are responsible for mechanosensation by regulating new adhesion assembly, which, in turn, directly controls cell spreading. This myosin II-independent mechanism of substrate stiffness sensing could potentially regulate a number of other stiffness-sensitive processes.

We investigated the cell behaviors that drive morphogenesis of the Drosophila follicular epithelium during expansion and elongation of early‐stage egg chambers. We found that cell division is not required for elongation of the early follicular epithelium, but drives the tissue toward optimal geometric packing. We examined the orientation of cell divisions with respect to the planar tissue axis and found a bias toward the primary direction of tissue expansion. However, interphase cell shapes demonstrate the opposite bias. Hertwig's rule, which holds that cell elongation determines division orientation, is therefore broken in this tissue. This observation cannot be explained by the anisotropic activity of the conserved Pins/Mud spindle‐orienting machinery, which controls division orientation in the apical–basal axis and planar division orientation in other epithelial tissues. Rather, cortical tension at the apical surface translates into planar division orientation in a manner dependent on Canoe/Afadin, which links actomyosin to adherens junctions. These findings demonstrate that division orientation in different axes—apical–basal and planar—is controlled by distinct, independent mechanisms in a proliferating epithelium.

The ability of adherent cells to form adhesions is critical to numerous phases of their physiology. The assembly of adhesions is mediated by several types of integrins. These integrins differ in physical properties, including rate of diffusion on the plasma membrane, rapidity of changing conformation from bent to extended, affinity for extracellular matrix ligands, and lifetimes of their ligand-bound states. However, the way in which nanoscale physical properties of integrins ensure proper adhesion assembly remains elusive. We observe experimentally that both β-1 and β-3 integrins localize in nascent adhesions at the cell leading edge. In order to understand how different nanoscale parameters of β-1 and β-3 integrins mediate proper adhesion assembly, we therefore develop a coarse-grained computational model. Results from the model demonstrate that morphology and distribution of nascent adhesions depend on ligand binding affinity and strength of pairwise interactions. Organization of nascent adhesions depends on the relative amounts of integrins with different bond kinetics. Moreover, the model shows that the architecture of an actin filament network does not perturb the total amount of integrin clustering and ligand binding; however, only bundled actin architectures favor adhesion stability and ultimately maturation. Together, our results support the view that cells can finely tune the expression of different integrin types to determine both structural and dynamic properties of adhesions.

The ability of adherent cells to sense changes in the mechanical properties of their extracellular environments is critical to numerous aspects of their physiology. It has been well documented that cell attachment and spreading are sensitive to substrate stiffness. Here we demonstrate that this behavior is actually biphasic, with a transition that occurs around a Young's modulus of ~7 kPa. Furthermore, we demonstrate that, contrary to established assumptions, this property is independent of myosin II activity. Rather, we find that cell spreading on soft substrates is inhibited due to reduced nascent adhesion formation within the lamellipodium. Cells on soft substrates display normal leading edge protrusion activity, but these protrusions are not stabilized due to impaired adhesion assembly. Enhancing integrin-ECM affinity through addition of Mn 2+ recovers nascent adhesion assembly and cell spreading on soft substrates. Using a computational model to simulate nascent adhesion assembly, we find that biophysical properties of the integrin-ECM bond are optimized to stabilize interactions above a threshold matrix stiffness that is consistent with the experimentally observations. Together these results suggest that myosin II-independent forces in the lamellipodium are responsible for mechanosensation by regulating new adhesion assembly, which in turn, directly controls cell spreading. This myosin II-independent mechanism of substrate stiffness sensing could potentially regulate a number of other stiffness sensitive processes. Significance StatementCell physiology can be regulated by the mechanics of the extracellular environment. Here, we demonstrate that cell spreading is a mechanosensitive process regulated by weak forces generated at the cell periphery and independent of motor activity. We show that stiffness sensing depends on the kinetics of the initial adhesion bonds that are subjected to forces driven by protein polymerization. This work demonstrates how the binding kinetics of adhesion molecules are sensitively tuned to a range of forces that enable mechanosensation.All rights reserved. No reuse allowed without permission.was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

36The ability of adherent cells to form adhesions is critical to several phases of their physiology. The 37 assembly of adhesions is mediated by several types of integrins. These integrins differ in physical 38 properties, including rate of diffusion on the plasma membrane, rapidity of changing conformation 39 from bent to extended, affinity for extracellular matrix ligands, and lifetimes of their ligand-bound 40 states. However, the way in which nanoscale physical properties of integrins ensure proper 41 adhesion assembly remains elusive. We observe experimentally that both b-1 and b-3 integrins 42 localize in nascent adhesions at the cell leading edge. In order to understand how different 43 nanoscale parameters of b-1 and b-3 integrins mediate proper adhesion assembly, we therefore 44 develop a coarse-grained computational model. Results from the model demonstrate that 45 morphology and distribution of nascent adhesions depend on ligand binding affinity and strength 46 of pairwise interactions. Organization of nascent adhesions depends on the relative amounts of 47 integrins with different bond kinetics. Moreover, the model shows that the architecture of an actin 48 filament network does not perturb the total amount of integrin clustering and ligand binding; 49 however, only bundled actin architectures favor adhesion stability and ultimately maturation.50Together, our results support the view that cells can finely tune the expression of different integrin 51 types to determine both structural and dynamic properties of adhesions. 52 53 3 Author summary 54 Integrin-mediated cell adhesions to the extracellular environment contribute to various cell 55 activities and provide cells with vital environmental cues. Cell adhesions are complex structures 56 that emerge from a number of molecular and macromolecular interactions between integrins and 57 cytoplasmic proteins, between integrins and extracellular ligands, and between integrins 58 themselves. How the combination of these interactions regulate adhesions formation remains 59 poorly understood because of limitations in experimental approaches and numerical methods.60 Here, we develop a multiscale model of adhesion assembly that treats individual integrins and 61 elements from both the cytoplasm and the extracellular environment as single coarse-grained (CG) 62 point particles, thus simplifying the description of the main macromolecular components of 63 adhesions. The CG model implements sequential interactions and dependencies between the 64 components and ultimately allows one to characterize various regimes of adhesions formation 65 based on experimentally detected parameters. The results reconcile a number of independent 66 experimental observations and provide important insights into the molecular basis of adhesion 67 assembly from various integrin types. 68 69 As the linker between cytoskeletal adhesion proteins and extracellular matrix ligands, integrins 70 play a vital role in the formation of adhesions and profoundly influence different phases of cell...