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Publication Role of lateral cell–cell border location and extracellular/transmembrane domains in PECAM/CD31 mechanosensation(2004-08-06) Kaufman, David A.; Albelda, Steven M.; Sun, Jing; Davies, Peter F.Phosphorylation of tyrosine residues on platelet–endothelial cell adhesion molecule-1 (PECAM-1), followed by signal trans- 13 duction events, has been described in endothelial cells following exposure to hyperosmotic and fluid shear stress. However, it is 14 unclear whether PECAM-1 functions as a primary mechanosensor in this process. Utilizing a PECAM-1–null EC-like cell line, we 15 examined the importance of cellular localization and the extracellular and transmembrane domains in PECAM-1 phosphorylation 16 responses to mechanical stress. Tyrosine phosphorylation of PECAM-1 was stimulated in response to mechanical stress in null cells 17 transfected either with full length PECAM-1 or with PECAM-1 mutants that do not localize to the lateral cell–cell adhesion site and 18 that do not support homophilic binding between PECAM-1 molecules. Furthermore, null cells transfected with a construct that 19 contains the intact cytoplasmic domain of PECAM-1 fused to the extracellular and transmembrane domains of the interleukin-2 20 receptor also underwent mechanical stress-induced PECAM-1 tyrosine phosphorylation. These findings suggest that mechano- 21 sensitive PECAM-1 may lie downstream of a primary mechanosensor that activates a tyrosine kinase.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 Signal-to-noise measurements utilizing a novel dual-energy multimedia detector(2001-08-01) Giakos, G. C; Chowdhury, S.; Shah, N.; Vedantham, Srinivasan; Passerini, A. G; Suryanarayanan, Sankararaman; Nemer, R.; Mehta, K.; Patnekar, N.; Sumrain, S.; Nataraj, K.; Scheiber, ChristianDual-energy measurements are presented utilizing a novel slot-scan digital radiographic imaging detector, operating on gaseous solid state ionization principles. The novel multimedia detector has two basic functional components: a noble gas-filled detector volume operating on gas microstrip principles, and a solid state detector volume. The purpose of this study is to investigate the potential use of this multimedia detector for enhanced dual-energy imaging. The experimental results indicate that the multimedia detector exhibits a large subtracted signal-to-noise ratio. Although the intrinsic merit of this device is being explored for medical imaging, potential applications of the multimedia detector technology in other industrial areas, such as aerospace imaging, aviation security, and surveillance, are also very promising.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 Cholesterol Depletion Increases Membrane Stiffness of Aortic Endothelial Cells(2004-11-01) Byfield, Fitzroy H; Aranda-Espinoza, Helim; Romanenko, Victor G.; Rothblat, George H.; Levitan, IrenaThis study has investigated the effect of cellular cholesterol on membrane deformability of bovine aortic endothelial cells. Cellular cholesterol content was depleted by exposing the cells to methyl-ß-cyclodextrin or enriched by exposing the cells to methyl-ß-cyclodextrin saturated with cholesterol. Control cells were treated with methyl-ß-cyclodextrincholesterol at a molar ratio that had no effect on the level of cellular cholesterol. Mechanical properties of the cells with different cholesterol contents were compared by measuring the degree of membrane deformation in response to a step in negative pressure applied to the membrane by a micropipette. The experiments were performed on substrate-attached cells that maintained normal morphology. The data were analyzed using a standard linear elastic half-space model to calculate Young elastic modulus. Our observations show that, in contrast to the known effect of cholesterol on membrane stiffness of lipid bilayers, cholesterol depletion of bovine aortic endothelial cells resulted in a significant decrease in membrane deformability and a corresponding increase in the value of the elastic coefficient of the membrane, indicating that cholesterol-depleted cells are stiffer than control cells. Repleting the cells with cholesterol reversed the effect. An increase in cellular cholesterol to a level higher than that of normal cells, however, had no effect on the elastic properties of bovine aortic endothelial cells. We also show that although cholesterol depletion had no apparent effect on the intensity of F-actin-specific fluorescence, disrupting F-actin with latrunculin A abrogated the stiffening effect. We suggest that cholesterol depletion increases the stiffness of the membrane by altering the properties of the submembrane F-actin and/or its attachment to the membrane.Publication The convergence of haemodynamics, genomics, and endothelial structure in studies of the focal origin of atherosclerosis(2002-04-01) Davies, Peter F; Shi, Congzhu; Polacek, Denise C; Helmke, Brian PThe 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.Publication The Cytoskeleton Under External Fluid Mechanical Forces: Hemodynamic Forces Acting on the Endothelium(2002-03-01) Helmke, Brian P; Davies, Peter FThe 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.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 Hypoxia-inducible Factor Regulates αvß3 Integrin Cell Surface Expression(2005-04-01) Cowden Dahl, Karen D.; Weaver, Valerie M.; Robertson, Sarah E.; Simon, M. CelesteHypoxia-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.Publication Contrast Adaptation in Subthreshold and Spiking Responses of Mammalian Y-Type Retinal Ganglion Cells(2005-01-26) Zaghloul, Kareem A; Boahen, Kwabena A; Demb, Jonathan BRetinal 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.