Institute for Medicine and Engineering Papers Copyright (c) 2013 University of Pennsylvania All rights reserved. http://repository.upenn.edu/ime_papers Recent documents in Institute for Medicine and Engineering Papers en-us Wed, 23 Jan 2013 20:33:43 PST 3600 The Cytoskeleton Under External Fluid Mechanical Forces: Hemodynamic Forces Acting on the Endothelium http://repository.upenn.edu/ime_papers/35 http://repository.upenn.edu/ime_papers/35 Mon, 11 Aug 2008 13:41:02 PDT The endothelium, a single layer of cells that lines all blood vessels, is the focus of intense interest in biomechanics because it is the principal recipient of hemodynamic shear stress. In arteries, shear stress has been demonstrated to regulate both acute vasoregulation and chronic adaptive vessel remodeling and is strongly implicated in the localization of atherosclerotic lesions. Thus, endothelial biomechanics and the associated mechanotransduction of shear stress are of great importance in vascular physiology and pathology. Here we discuss the important role of the cytoskeleton in a decentralization model of endothelial mechanotransduction. In particular, recent studies of four-dimensional cytoskeletal motion in living cells under external fluid mechanical forces are summarized together with new data on the spatial distribution of cytoskeletal strain. These quantitative studies strongly support the decentralized distribution of luminally imposed forces throughout the endothelial cell.

]]> Brian P. Helmke et al. The convergence of haemodynamics, genomics, and endothelial structure in studies of the focal origin of atherosclerosis http://repository.upenn.edu/ime_papers/34 http://repository.upenn.edu/ime_papers/34 Mon, 11 Aug 2008 12:55:08 PDT The completion of the Human Genome Project and ongoing sequencing of mouse, rat and other genomes has led to an explosion of genetics-related technologies that are finding their way into all areas of biological research; the field of biorheology is no exception. Here we outline how two disparate modern molecular techniques, microarray analyses of gene expression and real-time spatial imaging of living cell structures, are being utilized in studies of endothelial mechanotransduction associated with controlled shear stress in vitro and haemodynamics in vivo. We emphasize the value of such techniques as components of an integrated understanding of vascular rheology. In mechanotransduction, a systems approach is recommended that encompasses fluid dynamics, cell biomechanics, live cell imaging, and the biochemical, cell biology and molecular biology methods that now encompass genomics. Microarrays are a useful and powerful tool for such integration by identifying simultaneous changes in the expression of many genes associated with interconnecting mechanoresponsive cellular pathways.

]]> Peter F. Davies et al. The Interaction of Neurofilaments with the Microtubule Motor Cytoplasmic Dynein http://repository.upenn.edu/ime_papers/33 http://repository.upenn.edu/ime_papers/33 Fri, 18 Jan 2008 07:15:18 PST Neurofilaments 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.

]]> Oliver I. Wagner et al. Contrast Adaptation in Subthreshold and Spiking Responses of Mammalian Y-Type Retinal Ganglion Cells http://repository.upenn.edu/ime_papers/32 http://repository.upenn.edu/ime_papers/32 Fri, 18 Jan 2008 07:11:56 PST Retinal ganglion cells adapt their responses to the amplitude of fluctuations around the mean light level, or the "contrast." But, in mammalian retina, it is not known whether adaptation arises exclusively at the level of synaptic inputs or whether there is also adaptation in the process of ganglion cell spike generation. Here, we made intracellular recordings from guinea pig Y-type ganglion cells and quantified changes in contrast sensitivity (gain) using a linear-nonlinear analysis. This analysis allowed us to measure adaptation in the presence of nonlinearities, such as the spike threshold, and to compare adaptation in subthreshold and spiking responses. At high contrast (0.30), relative to low contrast (0.10), gain reduced to 0.82 ± 0.016 (mean ± SEM) for the subthreshold response and to 0.61 ± 0.011 for the spiking response. Thus, there was an apparent reduction in gain between the subthreshold and spiking response of 0.74 ± 0.013. Control experiments suggested that the above effects could not be explained by an artifact of the intracellular recording conditions: extracellular recordings showed a gain change of 0.58 ± 0.022. For intracellular recordings, negative current reduced the spike output but did not affect the gain change in the subthreshold response: 0.80 ± 0.051. Thus, adaptation in the subthreshold response did not require spike-dependent conductances. We conclude that the contrast-dependent gain change in the spiking response can be explained by both a synaptic mechanism, as reflected by responses in the subthreshold potential, and an intrinsic mechanism in the ganglion cell related to spike generation.

]]> Kareem A. Zaghloul et al. Hypoxia-inducible Factor Regulates αvß3 Integrin Cell Surface Expression http://repository.upenn.edu/ime_papers/31 http://repository.upenn.edu/ime_papers/31 Tue, 15 Jan 2008 12:44:24 PST Hypoxia-inducible factor (HIF)-deficient placentas exhibit a number of defects, including changes in cell fate adoption, lack of fetal angiogenesis, hypocellularity, and poor invasion into maternal tissue. HIF is a heterodimeric transcription factor consisting of α and ß aryl hydrocarbon receptor nuclear translocator or ARNT) subunits. We used undifferentiated trophoblast stem (TS) cells to characterize HIF-dependent adhesion, migration, and invasion. Arnt-/- and Hifα-/- TS cells exhibit reduced adhesion and migration toward vitronectin compared with wild-type cells. Furthermore, this defect is associated with decreased cell surface expression of integrin αvß3 and significantly decreased expression of this integrin in focal adhesions. Because of the importance of adhesion and migration in tumor progression (in addition to placental development), we examined the affect of culturing B16F0 melanoma cells in 1.5% oxygen (O2). Culturing B16F0 melanoma cells at 1.5% O2 resulted in increased αvß3 integrin surface expression and increased adhesion to and migration toward vitronectin. Together, these data suggest that HIF and O2 tension influence placental invasion and tumor migration by increasing cell surface expression of αvß3 integrin.

]]> Karen D. Cowden Dahl et al. Phosphatidylinositol-4,5 Bisphosphate Produced by PIP5K(gamma) Regulates Gelsolin, Actin Assembly, and Adhesion Strength of N-Cadherin Junctions http://repository.upenn.edu/ime_papers/30 http://repository.upenn.edu/ime_papers/30 Tue, 15 Jan 2008 12:39:41 PST Phosphoinositides regulate several actin-binding proteins but their role at intercellular adhesions has not been defined. We found that phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) was generated at sites of N-cadherin–mediated intercellular adhesion and was a critical regulator of intercellular adhesion strength. Immunostaining for PI(4,5)P2 or transfection with GFP-PH-PLCδ showed that PI(4,5)P2 was enriched at sites of N-cadherin adhesions and this enrichment required activated Rac1. Isoform-specific immunostaining for type I phosphatidylinositol 4-phosphate 5 kinase (PIP5KI) showed that PIP5KIγ was spatially associated with N-cadherin–Fc beads. Association of PIP5KIγ with N-cadherin adhesions was in part dependent on the activation of RhoA. Transfection with catalytically inactive PIP5KIγ blocked the enrichment of PI(4,5)P2 around beads. Catalytically inactive PIP5KIγ or a cell-permeant peptide that mimics and competes for the PI(4,5)P2-binding region of the actin-binding protein gelsolin inhibited incorporation of actin monomers in response to N-cadherin ligation and reduced intercellular adhesion strength by more than twofold. Gelsolin null fibroblasts transfected with a gelsolin severing mutant containing an intact PI(4,5)P2 binding region, demonstrated intercellular adhesion strength similar to wild-type transfected controls. We conclude that PIP5KIγ-mediated generation of PI(4,5)P2 at sites of N-cadherin contacts regulates intercellular adhesion strength, an effect due in part to PI(4,5)P2-mediated regulation of gelsolin.

]]> T. Y. El Sayegh et al. Regulation and Function of Tenascin-C in Heart Valve Homeostasis http://repository.upenn.edu/ime_papers/29 http://repository.upenn.edu/ime_papers/29 Tue, 15 Jan 2008 12:23:38 PST Tenascin-C (TN-C) interacts with extracellular molecules and plays a role in cell adhesion, signaling, and differentiation. There is evidence that TN-C is regulated by mechanical forces and since the heart valve is a highly dynamic structure, it is possible that TN-C is expressed within specific sites within the aortic valve, as these sites are subject to unique mechanical forces. It is also reasonable to expect differences in TN-C expression between the pulmonary and the aortic valve because these two locations experience different levels of mechanical force. Here we compare TN-C expression levels in adult pulmonary versus aortic valves and determine if different cell types within aortic valves express different amounts of TNC. Our results indicate that TN-C is expressed in normal post-natal heart valves and expression is higher on the aortic versus ventricular side of the aortic valve. TN-C expression may be higher in aortic versus pulmonary valve. SMC, fibroblasts or myofibroblasts may produce TN-C, but endothelial cells appear not to produce TN-C. These studies will help us in determining the role TN-C plays in normal heart valve tissue homeostasis and how different cell types react to different mechanical forces.

]]> Anand Kalola Impaired Notch Signaling Promotes <em>De novo</em> Squamous Cell Carcinoma Formation http://repository.upenn.edu/ime_papers/28 http://repository.upenn.edu/ime_papers/28 Tue, 15 Jan 2008 12:17:11 PST Signaling through Notch receptors in the skin has been implicated in the differentiation, proliferation, and survival of keratinocytes, as well as in the pathogenesis of basal cell carcinoma (BCC). To determine the composite function of Notch receptor–mediated signaling in the skin and overcome potential redundancies between receptors, conditional transgenic mice were generated that express the pan-Notch inhibitor, dominant-negative Mastermind Like 1 (DNMAML1), to repress all canonical [CBF-1/Suppressor of hairless/LAG-1 (CSL)–dependent] Notch signaling exclusively in the epidermis. Here, we report that DNMAML1 mice display hyperplastic epidermis and spontaneously develop cutaneous squamous cell carcinoma (SCC) as well as dysplastic precursor lesions, actinic keratoses. Mice expressing epidermal DNMAML1 display enhanced accumulation of nuclear ß-catenin and cyclin D1 in suprabasilar keratinocytes and in lesional cells from SCCs, which was also observed in human cutaneous SCC. These results suggest a model wherein CSL-dependent Notch signaling confers protection against cutaneous SCC. The demonstration that inhibition of canonical Notch signaling in mice leads to spontaneous formation of SCC and recapitulates the disease in humans yields fundamental insights into the pathogenesis of SCC and provides a unique in vivo animal model to examine the pathobiology of cutaneous SCC and for evaluating novel therapies.

]]> Aaron Proweller et al. Counterion-Mediated Attraction and Kinks on Loops of Semiflexible Polyelectrolyte Bundles http://repository.upenn.edu/ime_papers/27 http://repository.upenn.edu/ime_papers/27 Tue, 15 Jan 2008 10:11:08 PST The 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.

]]> A. Cebers et al. Antibacterial Activities of Rhodamine B-Conjugated Gelsolin-Derived Peptides Compared to Those of the Antimicrobial Peptides Cathelicidin LL37, Magainin II, and Melittin http://repository.upenn.edu/ime_papers/26 http://repository.upenn.edu/ime_papers/26 Tue, 15 Jan 2008 09:19:01 PST The growing number of antibiotic-resistant bacteria necessitates the search for new antimicrobial agents and the principles by which they work. We report that cell membrane-permeant rhodamine B (RhB)-conjugated peptides based on the phosphatidylinositol-4,5-bisphosphate binding site of gelsolin can kill the gram-negative organisms Escherichia coli and Pseudomonas aeruginosa and the gram-positive organism Streptococcus pneumoniae. RhB linkage to the QRLFQVKGRR sequence in gelsolin was essential for the antibacterial function, since the unconjugated peptide had no effect on the bacteria tested. Because RhB-QRLFQVKGRR (also termed PBP10), its scrambled sequence (RhB-FRVKLKQGQR), and PBP10 synthesized from D-isomer amino acids show similar antibacterial properties, the physical and chemical properties of these derivatives appear to be more important than specific peptide folding for their antibacterial functions. The similar activities of PBP10 and all-D-amino-acid PBP10 also indicate that a specific interaction between RhB derivatives and bacterial proteins is unlikely to be involved in the bacterial killing function of PBP10. By using a phospholipid monolayer system, we found a positive correlation between the antibacterial function of PBP10, as well as some naturally occurring antibacterial peptides, and the intrinsic surface pressure activity at the hydrophobic-hydrophilic interface. Surprisingly, we observed little or no dependence of the insertion of these peptides into lipid monolayers on the phospholipid composition. These studies show that an effective antimicrobial agent can be produced from a peptide sequence with specificity to a phospholipid not found in bacteria, and comparisons with other antimicrobial agents suggest that the surface activities of these peptides are more important than specific binding to bacterial proteins or lipids for their antimicrobial functions.

]]> Robert Bucki et al. Interaction of the Gelsolin-Derived Antibacterial PBP 10 Peptide with Lipid Bilayers and Cell Membranes http://repository.upenn.edu/ime_papers/25 http://repository.upenn.edu/ime_papers/25 Tue, 15 Jan 2008 09:14:48 PST PBP 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.

]]> Robert Bucki et al. The requirement for Notch signaling at the β-selection checkpoint in vivo is absolute and independent of the pre–T cell receptor http://repository.upenn.edu/ime_papers/24 http://repository.upenn.edu/ime_papers/24 Wed, 02 Jan 2008 13:29:43 PST Genetic inactivation of Notch signaling in CD4−CD8− double-negative (DN) thymocytes was previously shown to impair T cell receptor (TCR) gene rearrangement and to cause a partial block in CD4+CD8+ double-positive (DP) thymocyte development in mice. In contrast, in vitro cultures suggested that Notch was absolutely required for the generation of DP thymocytes independent of pre-TCR expression and activity. To resolve the respective role of Notch and the pre-TCR, we inhibited Notch-mediated transcriptional activation in vivo with a green fluorescent protein–tagged dominant-negative Mastermind-like 1 (DNMAML) that allowed us to track single cells incapable of Notch signaling. DNMAML expression in DN cells led to decreased production of DP thymocytes but only to a modest decrease in intracellular TCRβ expression. DNMAML attenuated the pre-TCR–associated increase in cell size and CD27 expression. TCRα or TCRαβ transgenes failed to rescue DNMAML-related defects. Intrathymic injections of DNMAML− or DNMAML+ DN thymocytes revealed a complete DN/DP transition block, with production of DNMAML+ DP thymocytes only from cells undergoing late Notch inactivation. These findings indicate that the Notch requirement during the β-selection checkpoint in vivo is absolute and independent of the pre-TCR, and it depends on transcriptional activation by Notch via the CSL/RBP-J–MAML complex.

]]> Ivan Maillard et al. Sensitivity of Volume-regulated Anion Current to Cholesterol Structural Analogues http://repository.upenn.edu/ime_papers/23 http://repository.upenn.edu/ime_papers/23 Wed, 02 Jan 2008 12:58:59 PST Depletion of membrane cholesterol and substitution of endogenous cholesterol with its structural analogues was used to analyze the mechanism by which cholesterol regulates volume-regulated anion current (VRAC) in endothelial cells. Depletion of membrane cholesterol enhanced the development of VRAC activated in a swelling-independent way by dialyzing the cells either with GTPγS or with low ionic strength solution. Using MβCD–sterol complexes, 50–80% of endogenous cholesterol was substituted with a specific analogue, as verified by gas-liquid chromatography. The effects of cholesterol depletion were reversed by the substitution of endogenous cholesterol with its chiral analogue, epicholesterol, or with a plant sterol, β-sitosterol, two analogues that mimic the effect of cholesterol on the physical properties of the membrane bilayer. Alternatively, when cholesterol was substituted with coprostanol that has only minimal effect on the membrane physical properties it resulted in VRAC enhancement, similar to cholesterol depletion. In summary, our data show that these channels do not discriminate between the two chiral analogues of cholesterol, as well as between the two cholesterols and β-sitosterol, but discriminate between cholesterol and coprostanol. These observations suggest that endothelial VRAC is regulated by the physical properties of the membrane.

]]> Victor G. Romanenko et al. Separate Functions of Gelsolin Mediate Sequential Steps of Collagen Phagocytosis http://repository.upenn.edu/ime_papers/22 http://repository.upenn.edu/ime_papers/22 Wed, 12 Dec 2007 07:33:43 PST Collagen 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.

]]> P. D. Arora et al. Notch Signaling in Cancer http://repository.upenn.edu/ime_papers/21 http://repository.upenn.edu/ime_papers/21 Wed, 21 Nov 2007 06:39:06 PST Notch signaling plays a key role in the normal development of many tissues and cell types, through diverse effects on differentiation, survival, and/or proliferation that are highly dependent on signal strength and cellular context. Because perturbations in the regulation of differentiation, survival, and/or proliferation underlie malignant transformation, pathophysiologic Notch signals potentially contribute to cancer development in several different ways.

Notch signaling was first linked to tumorigenesis through identification of a recurrent t(7;9)(q34;q34.3) chromosomal translocation involving the human Notch 1 gene that is found in a small subset of human pre-T-cell acute lymphoblastic leukemias (T-ALL).1 Since this discovery, aberrant Notch signaling has been suggested to be involved in a wide variety of human neoplasms. In this review, we will focus on recent studies linking aberrant Notch signaling with cancer. First, we discuss various mechanisms through which Notch signaling may influence cellular transformation. Then, we critically review literature pertaining to the role of Notch signaling in several cancers, and discuss possible therapeutic targets in the Notch pathway.

]]> Eric J. Allenspach et al. Biopolymer Networks and Cellular Mechanosensing http://repository.upenn.edu/ime_papers/20 http://repository.upenn.edu/ime_papers/20 Tue, 20 Nov 2007 09:14:30 PST Cells 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.

]]> Penelope Georges et al. Autocrine laminin-5 ligates {alpha}6{beta}4 integrin and activates RAC and NF{kappa}B to mediate anchorange-independent survival of mammary tumors http://repository.upenn.edu/ime_papers/19 http://repository.upenn.edu/ime_papers/19 Wed, 07 Nov 2007 12:58:27 PST Invasive carcinomas survive and evade apoptosis despite the absence of an exogenous basement membrane. How epithelial tumors acquire anchorage independence for survival remains poorly defined. Epithelial tumors often secrete abundant amounts of the extracellular matrix protein laminin 5 (LM-5) and frequently express α6β4 integrin. Here, we show that autocrine LM-5 mediates anchorage independent survival in breast tumors through ligation of a wild-type, but not a cytoplasmic tail–truncated α6β4 integrin. α6β4 integrin does not mediate tumor survival through activation of ERK or AKT. Instead, the cytoplasmic tail of β4 integrin is necessary for basal and epidermal growth factor–induced RAC activity, and RAC mediates tumor survival. Indeed, a constitutively active RAC sustains the viability of mammary tumors lacking functional β1 and β4 integrin through activation of NFκB, and overexpression of NFκB p65 mediates anchorage-independent survival of nonmalignant mammary epithelial cells. Therefore, epithelial tumors could survive in the absence of exogenous basement membrane through autocrine LM-5–α6β4 integrin–RAC–NFκB signaling.

]]> Nastaran Zahir et al. Kinetics of random aggregation-fragmentation processes with multiple components http://repository.upenn.edu/ime_papers/18 http://repository.upenn.edu/ime_papers/18 Wed, 24 Oct 2007 06:38:15 PDT A computationally efficient algorithm is presented for exact simulation of the stochastic time evolution of spatially homogeneous aggregation-fragmentation processes featuring multiple components or conservation laws. The algorithm can predict the average size and composition distributions of aggregating particles as well as their fluctuations, regardless of the functional form (e.g., composition dependence) of the aggregation or fragmentation kernels. Furthermore, it accurately predicts the complete time evolutions of all moments of the size and composition distributions, even for systems that exhibit gel transitions. We demonstrate the robustness and utility of the algorithm in case studies of linear and branched polymerization processes, the last of which is a two-component process. These simulation results provide the stochastic description of these processes and give new insights into their gel transitions, fluctuations, and long-time behavior when deterministic approaches to aggregation kinetics may not be reliable.

]]> Ian J. Laurenzi et al. α6ß4 integrin regulates keratinocyte chemotaxis through differential GTPase activation and antagonism of α3ß1 integrin http://repository.upenn.edu/ime_papers/17 http://repository.upenn.edu/ime_papers/17 Wed, 24 Oct 2007 06:08:48 PDT Growth factor-induced cell migration and proliferation are essential for epithelial wound repair. Cell migration during wound repair also depends upon expression of laminin-5, a ligand for α6ß4 integrin. We investigated the role of α6ß4 integrin in laminin-5-dependent keratinocyte migration by re-expressing normal or attachment-defective ß4 integrin in ß4 integrin null keratinocytes. We found that expression of ß4 integrin in either a ligand bound or ligand unbound state was necessary and sufficient for EGF-induced cell migration. In a ligand bound state, ß4 integrin supported EGF-induced cell migration though sustained activation of Rac1. In the absence of α6ß4 integrin ligation, Rac1 activation became tempered and EGF chemotaxis proceeded through an alternate mechanism that depended upon α3ß1 integrin and was characterized by cell scattering. α3ß1 integrin also relocalated from cell-cell contacts to sites of basal clustering where it displayed increased conformational activation. The aberrant distribution and activation of α3ß1 integrin in attachment-defective ß4 cells could be reversed by the activation of Rac1. Conversely, in WT ß4 cells the normal cell-cell localization of α3ß1 integrin became aberrant after the inhibition of Rac1. These studies indicate that the extracellular domain of ß4 integrin, through its ability to bind ligand, functions to integrate the divergent effects of growth factors on the cytoskeleton and adhesion receptors so that coordinated keratinocyte migration can be achieved.

]]> Alan J. Russell et al. Modulation of Endothelial Inward-Rectifier K+ Current by Optical Isomers of Cholesterol http://repository.upenn.edu/ime_papers/16 http://repository.upenn.edu/ime_papers/16 Wed, 24 Oct 2007 05:55:10 PDT Membrane potential of aortic endothelial cells under resting conditions is dominated by inward-rectifier K+ channels belonging to the Kir 2 family. Regulation of endothelial Kir by membrane cholesterol was studied in bovine aortic endothelial cells by altering the sterol composition of the cell membrane. Our results show that enriching the cells with cholesterol decreases the Kir current density, whereas depleting the cells of cholesterol increases the density of the current. The dependence of the Kir current density on the level of cellular cholesterol fits a sigmoid curve with the highest sensitivity of the Kir current at normal physiological levels of cholesterol. To investigate the mechanism of Kir regulation by cholesterol, endogenous cholesterol was substituted by its optical isomer, epicholesterol. Substitution of ~50% of cholesterol by epicholesterol results in an early and significant increase in the Kir current density. Furthermore, substitution of cholesterol by epicholesterol has a stronger facilitative effect on the current than cholesterol depletion. Neither single channel properties nor membrane capacitance were significantly affected by the changes in the membrane sterol composition. These results suggest that 1) cholesterol modulates cellular K+ conductance by changing the number of the active channels and 2) that specific cholesterol-protein interactions are critical for the regulation of endothelial Kir.

]]> Victor G. Romanenko et al.