Discher, Dennis E
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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, CatherineSurface 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 Polymersomes(2006-08-01) Discher, Dennis E; Ahmed, FariyalPolymersomes 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 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 EVesicles 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 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. LeeContractile 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 Patterning, Prestress, and Peeling Dynamics of Myocytes(2004-02-01) Engler, Adam J.; Griffin, Maureen A.; Sweeney, H. Lee; Barber, Thomas A.; Discher, Dennis E; Healy, Kevin E.As typical anchorage-dependent cells myocytes must balance contractility against adequate adhesion. Skeletal myotubes grown as isolated strips from myoblasts on micropatterned glass exhibited spontaneous peeling after one end of the myotube was mechanically detached. Such results indicate the development of a prestress in the cells. To assess this prestress and study the dynamic adhesion strength of single myocytes, the shear stress of fluid aspirated into a large-bore micropipette was then used to forcibly peel myotubes. The velocity at which cells peeled from the surface, Vpeel, was measured as a continuously increasing function of the imposed tension, Tpeel, which ranges from ~0 to 50 nN/μm. For each cell, peeling proved highly heterogeneous, with Vpeel fluctuating between 0 μm/s (~80% of time) and ~10 μm/s. Parallel studies of smooth muscle cells expressing GFP-paxillin also exhibited a discontinuous peeling in which focal adhesions fractured above sites of strong attachment (when pressure peeled using a small-bore pipette). The peeling approaches described here lend insight into the contractile-adhesion balance and can be used to study the real-time dynamics of stressed adhesions through both physical detection and the use of GFP markers; the methods should prove useful in comparing normal versus dystrophic muscle cells.Publication Adhesively-Tensed Cell Membranes: Lysis Kinetics and Atomic Force Microscopy Probing(2003-10-01) Hategan, Alina; Kahn, Samuel; Law, Richard; Discher, Dennis EMembrane 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 Actin Protofilament Orientation in Deformation of the Erythrocyte Membrane Skeleton(2000-12-01) Picart, Catherine; Dalhaimer, Paul M; Discher, Dennis EThe 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 Indentation and adhesive probing of a cell membrane with AFM: Theoretical model and experiments(2005-11-01) Sen, Shamik; Subramanian, Shyamsundar; Discher, Dennis EIn probing adhesion and cell mechanics by atomic force microscopy (AFM), the mechanical properties of the membrane have an important if neglected role. Here we theoretically model the contact of an AFM tip with a cell membrane, where direct motivation and data are derived from a prototypical ligand-receptor adhesion experiment. An AFM tip is functionalized with a prototypical ligand, SIRPα, and then used to probe its native receptor on red cells, CD47. The interactions prove specific and typical in force, and also show in detachment, a sawtooth-shaped disruption process that can extend over hundreds of nm. The theoretical model here that accounts for both membrane indentation as well as membrane extension in tip retraction incorporates membrane tension and elasticity as well as AFM tip geometry and stochastic disruption. Importantly, indentation depth proves initially proportional to membrane tension and does not follow the standard Hertz model. Computations of detachment confirm nonperiodic disruption with membrane extensions of hundreds of nm set by membrane tension. Membrane mechanical properties thus clearly influence AFM probing of cells, including single molecule adhesion experiments.Publication Domain unfolding in neurofilament sidearms: effects of phosphorylation and ATP(2002-10-10) Aranda-Espinoza, Helim; Carl, Philippe; Janmey, Paul; Leterrier, Jean-François; Discher, Dennis ELateral projections of neuro¢laments (NF) called sidearms (SA) a¡ect axon stability and caliber. SA phosphorylation is thought to modulate inter-NF distance and interactions between NF and other subcellular organelles. SA were probed by atomic force microscopy (AFM) and dynamic light scattering (DLS) as a function of phosphorylation and ATP content. DLS shows SA are larger when phosphorylated, and AFM shows four unfoldable domains in SA regardless of phosphorylation state or the presence of ATP. However, the native phosphorylated SA requires three-fold higher force to unfold by AFM than dephosphorylated SA, suggesting a less pliant as well as larger structure when phosphorylated.
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