Discher, Dennis E
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Publication Adhesion-contractile balance in myocyte differentiation(2004-11-02) Griffin, Maureen A.; Sen, Shamik; Sweeney, H. Lee; Discher, Dennis ETissue cells generally pull on their matrix attachments and balance a quasi-static contractility against adequate adhesion, but any correlation with and/or influence on phenotype are not yet understood. Here, we begin to demonstrate how differentiation state couples to actomyosin-based contractility through adhesion and substrate compliance. Myotubes are differentiated from myoblasts on collagen-patterned coverslips that allow linear fusion but prevent classic myotube branching. Postfusion, myotubes adhere to the micro-strips but lock into a stress fiber-rich state and do not differentiate significantly further. In contrast, myotubes grown on top of such cells do progress through differentiation, exhibiting actomyosin striations within one week. A compliant adhesion to these lower cells is suggested to couple to contractility and accommodate the reorganization needed for upper cell striation. Contractility is assessed in these adherent cells by mechanically detaching one end of the myotubes. All myotubes, whether striated or not, shorten with an exponential decay. The cell-on-cell myotubes relax more, which implies a greater contractile stress. The non-muscle myosin II inhibitor blebbistatin inhibits relaxation for either case. Myotubes in culture are thus clearly prestressed by myosin II, and this contractility couples to substrate compliance and ultimately influences actomyosin striation.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 LAmphiphilic 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.Publication Topographical Pattern Dynamics in Passive Adhesion of Cell Membranes(2004-11-01) Hategan, Alina; Kahn, Samuel; Sengupta, Kheya; Discher, Dennis E; Sackmann, ErichStrong adhesion of highly active cells often nucleates focal adhesions, synapses, and related structures. Red cells lack such complex adhesion systems and are also nonmotile, but they are shown here to dynamically evolve complex spatial patterns beyond an electrostatic threshold for strong adhesion. Spreading of the cell onto a dense, homogeneous poly-L-lysine surface appears complete in <1 s with occasional blisters that form and dissipate on a similar timescale; distinct rippled or stippled patterns in fluorescently labeled membrane components emerge later, however, on timescales more typical of long-range lipid diffusion (approximately minutes). Within the contact zone, the anionic fluorescent lipid fluorescein phosphoethanolamine is seen to rearrange, forming worm-like rippled or stippled domains of <500 nm that prove independent of whether the cell is intact and sustaining a tension or ruptured. Lipid patterns are accompanied by visible perturbations in Band 3 distribution and weaker perturbations in membrane skeleton actin. Pressing down on the membrane quenches the lipid patterns, revealing a clear topographical basis for pattern formation. Counterion screening and membrane fluctuations likely contribute, but the results primarily highlight the fact that even in adhesion of a passive red cell, regions of strong contact slowly evolve to become interspersed with regions where the membrane is more distant from the surface.Publication Elongation and Fluctuations of Semi-flexible Polymers in a Nematic Solvent(2004-03-26) Dogic, Z.; Zhang, J.; Discher, Dennis E; Lau, A. W.C.; Janmey, Paul; Aranda-Espinoza, Helim; Kamien, Randall; Dalhaimer, Paul M; Lubensky, Thomas C.; Yodh, ArjunWe directly visualize single polymers with persistence lengths ranging from lp = 0:05 to 16 µm, dissolved in the nematic phase of rod-like fd virus. Polymers with sufficiently large persistence length undergo a coil-rod transition at the isotropic-nematic transition of the background solvent. We quantitatively analyze the transverse fluctuations of semi-flexible polymers and show that at long wavelengths they are driven by the fluctuating nematic background. We extract both the Odijk deflection length and the elastic constant of the background nematic phase from the data.Publication Efficient Nuclear Delivery and Nuclear Body Localization of Antisense Oligo-Nucleotides using Degradable Polymersomes(2006-08-30) Kim, Younghoon; Tewari, Manu; Pajerowski, J. David; Sen, Shamik; Jason, Williams; Sirsi, Shashank; Lutz, Gordon; Discher, Dennis EDelivery of antisense oligonucleotides, AON, presents many of the same challenges as delivery of any nucleic acid: charge, stability, cell uptake, endolysosomal escape, and entry into the nucleus. Here we demonstrate efficient delivery of AON after loading into biodegradable polymer vesicles or 'polymersomes'. We focus on AON delivery to muscle cells in vitro and in vivo because of the emergence of AON in therapeutic strategies directed at muscular dystrophies. To first clarify uptake kinetics without the complications of typical multi-layered myotube cultures, we use micro-patterned C2C12 cells and show efficient uptake of AON-polymersomes. The biodegradable polymersomes break down and foster AON escape with the binding of fluorescent-AON into the nuclear bodies. Intramuscular injections of the polymersome-AON into the hind limbs of mdx-dystrophic mice show more efficient nuclear uptake than AON alone and also lead to dystrophin expression in the mdx mice. In sum, these neutral, degradable carriers of AON show promise in vivo.Publication Physical plasticity of the nucleus in stem cell differentiation(2007-10-02) Pajerowski, J. David; Dahl, Kris Noel; Zhong, Franklin L; Sammak, Paul J; Discher, Dennis ECell differentiation in embryogenesis involves extensive changes in gene expression structural reorganization within the nucleus, including chromatin condensation and nucleoprotein immobilization. We hypothesized that nuclei in naive stem cells would therefore prove to be physically plastic and also more pliable than nuclei in differentiated cells. Micromanipulation methods indeed show that nuclei in human embryonic stem cells are highly deformable and stiffen 6-fold through terminal differentiation, and that nuclei in human adult stem cells possess an intermediate stiffness and deform irreversibly. Because the nucleo-skeletal component Lamin A/C is not expressed in either type of stem cell, we knocked down Lamin A/C in human epithelial cells and measured a deformability similar to that of adult hematopoietic stem cells. Rheologically, lamin-deficient states prove to be the most fluidlike, especially within the first ≈10 sec of deformation. Nuclear distortions that persist longer than this are irreversible, and fluorescence- imaged microdeformation with photobleaching confirms that chromatin indeed flows, distends, and reorganizes while the lamina stretches. The rheological character of the nucleus is thus set largely by nucleoplasm/chromatin, whereas the extent of deformation is modulated by the lamina.Publication Deformation-Enhanced Fluctuations in the Red Cell Skeleton with Theoretical Relations to Elasticity, Connectivity, and Spectrin Unfolding(2001-12-01) Lee, James C-M.; Discher, Dennis ETo assess local elasticity in the red cell’s spectrin-actin network, nano-particles were tethered to actin nodes and their constrained thermal motions were tracked. Cells were both immobilized and controllably deformed by aspiration into a micropipette. Since the network is well-appreciated as soft, thermal fluctuations even in an unstressed portion of network were expected to be many tens of nanometers based on simple equipartition ideas. Real-time particle tracking indeed reveals such root-mean-squared motions for 40-nm fluorescent beads either tethered to actin directly within a cell ghost or connected to actin from outside a cell via glycophorin. Moreover, the elastically constrained displacements are significant on the scale of the network’s internodal distance of ~60-80 nm. Surprisingly, along the aspirated projection—where the network is axially extended by as much as twofold or more—fluctuations in the axial direction are increased by almost twofold relative to motions in the unstressed network. The molecular basis for such strain softening is discussed broadly in terms of force-driven transitions. Specific considerations are given to 1) protein dissociations that reduce network connectivity, and 2) unfolding kinetics of a localized few of the red cell’s ~107 spectrin repeats.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 EPhagocytosis 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 Cooperativity in Forced Unfolding of Tandem Spectrin Repeats(2003-01-01) Law, Richard; Carl, Philippe; Harper, Sandy; Dalhaimer, Paul M; Speicher, David W; Discher, Dennis EForce-driven conformational changes provide a broad basis for protein extensibility, and multidomain proteins broaden the possibilities further by allowing for a multiplicity of forcibly extended states. Red cell spectrin is prototypical in being an extensible, multidomain protein widely recognized for its contribution to erythrocyte flexibility. Atomic force microscopy has already shown that single repeats of various spectrin family proteins can be forced to unfold reversibly under extension. Recent structural data indicates, however, that the linker between triple-helical spectrin repeats is often a contiguous helix, thus raising questions as to what the linker contributes and what defines a domain mechanically. We have examined the extensible unfolding of red cell spectrins as monomeric constructs of just two, three, or four repeats from the actin-binding ends of both α- and β-chains, i.e., α18–21 and β1–4 or their subfragments. In addition to single repeat unfolding evident in sawtooth patterns peaked at relatively low forces (<50 pN at 1>nm/ms extension rates), tandem repeat unfolding is also demonstrated in ensemble-scale analyses of thousands of atomic force microscopy contacts. Evidence for extending two chains and loops is provided by force versus length scatterplots which also indicate that tandem repeat unfolding occurs at a significant frequency relative to single repeat unfolding. Cooperativity in forced unfolding of spectrin is also clearly demonstrated by a common force scale for the unfolding of both single and tandem repeats.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.