Paul A. Janmey

Tissue Cells Feel and Respond to the Stiffness of Their Substrate

Normal tissue cells are generally not viable when suspended in a fluid and are therefore said to be anchorage dependent. Such cells must adhere to a solid, but a solid can be as rigid as glass or softer than a babys skin. The behavior of some cells on soft materials is characteristic of important phenotypes; for example, cell growth on soft agar gels is used to identify cancer cells. However, an understanding of how tissue cells—including fibroblasts, myocytes, neurons, and other cell types—sense matrix stiffness is just emerging with quantitative studies of cells adhering to gels (or to other cells) with which elasticity can be tuned to approximate that of tissues. Key roles in molecular pathways are played by adhesion complexes and the actinmyosin cytoskeleton, whose contractile forces are transmitted through transcellular structures. The feedback of local matrix stiffness on cell state likely has important implications for development, differentiation, disease, and regeneration.

Nonlinear elasticity in biological gels

The mechanical properties of soft biological tissues are essential to their physiological function and cannot easily be duplicated by synthetic materials. Unlike simple polymer gels, many biological materials—including blood vessels, mesentery tissue, lung parenchyma, cornea and blood clots—stiffen as they are strained, thereby preventing large deformations that could threaten tissue integrity. The molecular structures and design principles responsible for this nonlinear elasticity are unknown. Here we report a molecular theory that accounts for strain-stiffening in a range of molecularly distinct gels formed from cytoskeletal and extracellular proteins and that reveals universal stress–strain relations at low to intermediate strains. The input to this theory is the force–extension curve for individual semi-flexible filaments and the assumptions that biological networks composed of these filaments are homogeneous, isotropic, and that they strain uniformly. This theory shows that systems of filamentous proteins arranged in an open crosslinked mesh invariably stiffen at low strains without requiring a specific architecture or multiple elements with different intrinsic stiffness.

Thrombin receptor ligation and activated rac uncap actin filament barbed ends through phosphoinositide synthesis in permeabilized human platelets

Cells respond to diverse external stimuli by polymerizing cytoplasmic actin, and recent evidence indicates that GTPases can specify where this polymerization takes place. Actin assembly in stimulated blood platelets occurs where sequestered monomers add onto the fast-growing (barbed) ends of actin filaments (F-actin), which are capped in the resting cells. We report that D3 and D4 polyphosphoinositides, Pl(4)P, Pl(4,5)P2, Pl(3,4)P2, and Pl(3,4,5)P3, uncap F-actin in resting permeabilized platelets. The thrombin receptor-activating peptide (TRAP), GTP, and GTP gamma S, but not GDP beta S, also uncap F-actin in permeabilized platelets. GDP beta S inhibits TRAP-induced F-actin uncapping, and Pl(4,5)P2 overcomes this inhibition. Constitutively active mutant Rac, but not Rho, activates uncapping of F-actin. Pl(4,5)P2-binding peptides derived from gelsolin inhibit F-actin uncapping by TRAP, Rac, and GTP gamma S. TRAP and Rac induce rapid Pl(4,5)P2 synthesis in permeabilized platelets. The findings establish a signaling pathway for actin assembly involving Rac in which the final message is phosphoinositide-mediated F-actin uncapping.

Elasticity of semiflexible biopolymer networks.

We develop a model for cross-linked gels and sterically entangled solutions of semiflexible biopolymers such as F-actin. Such networks play a crucial structural role in the cytoskeleton of cells. We show that the rheologic properties of these networks can result from nonclassical rubber elasticity. This model can explain a number of elastic properties of such networks in vitro, including the concentration dependence of the storage modulus and yield strain.

Soft biological materials and their impact on cell function

Most organs and biological tissues are soft viscoelastic materials with elastic moduli ranging from on the order of 100 Pa for the brain to 100 000 Pa for soft cartilage. Biocompatible synthetic materials already have many applications, but combining chemical compatibility with physiologically appropriate mechanical properties will increase their potential for use both as implants and as substrates for tissue engineering. Understanding and controlling mechanical properties, specifically softness, is important for appropriate physiological function in numerous contexts. The mechanical properties of the substrate on which, or within which, cells are placed can have as large an impact as chemical stimuli on cell morphology, differentiation, motility, and commitment to live or die.

Viscoelastic properties of individual glial cells and neurons in the CNS

One hundred fifty years ago glial cells were discovered as a second, non-neuronal, cell type in the central nervous system. To ascribe a function to these new, enigmatic cells, it was suggested that they either glue the neurons together (the Greek word “γλια” means “glue”) or provide a robust scaffold for them (“support cells”). Although both speculations are still widely accepted, they would actually require quite different mechanical cell properties, and neither one has ever been confirmed experimentally. We investigated the biomechanics of CNS tissue and acutely isolated individual neurons and glial cells from mammalian brain (hippocampus) and retina. Scanning force microscopy, bulk rheology, and optically induced deformation were used to determine their viscoelastic characteristics. We found that (i) in all CNS cells the elastic behavior dominates over the viscous behavior, (ii) in distinct cell compartments, such as soma and cell processes, the mechanical properties differ, most likely because of the unequal local distribution of cell organelles, (iii) in comparison to most other eukaryotic cells, both neurons and glial cells are very soft (“rubber elastic”), and (iv) intriguingly, glial cells are even softer than their neighboring neurons. Our results indicate that glial cells can neither serve as structural support cells (as they are too soft) nor as glue (because restoring forces are dominant) for neurons. Nevertheless, from a structural perspective they might act as soft, compliant embedding for neurons, protecting them in case of mechanical trauma, and also as a soft substrate required for neurite growth and facilitating neuronal plasticity.

Fibrin gels and their clinical and bioengineering applications

Fibrin gels, prepared from fibrinogen and thrombin, the key proteins involved in blood clotting, were among the first biomaterials used to prevent bleeding and promote wound healing. The unique polymerization mechanism of fibrin, which allows control of gelation times and network architecture by variation in reaction conditions, allows formation of a wide array of soft substrates under physiological conditions. Fibrin gels have been extensively studied rheologically in part because their nonlinear elasticity, characterized by soft compliance at small strains and impressive stiffening to resist larger deformations, appears essential for their function as haemostatic plugs and as matrices for cell migration and wound healing. The filaments forming a fibrin network are among the softest in nature, allowing them to deform to large extents and stiffen but not break. The biochemical and mechanical properties of fibrin have recently been exploited in numerous studies that suggest its potential for applications in medicine and bioengineering.

THE POLYELECTROLYTE NATURE OF F-ACTIN AND THE MECHANISM OF ACTIN BUNDLE FORMATION

Polymerized (F-)actin is induced to form bundles by a number of polycations including divalent metal ions, Co(NH), and basic polypeptides. The general features of bundle formation are largely independent of the specific structure of the bundling agent used. A threshold concentration of polycation is required to form lateral aggregates of actin filaments. The threshold concentration varies strongly with the valence of the cation and increases with the ionic strength of the solution. Polyanions such as nucleoside phosphates or oligomers of acidic amino acids disaggregate actin bundles into single filaments. These features are similar to the phenomenon of DNA condensation and can be explained analogously by polyelectrolyte theories. Similar results were found when F-actin was bundled by the peptide corresponding to the actin binding site of myristoylated alanine-rich protein kinase C substrate protein (MARCKS) or by smooth muscle calponin, suggesting that a broad class of actin bundling factors may function in a common manner. Physiologic concentrations of both small ions and large proteins can induce actin interfilament association independent of a requirement for specific binding sites.

Bone marrow-derived human mesenchymal stem cells become quiescent on soft substrates but remain responsive to chemical or mechanical stimuli.

The microenvironment of bone marrow-derived human mesenchymal stem cells (hMSCs) strictly regulates their self-renewal and differentiation. Culturing these cells ex vivo leads to a rapid expansion followed by senescence, which is characterized by a lack of proliferation and differentiation. In this study, 250-Pa polyacrylamide gels, which mimics the elasticity of bone marrow and fat tissues, were coated with a mixture of collagen type 1 and fibronectin. When hMSCs were seeded sparsely on these gels, they halted progression through the cell cycle despite the presence of serum, but when presented with a stiff substrate, these non-proliferative cells reentered the cell cycle. Non-proliferative hMSCs on 250-Pa gels also exhibited the capability to differentiate into adipocytes when cultured in adipogenic induction medium or into osteoblasts if transferred to a stiff substrate and incubated with osteoblast induction medium. These results demonstrate that hMSCs on 250-Pa gels are quiescent but competent to resume proliferation or initiate terminal differentiation with appropriate cues. These observations suggest that mechanical signals from the elasticity of the extracellular matrix may be one of the factors that enable the bone marrow niche to maintain MSCs as a reservoir for a long period.

Cell Cycle Control by Physiological Matrix Elasticity and In Vivo Tissue Stiffening

BACKGROUND A number of adhesion-mediated signaling pathways and cell-cycle events have been identified that regulate cell proliferation, yet studies to date have been unable to determine which of these pathways control mitogenesis in response to physiologically relevant changes in tissue elasticity. In this report, we use hydrogel-based substrata matched to biological tissue stiffness to investigate the effects of matrix elasticity on the cell cycle. RESULTS We find that physiological tissue stiffness acts as a cell-cycle inhibitor in mammary epithelial cells and vascular smooth muscle cells; subcellular analysis in these cells, mouse embryonic fibroblasts, and osteoblasts shows that cell-cycle control by matrix stiffness is widely conserved. Remarkably, most mitogenic events previously documented as extracellular matrix (ECM)/integrin-dependent proceed normally when matrix stiffness is altered in the range that controls mitogenesis. These include ERK activity, immediate-early gene expression, and cdk inhibitor expression. In contrast, FAK-dependent Rac activation, Rac-dependent cyclin D1 gene induction, and cyclin D1-dependent Rb phosphorylation are strongly inhibited at physiological tissue stiffness and rescued when the matrix is stiffened in vitro. Importantly, the combined use of atomic force microscopy and fluorescence imaging in mice shows that comparable increases in tissue stiffness occur at sites of cell proliferation in vivo. CONCLUSIONS Matrix remodeling associated with pathogenesis is in itself a positive regulator of the cell cycle through a highly selective effect on integrin-dependent signaling to FAK, Rac, and cyclin D1.