EMSL: Galya Orr's Publications

Orr G, DJ Panther, KJ Cassens, JL Phillips, BJ Tarasevich, and JG Pounds. 2009. "Syndecan-1 Mediates the Coupling of Positively Charged Submicrometer Amorphous Silica Particles with Actin Filaments across the Alveolar Epithelial Cell Membrane." Toxicology and Applied Pharmacology 236(2):210-220. doi:10.1016/j.taap.2009.01.022 Abstract The cellular interactions and pathways of engineered submicro- and nano-scale particles dictate the cellular response and ultimately determine the level of toxicity or biocompatibility of the particles. Positive surface charge can increase particle internalization, and in some cases can also increase particle toxicity, but the underlying mechanisms are largely unknown. Here we identify the cellular interaction and pathway of positively charged submicrometer synthetic amorphous silica particles, which are used extensively in a wide range of industrial applications, and are explored for drug delivery and medical imaging and sensing. Using time lapse fluorescence imaging in living cells and other quantitative imaging approaches, it is found that heparan sulfate proteoglycans play a critical role in the attachment and internalization of the particles in alveolar type II epithelial cell line (C10), a potential target cell type bearing apical microvilli. Specifically, the transmembrane heparan sulfate proteoglycan, syndecan-1, is found to mediate the initial interactions of the particles at the cell surface, their coupling with actin filaments across the cell membrane, and their subsequent internalization via macropinocytosis. The observed interaction of syndecan molecules with the particle prior to their engagement with actin filaments suggests that the particles initiate their own internalization by facilitating the clustering of the molecules, which is required for the actin coupling and subsequent internalization of syndecan. Our observations identify a new role for syndecan-1 in mediating the cellular interactions and fate of positively charged submicrometer amorphous silica particles in the alveolar type II epithelial cell, a target cell for inhaled particles. - 2

Orr G, DJ Panther, JL Phillips, BJ Tarasevich, A Dohnalkova, D Hu, JG Teeguarden, and JG Pounds. 2007. "Submicron and Nanoscale Inorganic Particles Exploit the Actin Machinery to be Propelled Along Microvillilike Structures into Alveolar Cells." ACS Nano 1(5):463-475. doi:10.1021/nn700149r Abstract The growing commerce in micro- and nanotechnology is expected to increase our exposure to submicron and nanoscale particles. One of the main targets of this exposure are the cells that line the respiratory tract, among them are the alveolar type II epithelial cells that have microvilli at their exposed apical surface. Here we show a pathway by which positively charged inorganic submicron and nanoscale particles take advantage of the actin turnover machinery within filopodia and microvilli-like structures to guide and advance their way into these cells. Our observations bring a new view of how submicron and nanoscale inorganic matter can be assimilated into the cellular environment and take advantage of its machinery. While the pathway that we describe can be exploited for a targeted drug delivery, it also points to properties of submicron or nanoscale particles that should be avoided in order to reduce particle internalization and possible toxicity.

Teeguarden JG, PM Hinderliter, G Orr, BD Thrall, and JG Pounds. 2007. "Particokinetics In Vitro: Dosimetry Considerations for In Vitro Nanoparticle Toxicity Assessments." Toxicological Sciences 95(2):300-312. doi:10.1093/toxsci/kfl165 Abstract The rapid growth in the use of in vitro methods for nanoparticle toxicity assessment has proceeded with limited consideration of the unique kinetics of these materials in solution. Particles in general and nanoparticles specifically, diffuse, settle and agglomerate in cell culture media as a function of several systemic and particle factors: media density and viscosity, particle size, shape and density. Cellular dose then is also a function of these factors as they determine the rate of transport of nanoparticles to cells in culture. Here we develop and apply the principles of particokinetics and dosimetry in vitro and outline an approach for simulation of nanoparticle particokinetics in cell culture systems. We illustrate that where equal mass concentrations (μg/ml) imply equal doses for dissimilar materials, the corresponding particle number or surface area concentration doses differ by orders of magnitude. When rates of diffusional and gravitational particle delivery are accounted for, the trends and magnitude of cellular dose as a function of particle size and density differ significantly from those implied by NMC doses. Fifteen nm Ag nanoparticles appear ~4000 times more potent than micron sized cadmium oxide particles on a cm2/ml media basis, but are only ~50 times more potent when differences in nanoparticle delivery to adherent cells are considered. We conclude that simple surrogates of dose can cause significant misinterpretation of response and uptake data for nanoparticles in vitro. Incorporating particokinetics and principles of dosimetry would significantly improve the basis for or nanoparticles toxicity assessment, increasing the predictive power and scalability of such assays.

Oehmen CS, TP Straatsma, GA Anderson, G Orr, BJM Webb-Robertson, RC Taylor, RW Mooney, DJ Baxter, DR Jones, and DA Dixon. 2006. "New Challenges Facing Integrative Biological Science in the Post-Genomic Era." Journal of Biological Systems 14(2):275-293. Abstract The future of biology will be increasingly driven by the fundamental paradigm shift from hypothesis-driven research to data-driven discovery research employing the massive amounts of available biological data. We identify key technological developments needed to enable this paradigm shift involving (1) the ability to store and manage extremely large datasets which are dispersed over a wide geographical area, (2) development of novel analysis and visualization tools which are capable of operating on enormous data resources without overwhelming researchers with unusable information, and (3) formalisms for integrating mathematical models of biosystems from the molecular level to the organism population level. This will require the development of tools which efficiently utilize high-performance compute power, large storage infrastructures and large aggregate memory architectures. The end result will be the ability of a researcher to integrate complex data from many different sources with simulations to analyze a given system at a wide range of temporal and spatial scales in a single conceptual model.

Dong J, L Opresko, WB Chrisler, G Orr, RD Quesenberry, DA Lauffenburger, and HS Wiley. 2005. "The Membrane-anchoring Domain of Epidermal Growth Factor Receptor Ligands Dictates Their Ability to Operate in Juxtacrine Mode." Molecular Biology of the Cell 16(6):2984-2998. Abstract All ligands of the epidermal growth factor receptor (EGFR) are synthesized as membrane-anchored precursors. Previous work has suggested that some ligands, such as EGF, must be proteolytically released to be active, whereas others, such as heparinbinding EGF-like growth factor (HB-EGF) can function while still anchored to the membrane (i.e., juxtacrine signaling). To explore the structural basis for these differences in ligand activity, we engineered a series of membrane-anchored ligands in which the core, receptor-binding domain of EGF was combined with different domains of both EGF and HB-EGF. We found that ligands having the N-terminal extension of EGF could not bind to the EGFR, even when released from the membrane. Ligands lacking an N-terminal extension, but possessing the membrane-anchoring domain of EGF still required proteolytic release for activity, whereas ligands with the membraneanchoring domain of HB-EGF could elicit full biological activity while still membraneanchored. Ligands containing the HB-EGF membrane anchor, but lacking an N-terminal extension, activated EGFR during their transit through the Golgi apparatus . However, cell-mixing experiments and fluorescence resonance energy transfer (FRET) studies showed that juxtacrine signaling typically occurred in trans at the cell surface, at points of cell-cell contact. Our data suggest that the membrane-anchoring domain of ligands selectively controls their ability to participate in juxtacrine signaling and thus, only a subclass of EGFR ligands can act in a juxtacrine mode.

Hendriks BS, G Orr, AH Wells, HS Wiley, and DA Lauffenburger. 2005. "Parsing ERK Activation Reveals Quantitatively Equivalent Contributions From Epidermal Growth Factor Receptor and HER2 In Human Mammary Epithelial Cells." Journal of Biological Chemistry 280(7):6157-6169. Abstract HER2, a member of the EGFR tyrosine kinase family, functions as an accessory EGFR signaling component and alters EGFR trafficking by heterodimerization. HER2 overexpression leads to aberrant cell behavior including enhanced proliferation and motility. Here we apply a combination of computational modeling and quantitative experimental studies of the dynamic interactions between EGFR and HER2, and their downstream activation of extracellular signal-related kinase (ERK) to understand this complex signaling system. Using cells expressing different levels of HER2 relative to the EGFR, we can separate relative contributions of EGFR and HER2 to signaling amplitude and duration. Based on our model calculations, we demonstrate that, in contrast with previous suggestions in the literature, the intrinsic capabilities of EGFR and HER2 to activated ERK are quantitatively equivalent . We find that HER2-mediated effects on EGFR dimerization and trafficking are sufficient to explain the detected HER2-mediated amplification of EGF-induced ERK signaling. Our model suggests that transient amplification of ERK activity by HER2 arises predominantly from the 2-to-1 stoichiometry of receptor kinase to bound ligand in EGFR/HER2 heterodimers compared to the 1-to-1 stoichiometry of the EGFR homodimer, but alterations in receptor trafficking, with resultant EGFR sparing, cause the sustained HER2-mediated enhancement of ERK signaling.

Orr G, D Hu, S Ozcelik, L Opresko, HS Wiley, and SD Colson. 2005. "Cholesterol Dictates the Freedom of EGF Receptors and HER2 in the Plane of the Membrane." Biophysical Journal 89(2):1362-1373. doi:10.1529/biophysj.104.056192 Abstract The flow of information through the EGF receptor (EGFR) is shaped by molecular interactions in the plasma membrane. The EGFR is associated with lipid rafts, but their role in modulating receptor mobility and subsequent interactions is unclear. To investigate the role of nanoscale rafts in EGFR dynamics, we used single-molecule fluorescence imaging to track individual receptors and their dimerization partner, HER2, in the membrane of human mammary epithelial cells. We found that the motion of both receptors was interrupted by dwellings within nanodomains. EGFR was significantly less mobile than HER2. This difference was likely due to F-actin because its deploymerization led to similar diffusion patterns between the EGFR and HER2. Manipulations of membrane cholesterol content dramatically altered the diffusion pattern of both receptors. Cholesterol depletion led to almost complete confinement of the receptors, whereas cholesterol enrichment extended the boundaries of the restricted areas. Interestingly, F-actin deploymerization partially restored receptor mobility in cholesterol depleted membranes. Our observations suggest that membrane cholesterol provides a dynamic environment that facilitates the free motion of EGFR and HER2, possible by modulating the dynamic state of F-actin. The association of the receptors with lipid rafts could therefore promote their rapid interactions only upon ligand stimulation.

Harms GS, G Orr, and HP Lu. 2004. "Probing Ion Channel Conformational Dynamics Using Simultaneous Single-Molecule Ultrafast Spectroscopy and Patch-Champ Electric Recording." Applied Physics Letters 84(10):1792-1794. Abstract A new approach to probing single-molecule ion channel kinetics and conformational dynamics, patch-clamp confocal fluorescence microscopy (PCCFM), uses simultaneous ultrafast fluorescence spectroscopy and single-channel electric current recording.

Harms GS, G Orr, M Montal, BD Thrall, SD Colson, and HP Lu. 2003. "Probing conformational changes of gramicidin ion channels by single-molecule patch-clamp fluorescence microscopy." Biophysical Journal 85(3):1826-1838. Abstract Stochastic and inhomogeneous conformational changes often regulate the dynamics of ion channels. Such inhomegeniety makes it difficult, if not impossible; to be characterized not only by ensemble-averaged experiments by also by single-channel patch recording that does not specifically probe the associated conformational changes. Here, we report on our work using a new approach combining single-molecule fluorescence spectroscopy and single-channel patch recording to investigate conformational changes of individual gramicidin ion channels. We observed fluorescence self-quenching and single-pair fluorescence resonance energy transfer (spFRET) from dye-labeled gramicidin dimmers within the channel was open. We also observed that the efficiency of self-quenching and spFRETS is widely distributed when the channel is closed. Our results strongly suggest a hitherto undetectable correlation of multiple conformational states of the gramicidin channel associated with closed and open states under physiologically-related conditions.

Harms GS, G Orr, and HP Lu. 2003. "Probing Single-Molecule Ion Channel Conformational Dynamics Using Fluorescence Imaging, Combined Ultrafast Spectroscopy, and Patch-Clamp Recording." Biophysical Journal 84(2 Pt. 2 Supp.):123-A. Abstract Stochastic and inhomogeneous conformational changes often regulate the dynamics of ion channels. Such inhomegeniety makes it difficult, if not impossible; to be characterized not only by ensemble-averaged experiments by also by single-channel patch recording that does not specifically probe the associated conformational changes.