Cells test substrate rigidity by local contractions on submicrometer pillars

Abstract

Cell growth and differentiation are critically dependent upon matrix rigidity, yet many aspects of the cellular rigidity-sensing mechanism are not understood. Here, we analyze matrix forces after initial cell-matrix contact, when early rigidity-sensing events occur, using a series of elastomeric pillar arrays with dimensions extending to the submicron scale (2, 1, and 0.5 μm in diameter covering a range of stiffnesses). We observe that the cellular response is fundamentally different on micron-scale and submicron pillars. On 2-μm diameter pillars, adhesions form at the pillar periphery, forces are directed toward the center of the cell, and a constant maximum force is applied independent of stiffness. On 0.5-μm diameter pillars, adhesions form on the pillar tops, and local contractions between neighboring pillars are observed with a maximum displacement of ∼60 nm, independent of stiffness. Because mutants in rigidity sensing show no detectable displacement on 0.5-μm diameter pillars, there is a correlation between local contractions to 60 nm and rigidity sensing. Localization of myosin between submicron pillars demonstrates that submicron scale myosin filaments can cause these local contractions. Finally, submicron pillars can capture many details of cellular force generation that are missed on larger pillars and more closely mimic continuous surfaces.cell mechanics | mechanotransduction | nanofabrication T he rigidity of matrix substrates provides important signals that determine cell growth (1), differentiation (2, 3), adhesion (4), or motility (5), among others. How the cellular motility machinery can sense matrix rigidity is unknown, but the mechanism(s) of rigidity sensing must be constrained by the size of the rigidity sensing machinery and the physical quantity "measured" by the cell (6). Arrays of elastomeric micropillars have proven to be a valuable tool in measuring cellular forces: optical microscopy can be used to precisely measure pillar displacement and generate real-time force maps across entire cells (7, 8). For example, over the time scale of hours to days, fibroblasts on arrays of 1-and 2-μm diameter pillars generate average displacements on the order of 100 nm independent of the pillar stiffness over a range of 2-130 nN/μm, i.e., the cells respond to rigidity by measuring the force required to produce a constant displacement (9). However, no studies have examined forces during the initial contact between the cell and the substrate, when the first rigiditysensing events take place (10). Moreover, in studies of the minimal cell-substrate contact area needed to sense rigidity and assemble adhesions, fibroblasts assembled adhesion contacts at the edges of beads with contact areas of more than ∼1 μm 2 , whereas with submicron beads, adhesion contacts only assembled after force from a rigid laser tweezers was applied (11). Analysis of bead displacement with laser tweezers also suggests that cells measure the force required for local displacements of ∼100 nm to deduce rigidity, i.e., a constant displ...

Topography Design Concept of a Tissue Engineering Scaffold for Controlling Cell Function and Fate Through Actin Cytoskeletal Modulation

“…3B, Micropillar-2), which closely correlates with the minimum size of the actin-myosin contractile complex: a minimum length of *1 mm. 83,84 Topography on the scale of ten to hundreds of nanometers: constraints on an integrin cluster Topographical features ten to hundreds of nanometers work, as a direct constraint on integrins, as illustrated in Figure 3C. The critical size of the integrin cluster as a molecular clutch to transmit forces between ECM and the cytoplasm has been measured in an in vitro nanofabricated system.…”

“…In contrast, in cells on the 0.5 mm pillars, the paxillin is localized on the top of the pillars and actomyosin contractile complexes bridge the pillars. (Adapted with permission from Ghassemi et al 83 Copyright 2013 by the US National Academy of Sciences.) (C) Topography on the scale of ten to hundreds of nanometers as a constraint on an integrin cluster.…”

Pancreatic cancer: a mechanobiology approach

“…For example, Saez and colleagues developed an elastic pillar array that recapitulated the mechanical environment of epithelial sheets that allowed the study of the mechanical factors influencing mitosis with unprecedented detail [57]. Also, Ghassemi et al extended the applicability of these sensors by fabricating pillars of submicron sizes, which enabled many cellular processes that could not be observed with the micropillars [58]. Elastic pillars can be combined with fluorescent microscopy to correlate the force applied on the pillar at a particular time with the molecule involved in mechanosensing that is being recruited at this particular pillar (figures 4(e) and (f ) [59].…”

Neuronal Growth on a-Si and Au Nanopillars

“…[5][6][7][8] Smaller diameter pillars, nanopillars, are applied for different purposes, for example, examination of neuronal development 9 and pinning neurons. [10][11][12] Nanopillars have also been used to access cellular membranes for electroporation, 13,14 which led them to the successful measurement of neuronal intracellular action potentials. 15,16 Although these approaches intended to achieve new biological interfaces, there have not been much studies related to nanopillars used as scaffolds.…”