Discher, Dennis E

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2000 - 201025
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  • Publication
    Substrate Compliance versus Ligand Density in Cell on Gel Responses
    (2004-01-01) Engler, Adam; Bacakova, Lucie; Newman, Cynthia; Hategan, Alina; Discher, Dennis E; Griffin, Maureen
    Substrate stiffness is emerging as an important physical factor in the response of many cell types. In agreement with findings on other anchorage-dependent cell lineages, aortic smooth muscle cells are found to spread and organize their cytoskeleton and focal adhesions much more so on "rigid" glass or "stiff" gels than on "soft" gels. Whereas these cells generally show maximal spreading on intermediate collagen densities, the limited spreading on soft gels is surprisingly insensitive to adhesive ligand density. Bell-shaped cell spreading curves encompassing all substrates are modeled by simple functions that couple ligand density to substrate stiffness. Although smooth muscle cells spread minimally on soft gels regardless of collagen, GFP-actin gives a slight overexpression of total actin that can override the soft gel response and drive spreading; GFP and GFP-paxillin do not have the same effect. The GFP-actin cells invariably show an organized filamentous cytoskeleton and clearly indicate that the cytoskeleton is at least one structural node in a signaling network that can override spreading limits typically dictated by soft gels. Based on such results, we hypothesize a central structural role for the cytoskeleton in driving the membrane outward during spreading whereas adhesion reinforces the spreading.
  • Publication
    Inhibition of "self" engulfment through deactivation of myosin-II at the phagocytic synapse between human cells
    (2008-03-10) Tsai, Richard K; Discher, Dennis E
    Phagocytosis of foreign cells or particles by macrophages is a rapid process that is inefficient when faced with "self" cells that display CD47—although signaling mechanisms in self-recognition have remained largely unknown. With human macrophages, we show the phagocytic synapse at cell contacts involves a basal level of actin-driven phagocytosis that, in the absence of species-specific CD47 signaling, is made more efficient by phospho-activated myosin. We use "foreign" sheep red blood cells (RBCs) together with CD47-blocked, antibody-opsonized human RBCs in order to visualize synaptic accumulation of phosphotyrosine, paxillin, F-actin, and the major motor isoform, nonmuscle myosin-IIA. When CD47 is functional, the macrophage counter-receptor and phosphatase-activator SIRPα localizes to the synapse, suppressing accumulation of phosphotyrosine and myosin without affecting F-actin. On both RBCs and microbeads, human CD47 potently inhibits phagocytosis as does direct inhibition of myosin. CD47–SIRPα interaction initiates a dephosphorylation cascade directed in part at phosphotyrosine in myosin. A point mutation turns off this motor's contribution to phagocytosis, suggesting that self-recognition inhibits contractile engulfment.
  • Publication
    Surface probe measurements of the elasticity of sectioned tissue, thin gels and polyelectrolyte multilayer films : correlations between substrate stiffness and cell adhesion
    (2004-10-10) Engler, Adam J; Richert, Ludovic; Wong, Joyce; Discher, Dennis E; Picart, Catherine
    Surface probe measurements of the elasticity of thin-film matrices as well as biological samples prove generally important to understanding cell attachment across such systems. To illustrate this, sectioned arteries were probed by Atomic Force Microscopy (AFM) within the smooth muscle cell (SMC)-rich medial layer, yielding an apparent Young’s modulus Emedia ~ 5-8 kPa. Polyacrylamide gels with Egel spanning several-fold above and below this range were then cast 5-70 μm thick and coated with collagen: SMC spreading shows a hyperbolic dependence in projected cell area versus Egel. The modulus that gives half-max spreading is E1/2-spread ~ 8-10 kPa, proving remarkably close to Emedia. More complex, layer-by-layer micro-films of poly(L-lysine)/hyaluronic acid were also tested and show equivalent trends of increased SMC spreading with increased stiffness. Adhesive spreading of cells thus seems to correlate broadly with the effective stiffness of synthetic materials and tissues.
  • Publication
    Adhesively-Tensed Cell Membranes: Lysis Kinetics and Atomic Force Microscopy Probing
    (2003-10-01) Hategan, Alina; Kahn, Samuel; Law, Richard; Discher, Dennis E
    Membrane tension underlies a range of cell physiological processes. Strong adhesion of the simple red cell is used as a simple model of a spread cell with a finite membrane tension—a state which proves useful for studies of both membrane rupture kinetics and atomic force microscopy (AFM) probing of native structure. In agreement with theories of strong adhesion, the cell takes the form of a spherical cap on a substrate densely coated with poly-L-lysine. The spreading-induced tension, σ, in the membrane is ~1 mN/m, which leads to rupture over many minutes; and σ is estimated from comparable rupture times in separate micropipette aspiration experiments. Under the sharpened tip of an AFM probe, nano-Newton impingement forces (10–30 nN) are needed to penetrate the tensed erythrocyte membrane, and these forces increase exponentially with tip velocity (~nm/ms). We use the results to clarify how tapping-mode AFM imaging works at high enough tip velocities to avoid rupturing the membrane while progressively compressing it to a ~20-nm steric core of lipid and protein. We also demonstrate novel, reproducible AFM imaging of tension-supported membranes in physiological buffer, and we describe a stable, distended network consistent with the spectrin cytoskeleton. Additionally, slow retraction of the AFM tip from the tensed membrane yields tether-extended, multipeak sawtooth patterns of average force ~200 pN. In sum we show how adhesive tensioning of the red cell can be used to gain novel insights into native membrane dynamics and structure.
  • Publication
    Post-translational Regulation of Expression and Conformation of an Immunoglobulin Domain in Yeast Surface Display
    (2005-09-13) Parthasarathy, Ranganath; Boder, Eric T; Subramanian, Shyamsundar; Discher, Dennis E.
    Display of heterologous proteins on the surface of Saccharomyces cerevisiae is increasingly being exploited for directed evolution because of straightforward cell screens. However, yeast post-translationally modifies proteins in ways that must be factored into library engineering and refinement. Here, we express the extracellular immunoglobulin domain of an ubiquitous mammalian membrane protein, CD47, which is implicated in cancer, immunocompatibility, and motility. CD47 has multiple sites of glycosylation and a core disulfide bond. We assess the effects of both of these post-translational modifications on expression and antibody binding. CD47’s extracellular domain is fused to the yeast mating protein Aga2p on the cell wall, and the resulting fusion protein binds several key antibodies, including a conformation-sensitive antibody. Site-by-site mutagenesis of CD47’s five N-linked glycosylation sites progressively decreases expression levels on yeast, but folding appears stable. Cysteine mutations disrupt the expected core disulfide, and also decrease protein expression levels, though not to the extent seen with complete deglycosylation. However, with the core disulfide mutants, antibody binding proves to be lower than expression levels might indicate and glycosylation is clearly reduced compared to wild-type. The results indicate that glycosylation regulates heterologous display on yeast more than core disulfides do and thus suggest bounds on directed evolution by post-translational processing.
  • Publication
    Pore Stability and Dynamics in Polymer Membranes
    (2003-11-15) Bermudez, Harry; Hammer, Daniel A; Aranda-Espinoza, Helim; Discher, Dennis E
    Vesicles self-assembled from amphiphilic diblock copolymers exhibit a wide diversity of behavior upon poration, due to competitions between edge, surface and bending energies, while viscous dissipation mechanisms determine the time scales. The copolymers are essentially chemically identical, only varying in chain length (related to the membrane thickness d). For small d, we find large unstable pores and the resulting membrane fragments reassemble into vesicles within minutes. For large d, however, submicron pores form and are extremely long-lived. The results show that pore behavior depends strongly on d, suggesting that the relevant energies depend on d and pore size r in a more complexmanner than what is generally assumed. Further control over these systems would make them useful for numerous applications.
  • Publication
    Actin Protofilament Orientation in Deformation of the Erythrocyte Membrane Skeleton
    (2000-12-01) Picart, Catherine; Dalhaimer, Paul M; Discher, Dennis E
    The red cell’s spectrin-actin network is known to sustain local states of shear, dilation, and condensation, and yet the short actin filaments are found to maintain membrane-tangent and near-random azimuthal orientations. When calibrated with polarization results for single actin filaments, imaging of micropipette-deformed red cell ghosts has allowed an assessment of actin orientations and possible reorientations in the network. At the hemispherical cap of the aspirated projection, where the network can be dilated severalfold, filaments have the same membrane-tangent orientation as on a relatively unstrained portion of membrane. Likewise, over the length of the network projection pulled into the micropipette, where the network is strongly sheared in axial extension and circumferential contraction, actin maintains its tangent orientation and is only very weakly aligned with network extension. Similar results are found for the integral membrane protein Band 3. Allowing for thermal fluctuations, we deduce a bound for the effective coupling constant, α, between network shear and azimuthal orientation of the protofilament. The finding that α must be about an order of magnitude or more below its tight-coupling value illustrates how nanostructural kinematics can decouple from more macroscopic responses. Monte Carlo simulations of spectrin-actin networks at ~10-nm resolution further support this conclusion and substantiate an image of protofilaments as elements of a high-temperature spin glass.
  • Publication
    Myotubes differentiate optimally on substrates with tissue-like stiffness : pathological implications for soft or stiff microenvironments
    (2004-09-13) Engler, Adam J.; Sen, Shamik; Griffin, Maureen A.; Discher, Dennis E; Bönnemann, Carsten G.; Sweeney, H. Lee
    Contractile myocytes provide a test of the hypothesis that cells sense their mechanical as well as molecular microenvironment, altering expression, organization, and/or morphology accordingly. Here, myoblasts were cultured on collagen strips attached to glass or polymer gels of varied elasticity. Subsequent fusion into myotubes occurs independent of substrate flexibility. However, myosin/actin striations emerge later only on gels with stiffness typical of normal muscle (passive Young's modulus, E ~12 kPa). On glass and much softer or stiffer gels, including gels emulating stiff dystrophic muscle, cells do not striate. In addition, myotubes grown on top of a compliant bottom layer of glass-attached myotubes (but not softer fibroblasts) will striate, whereas the bottom cells will only assemble stress fibers and vinculin-rich adhesions. Unlike sarcomere formation, adhesion strength increases monotonically versus substrate stiffness with strongest adhesion on glass. These findings have major implications for in vivo introduction of stem cells into diseased or damaged striated muscle of altered mechanical composition.
  • Publication
    Polymersomes
    (2006-08-01) Discher, Dennis E; Ahmed, Fariyal
    Polymersomes are self-assembled polymer shells composed of block copolymer amphiphiles. These synthetic amphiphiles have amphiphilicity similar to lipids, but they have much larger molecular weights, so for this reason — along with others reviewed here — comparisons of polymersomes with viral capsids composed of large polypeptide chains are highly appropriate. We summarize the wide range of polymers used to make polymersomes along with descriptions of physical properties such as stability and permeability. We also elaborate on emerging studies of in vivo stealthiness, programmed disassembly for controlled release, targeting in vitro, and tumor-shrinkage in vivo. Comparisons of polymersomes with viral capsids are shown to encompass and inspire many aspects of current designs.
  • Publication
    Curvature-driven Molecular Demixing in the Budding and Breakup of Mixed Component Worm-like Miscelles
    (2010-01-04) Sharon, Loverde M; Ortiz, Vanessa; Kamien, Randall D; Discher, Dennis E; Klein, Michael L
    Amphiphilic block copolymers of suitable proportions can self-assemble into surprisingly long and stable worm-like micelles, but the intrinsic polydispersity of polymers as well as polymer blending efforts and the increasing use of degradable chains all raise basic questions of curvature–composition coupling and morphological stability of these high curvature assemblies. Molecular simulations here of polyethylene glycol (PEG) based systems show that a systematic increase in the hydrated PEG fraction, in both monodisperse and binary blends, induces budding and breakup into spherical and novel ‘dumbbell’ micelles—as seen in electron microscopy images of degradable worm-like micelles. Core dimension, d, in our large-scale, long-time dissipative particle dynamics (DPD) simulations is shown to scale with chain-length, N, as predicted theoretically by the strong segregation limit (d ≈ N2/3), but morphological transitions of binary mixtures are only crudely predicted by simple mixture rules. Here we show that for weakly demixing diblock copolymers, the coupling between local interfacial concentration and mean curvature can be described with a simple linear relationship. The computational methods developed here for PEG-based assemblies should be useful for many high curvature nanosystems.