Lamellipodium is a myosin-independent mechanosensor Patrick W. Oakesa,b,c,d,e,1, Tamara C. Bidonec,d,f, Yvonne Beckhamc,d,e, Austin V. Skeetersa, Guillermina R. Ramirez-San Juanc,d,e, Stephen P. Winterg, Gregory A. Vothc,d,f, and Margaret L. Gardelc,d,e,1 aDepartment of Physics and Astronomy, University of Rochester, Rochester, NY 14627; bDepartment of Biology, University of Rochester, Rochester, NY 14627; cInstitute for Biophysical Dynamics, University of Chicago, Chicago, IL 60637; dJames Franck Institute, University of Chicago, Chicago, IL 60637; eDepartment of Physics, University of Chicago, Chicago, IL 60637; fDepartment of Chemistry, University of Chicago, Chicago, IL 60637; and gInterdisciplinary Scientist Training Program, University of Chicago, Chicago, IL 60637 Edited by David A. Weitz, Harvard University, Cambridge, MA, and approved February 5, 2018 (received for review September 8, 2017) The ability of adherent cells to sense changes in the mechanical substrates. The extent of cell spreading is controlled by the properties of their extracellular environments is critical to numer- density and spatial organization of matrix ligands (38–40), as well ous aspects of their physiology. It has been well documented that as the rigidity of the substrate to which these ligands are attached cell attachment and spreading are sensitive to substrate stiffness. (4–12). It has also been suggested that the stress-relaxing properties Here, we demonstrate that this behavior is actually biphasic, with a of the matrix can contribute to cell spreading (41, 42). In the limit ’ < transition that occurs around a Young’s modulus of ∼7 kPa. Fur- of soft substrates with a Young s modulus 500 Pa, cell spreading is inhibited. As the substrate stiffness increases, the spread area thermore, we demonstrate that, contrary to established assump- – tions, this property is independent of myosin II activity. Rather, increases and ultimately plateaus (8 12). While previous reports we find that cell spreading on soft substrates is inhibited due to have differed on the exact range of relevant stiffness which reduced myosin-II independent nascent adhesion formation within regulates this behavior, likely due to variances in experimental approaches (43), cell spreading remains a robust metric to study the lamellipodium. Cells on soft substrates display normal leading- substrate stiffness sensing. edge protrusion activity, but these protrusions are not stabilized Here, we study the mechanism regulating substrate stiffness- due to impaired adhesion assembly. Enhancing integrin–ECM affin- 2+ dependent cell spreading. We found that NIH 3T3 cell spreading ity through addition of Mn recovers nascent adhesion assembly is acutely impacted as the Young’s modulus of the substrate and cell spreading on soft substrates. Using a computational model increases from 5 to 8 kPa. On substrates with a stiffness <5 kPa, SCIENCES to simulate nascent adhesion assembly, we find that biophysical cells spread poorly. Average cell spread area increased on sub- properties of the integrin–ECM bond are optimized to stabilize in- strates stiffer than 5 kPa, plateauing on substrates stiffer than APPLIED PHYSICAL teractions above a threshold matrix stiffness that is consistent with 8 kPa. Above this threshold, cell spread area remained constant. the experimental observations. Together, these results suggest Surprisingly, we found this stiffness-dependent change in cell that myosin II-independent forces in the lamellipodium are responsible spreading was independent of myosin II motor activity. Instead, for mechanosensation by regulating new adhesion assembly, which, we found that spreading on soft substrates is impaired by re- in turn, directly controls cell spreading. This myosin II-independent duced assembly of nascent, myosin-independent adhesions at the mechanism of substrate stiffness sensing could potentially regulate cell periphery. Enhancing integrin–ligand affinity through the 2+ a number of other stiffness-sensitive processes. addition of Mn was sufficient both to stabilize nascent adhe- BIOPHYSICS AND sions and increase cell spread area on soft substrates. We then COMPUTATIONAL BIOLOGY nascent adhesion | myosin-II | mechanosensing | integrin | catch-bond implemented a computational model to determine how changes in integrin–substrate catch-bond kinetics affected integrin binding on substrates of different stiffness. We found that the biophysical he ability of cells to sense mechanical forces and convert them properties of integrin–matrix catch-bonds were optimized to sense Tinto biochemical responses regulates a plethora of physiolog- ∼ – changes in substrate stiffness at 6 kPa, consistent with our ex- ical functions (1 3). In particular, cells respond to changes in the perimental results. Together, these results illustrate that nascent stiffness of the extracellular matrix (ECM) by altering a number of adhesion formation in the lamellipodium functions as a myosin adhesion-dependent behaviors, including spreading (4–12), mi- gration (4, 13, 14), proliferation (15), differentiation (16, 17), and Significance metastasis (18, 19). Matrix mechanosensing is thought to be me- diated by focal adhesions, hierarchical organelles comprising ∼150 proteins that facilitate dynamic and force-sensitive interac- Cell physiology can be regulated by the mechanics of the extra- tions between the ECM and the actin cytoskeleton (20–22). How cellular environment. Here, we demonstrate that cell spreading is these dynamic organelles mediate environmental sensing in a va- a mechanosensitive process regulated by weak forces generated riety of physiological contexts, however, is still largely unknown. at the cell periphery and independent of motor activity. We show Previous efforts have focused primarily on myosin II-mediated that stiffness sensing depends on the kinetics of the initial ad- mechanisms for substrate stiffness sensing (23–28). Stresses hesion bonds that are subjected to forces driven by protein po- generated by myosin motors on the actin cytoskeleton are lymerization. This work demonstrates how the binding kinetics transmitted to the ECM via focal adhesions. These stresses, of adhesion molecules are sensitively tuned to a range of forces coupled with the matrix rigidity, impact the deformation and that enable mechanosensation. binding affinity of proteins within the focal adhesion (29–32). Changes in the composition and kinetics of proteins within focal Author contributions: P.W.O. and M.L.G. designed research; P.W.O., Y.B., A.V.S., G.R.R.-S.J., adhesions are thought to variably regulate force transmission and S.P.W. performed research; P.W.O., T.C.B., and G.A.V. contributed new reagents/analytic from the actin cytoskeleton and the matrix (33–35), leading many tools; P.W.O., T.C.B., and A.V.S. analyzed data; and P.W.O., T.C.B., and M.L.G. wrote the paper. to describe focal adhesions as molecular clutches. Initial adhesion formation, however, occurs in the leading edge of the lamellipo- The authors declare no conflict of interest. dium and is a myosin-independent process (36, 37). These struc- This article is a PNAS Direct Submission. tures, known as nascent adhesions, are instead subject to forces Published under the PNAS license. that primarily originate from polymerization of actin filaments. 1To whom correspondence may be addressed. Email: [email protected] or gardel@ The contribution of nascent adhesions to mechanisms of substrate uchicago.edu. stiffness sensing has not been thoroughly explored. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. One of the best-characterized metrics of environmental sensing by 1073/pnas.1715869115/-/DCSupplemental. adherent cells is their ability to attach and spread on ligand-coated www.pnas.org/cgi/doi/10.1073/pnas.1715869115 PNAS Latest Articles | 1of6 II-independent mechanosensor to control cell adhesion and with blebbistatin had an increased spread area compared with spreading. control cells across all stiffnesses, but exhibited the same soft and stiff regimes. Morphologically, myosin-inhibited cells on all sub- Results strates showed more protrusions, but on stiff substrates, the cells Spread Area Is a Biphasic Response of Substrate Stiffness, Independent exhibited more spindle-like projections (Fig. 1D). Similar pheno- of Myosin Activity. To investigate the mechanisms that drive types were seen when cells were incubated with Rho-Kinase in- substrate stiffness sensing, we chose to measure the spread area hibitor (Y-27632; Fig. 1 E and F) and when cells were plated on of adherent cells. We first plated NIH 3T3 fibroblasts on a series other ECM proteins (Fig. S1). Thus, the change in cell spread area of polyacrylamide gels covalently coupled with fibronectin and that occurred between the soft and stiff regimes did not require with Young’s moduli ranging from 0.6 to 150 kPa (Fig. 1A). Cells myosin II activity. were also plated on glass absorbed with fibronectin as a control. Consistent with previous reports (4, 6, 8–11), we found that cells’ Substrate Stiffness Does Not Inhibit Lamellipodia Protrusion Dynamics. spread area was sensitive to substrate stiffness (Fig. 1A). In To understand how substrate stiffness impacts cell spread area, we contrast, however, we found that this response could be broken investigated the effects of substrate stiffness on protrusion dy- down into two regimes: There was poor spreading on soft (less namics. We tracked lamellipodia formation by taking time-lapse than ∼5 kPa) substrates