Discher, Dennis E

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Now showing 1 - 10 of 25
  • 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
    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 E
    Lateral 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.
  • Publication
    Pathway Shifts and Thermal Softening in Temperature-Coupled Forced Unfolding of Spectrin Domains
    (2003-11-01) Law, Richard; Liao, George; Harper, Sandy; Yang, Guoliang; Speicher, David W.; Discher, Dennis E
    Pathways of unfolding a protein depend in principle on the perturbation—whether it is temperature, denaturant, or even forced extension. Widely-shared, helical-bundle spectrin repeats are known to melt at temperatures as low as 40–45oC and are also known to unfold via multiple pathways as single molecules in atomic force microscopy. Given the varied roles of spectrin family proteins in cell deformability, we sought to determine the coupled effects of temperature on forced unfolding. Bimodal distributions of unfolding intervals are seen at all temperatures for the four-repeat β1–4 spectrin—an α-actinin homolog. The major unfolding length corresponds to unfolding of a single repeat, and a minor peak at twice the length corresponds to tandem repeats. Increasing temperature shows fewer tandem events but has no effect on unfolding intervals. As T approaches Tm, however, mean unfolding forces in atomic force microscopy also decrease; and circular dichroism studies demonstrate a nearly proportional decrease of helical content in solution. The results imply a thermal softening of a helical linker between repeats which otherwise propagates a helix-to-coil transition to adjacent repeats. In sum, structural changes with temperature correlate with both single-molecule unfolding forces and shifts in unfolding pathways.
  • Publication
    Micelles of Different Morphologies - Advantages of Worm-like Filomicelles of PEO-PCL in Paclitaxel Delivery
    (2007-06-01) Cai, Shenshen; Vijayan, Kandaswamy; Cheng, Debbie; Lima, Eliana M; Discher, Dennis E
    Worm-like and spherical micelles are both prepared here from the same amphiphilic diblock copolymer, poly(ethylene oxide)-b-poly (ε-caprolactone) (PEO [5 kDa]-PCL [6.5 kDa]) in order to compare loading and delivery of hydrophobic drugs. Worm-like micelles of this degradable copolymer are nanometers in cross-section and spontaneously assemble to stable lengths of microns, resembling filoviruses in some respects and thus suggesting the moniker "filomicelles". The highly flexible worm-like micelles can also be sonicated to generate kinetically stable spherical micelles composed of the same copolymer. The fission process exploits the finding that the PCL cores are fluid, rather than glassy or crystalline, and core-loading of the hydrophobic anticancer drug delivery, paclitaxel (TAX) shows that the worm-like micelles load and solubilize twice as much drug as spherical micelles. In cytotoxicity tests that compare to the clinically prevalent solubilizer, Cremophor® EL, both micellar carriers are far less toxic, and both types of TAX-loaded micelles also show 5-fold greater anticancer activity on A549 human lung cancer cells. PEO-PCL based worm-like filomicelles appear to be promising pharmaceutical nanocarriers with improved solubilization efficiency and comparable stability to spherical micelles, as well as better safety and efficacy in vitro compared to the prevalent Cremophor® EL TAX formulation.
  • Publication
    Electromechanical Limits of Polymersomes
    (2001-11-12) Aranda-Espinoza, Helim; Bermudez, Harry; Bates, Frank S; Discher, Dennis E
    Self-assembled membranes of amphiphilic diblock copolymers enable comparisons of cohesiveness with lipid membranes over the range of hydrophobic thicknesses d = 3-15 nm. At zero mechanical tension the breakdown potential Vc for polymersomes with d = 15 nm is 9 V, compared to 1 V for liposomes with d = 3 nm. Nonetheless, electromechanical stresses at breakdown universally exhibit a V2 c dependence, and membrane capacitance shows the expected strong d dependence, conforming to simple thermodynamic models. The viscous nature of the diblock membranes is apparent in the protracted postporation dynamics.
  • Publication
    Adhesion-contractile balance in myocyte differentiation
    (2004-11-02) Griffin, Maureen A.; Sen, Shamik; Sweeney, H. Lee; Discher, Dennis E
    Tissue 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 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.
  • Publication
    Topographical Pattern Dynamics in Passive Adhesion of Cell Membranes
    (2004-11-01) Hategan, Alina; Kahn, Samuel; Sengupta, Kheya; Discher, Dennis E; Sackmann, Erich
    Strong 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, Arjun
    We 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 E
    Delivery 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.