Janmey, Paul
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Publication Peering from the outside in: viscoelastic properties of the extracellular matrix dictate spatial organization and apoptosis resistance in mammary epithelial cells(2002-10-23) Zahir, N.; Yeung, T.; Janmey, Paul; Ming, W.; Weaver, Valerie M.The compliance of the extracellular matrix (ECM) differs between tissues and is altered in tumors. We examined the consequence of modifying the viscoelastic properties of the ECM on mammary epithelial cell (MEC) morphogenesis and apoptosis regulation. Results showed that the elastic modulus of the ECM exerts a profound effect on MEC tissue organization and gene expression that correlates with changes in actin organization and apoptosis resistance. Altering the rigidity of the ECM directly influences integrin expression and additionally modifies integrin-induced gene expression in association with actin reorganization. These data suggest that the compliance of the ECM may cooperatively regulate cell behavior by altering integrin function. Studies are now underway to investigate the possibility that these effects are mediated via changes in integrin-actin cytoskeletal dynamics.Publication Lamellar Phase of Stacked Two-Dimensional Rafts of Actin Filaments(2003-07-04) Wong, Gerard C.L.; Lin, Alison; Tang, Jay X.; Li, Youli; Janmey, Paul; Safinya, Cyrus RWe examined liquid crystalline phases of the cytoskeletal polyelectrolyte filamentous (F-)actin in the presence of multivalent counterions. As a function of increasing ion concentration, the F-actin rods in either an isotropic or a nematic phase will transform into a new and unexpected lamellar phase of crosslinked rafts (LXR phase), before condensing into a bundled phase of parallel, close-packed rods. This behavior is generic for alkali earth divalent ions Mg2+, Ca2+, Sr2+, and Ba2+, and the structural transitions are achieved without any architecture-specific actin-binding linker proteins.Publication Interaction of the Gelsolin-Derived Antibacterial PBP 10 Peptide with Lipid Bilayers and Cell Membranes(2006-09-01) Bucki, Robert; Janmey, PaulPBP 10, an antibacterial, cell membrane-permeant rhodamine B-conjugated peptide derived from the polyphosphoinositide binding site of gelsolin, interacts selectively with both lipopolysaccharides (LPS) and lipoteichoic acid (LTA), the distinct components of gram-negative and gram-positive bacteria, respectively. Isolated LPS and LTA decrease the antimicrobial activities of PBP 10, as well as other antimicrobial peptides, such as cathelicidin-LL37 (LL37) and mellitin. In an effort to elucidate the mechanism of bacterial killing by PBP 10, we compared its effects on artificial lipid bilayers and eukaryotic cell membranes with the actions of the mellitin, magainin II, and LL37 peptides. This study reveals that pore formation is unlikely to be involved in PBP 10-mediated membrane destabilization. We also investigated the effects of these peptides on platelets and red blood cells (RBCs). Comparison of these antimicrobial peptides shows that only mellitin has a toxic effect on platelets and RBCs in a concentration range concomitant with its bactericidal activity. The hemolytic activities of the PBP 10 and LL37 peptides significantly increase when RBCs are osmotically swollen in hypotonic solution, indicating that these antibacterial peptides may take advantage of the more extended form of bacterial membranes in exerting their killing activities. Additionally, we found that LL37 hemolytic activity was much higher when RBCs were induced to expose phosphatidylserine to the external leaflet of their plasma membranes. This finding suggests that asymmetrical distribution of phospholipids in the external membranes of eukaryotic cells may represent an important factor in determining the specificity of antibacterial peptides for targeting bacteria rather than eukaryotic cells.Publication Metal Ion-Induced Lateral Aggregation of Filamentous Viruses fd and M13(2002-07-01) Tang, Jay X.; Janmey, Paul; Lyubartsev, Alexander; Nordenskiöld, LarsWe report a detailed comparison between calculations of inter-filament interactions based on Monte-Carlo simulations and experimental features of lateral aggregation of bacteriophages fd and M13 induced by a number of divalent metal ions. The general findings are consistent with the polyelectrolyte nature of the virus filaments and confirm that the solution electrostatics account for most of the experimental features observed. One particularly interesting discovery is resolubilization for bundles of either fd or M13 viruses when the concentration of the bundle-inducing metal ion Mg2+ or Ca2+ is increased to large (>100 mM) values. In the range of Mg2+ or Ca2+ concentrations where large bundles of the virus filaments are formed, the optimal attractive interaction energy between the virus filaments is estimated to be on the order of 0.01 kT per net charge on the virus surface when a recent analytical prediction to the experimentally defined conditions of resolubilization is applied. We also observed qualitatively distinct behavior between the alkali-earth metal ions and the divalent transition metal ions in their action on the charged viruses. The understanding of metal ions-induced reversible aggregation based on solution electrostatics may lead to potential applications in molecular biology and medicine.Publication The Interaction of Neurofilaments with the Microtubule Motor Cytoplasmic Dynein(2004-11-01) Tokito, Mariko; Wagner, Oliver I; Ascaño, Jennifer; Janmey, Paul; Holzbaur, Erika; Leterrier, Jean-FrancoisNeurofilaments are synthesized in the cell body of neurons and transported outward along the axon via slow axonal transport. Direct observation of neurofilaments trafficking in live cells suggests that the slow outward rate of transport is due to the net effects of anterograde and retrograde microtubule motors pulling in opposition. Previous studies have suggested that cytoplasmic dynein is required for efficient neurofilament transport. In this study, we examine the interaction of neurofilaments with cytoplasmic dynein. We used fluid tapping mode atomic force microscopy to visualize single neurofilaments, microtubules, dynein/dynactin, and physical interactions between these neuronal components. AFM images suggest that neurofilaments act as cargo for dynein, associating with the base of the motor complex. Yeast two-hybrid and affinity chromatography assays confirm this hypothesis, indicating that neurofilament subunit M binds directly to dynein IC. This interaction is blocked by monoclonal antibodies directed either to NF-M or to dynein. Together these data suggest that a specific interaction between neurofilament subunit M and cytoplasmic dynein is involved in the saltatory bidirectional motility of neurofilaments undergoing axonal transport in the neuron.Publication Counterion-Mediated Attraction and Kinks on Loops of Semiflexible Polyelectrolyte Bundles(2006-06-21) Cēbers, A.; Dogic, Z.; Janmey, PaulThe formation of kinks in a loop of bundled polyelectrolyte filaments is analyzed in terms of the thermal fluctuations of charge density due to polyvalent counterions adsorbed on the polyelectrolyte filaments. It is found that the counterion-mediated attraction energy of filaments depends on their bending. By consideration of curvature elasticity energy and counterion-mediated attraction between polyelectrolyte filaments, the characteristic width of the kink and the number of kinks per loop is found to be in reasonable agreement with existing experimental data for rings of bundled actin filaments.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 Biopolymer Networks and Cellular Mechanosensing(2004-06-01) Georges, Penelope; Wagner, Oliver; Yeung, Tony; Janmey, PaulCells and tissues are mechanical as well as biochemical machines, and cellular response to mechanical cues can have as large an influence on structure and function as chemical signals. The mechanical properties of cells are largely determined by networks of semiflexible polymers forming the cytoskeleton, which has viscoelastic properties that differ in important ways from the viscoelasticity of common synthetic materials. Two such features are the high resistance to deformation achieved by a remarkably low volume fraction of protein, and the increase in stiffness that occurs when the cytoskeletal network is deformed. The actin filaments, microtubules and intermediate filaments that comprise the cytoskeleton of most cell types are linear polymers with some important similarities but also some fundamental differences. The stiffness of the individual polymer types is vastly different, with persistence lengths ranging from 1 mm for the 24 nm diameter microtubules to a few 100 nm for the 10-14 nm diameter intermediate filaments. The material properties of these biopolymer networks are proposed to function as part of the mechanosensing mechanism in cells, and the stiffness of cytoskeletal networks is similar to that of common extracellular protein networks such as those formed by collagen and fibrin in which many cell types function. Examples of the morphologic differences in fibroblasts and astrocytes grown on chemically identical surfaces overlying gels with elastic moduli spanning the range from 50 to 12,000 Pa illustrate the large effect of stiffness differences on cell structure and function.Publication Separate Functions of Gelsolin Mediate Sequential Steps of Collagen Phagocytosis(2005-11-01) Arora, P. D; Chan, M.W. C; Anderson, R. A; Janmey, Paul A; McCulloch, C. ACollagen phagocytosis is a critical mediator of extracellular matrix remodeling. Whereas the binding step of collagen phagocytosis is facilitated by Ca2+-dependent, gelsolin-mediated severing of actin filaments, the regulation of the collagen internalization step is not defined. We determined here whether phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] regulation of gelsolin is required for collagen internalization. In gelsolin null fibroblasts transfected with gelsolin severing mutants, actin severing and collagen binding were strongly impaired but internalization and actin monomer addition at collagen bead sites were much less affected. PI(4,5)P2 accumulated around collagen during internalization and was associated with gelsolin. Cell-permeable peptides mimicking the PI(4,5)P2 binding site of gelsolin blocked actin monomer addition, the association of gelsolin with actin at phagosomes, and collagen internalization but did not affect collagen binding. Collagen beads induced recruitment of type 1 γ phosphatidylinositol phosphate kinase (PIPK1γ661) to internalization sites. Dominant negative constructs and RNA interference demonstrated a requirement for catalytically active PIPK1γ661 for collagen internalization. We conclude that separate functions of gelsolin mediate sequential stages of collagen phagocytosis: Ca2+-dependent actin severing facilitates collagen binding, whereas PI(4,5)P2-dependent regulation of gelsolin promotes the actin assembly required for internalization of collagen fibrils.Publication Counterion-Mediated Attraction and Kinks on Loops of Semiflexible Polyelectrolyte Bundles(2006-06-21) Cebers, A.; Dogic, Z.; Janmey, PaulThe formation of kinks in a loop of bundled polyelectrolyte filaments is analyzed in terms of the thermal fluctuations of charge density due to polyvalent counterions adsorbed on the polyelectrolyte filaments. It is found that the counterion-mediated attraction energy of filaments depends on their bending. By consideration of curvature elasticity energy and counterion-mediated attraction between polyelectrolyte filaments, the characteristic width of the kink and the number of kinks per loop is found to be in reasonable agreement with existing experimental data for rings of bundled actin filaments.