Membrane Organization in Cell Signaling and Adhesion
Principal Investigator: T Buranda
Abstract: The overall objective of the proposed research is to understand processes and mechanisms involved in the organization and distribution of proteins and lipids in the plasma membrane of both resting and receptor activated cells. As model systems, we will focus on G-protein coupled receptors (GPCRs) and integrins. Chemokine/chemoattractant GPCRs, such as the N-Formyl peptide receptor (FPR), regulate numerous aspects of leukocyte function, including controlling hematopoiesis, chemotaxis and activation as in the event of physiological insult. GPCR activation has been suggested to lead to a redistribution of receptor into rafts in the plasma membrane. This activation also leads to alterations in the functions of integrins, resulting in clustering and conversion to a high affinity state. Integrin clustering has been suggested to be a critical determinant in regulating the avidity of its binding interactions and hence its physiological function. Thus, both the organization of the protein within the membrane (clustering) and the distribution of lipids about the protein (rafts) are likely to represent critical determinants in the proper functioning of membrane proteins. Although rafts are widely believed to play a critical role in mediating biological functions, it has proven exceedingly difficult to provide a robust characterization of rafts in terms of size, composition, and dynamics. It is noted that chemical composition of membranes is dynamic due to trafficking of vesicles, metabolism of lipids and insertion and removal of lipid components. We propose to use a combination of spectroscopic techniques to image real time changes in the lateral organization of lipid membranes in live cells by measuring energy transfer between fluorescently-labeled lipid probes and receptors bound to fluorescently labeled small molecule ligands. The data will be analyzed in terms of distances of closest approach between donors (on lipid probes or bound to proteins) and acceptors (on lipid probes or proteins). Distance of closest approach in membranes reflect the size of a protein, or conformation (integrins), the presence of boundary lipid which excludes the acceptor, or the distance of the donor above the membrane. The aims are: Aim 1. To characterize the structure function relationship associated with integrin activation and connection to membrane organization. Aim 2. To assess in real time the relationship between GPCR signaling and membrane reorganization. Aim 3. To generate integrin GFP chimerae and cytoplasmic tail alpha/L and beta2 mutants of alpha4beta1 and assess lateral organization in K562 cells. The proposed work provides a unique training opportunity to use biophysical tools and concepts combined with molecular and cell biological approaches to address problems of immense interest to cell biology. Completion of the proposed work will yield a better mechanistic grasp of the dynamics of membrane organization as a factor in signaling and cell adhesion.
Funding Period: 2004-03-01 - 2010-02-28
more information: NIH RePORT
- Some mechanistic insights into GPCR activation from detergent-solubilized ternary complexes on beadsTione Buranda
Department of Pathology and Cancer Center, University of New Mexico Health Science Center, Albuquerque, New Mexico 87131, USA
Adv Protein Chem 74:95-135. 2007..The data and concepts are discussed in the context of a review of current literature on signaling mechanism based on structural and spectroscopic (FRET) studies of ternary complex components...
- Recognition of decay accelerating factor and alpha(v)beta(3) by inactivated hantaviruses: Toward the development of high-throughput screening flow cytometry assaysTione Buranda
Department of Pathology, University of New Mexico, Albuquerque, 87131, USA
Anal Biochem 402:151-60. 2010..This is a first step toward developing HTS format assays for small molecule inhibitors of viral-cell interactions as well as dissecting the mechanism of infection in a BSL-2 environment...
- Real-time partitioning of octadecyl rhodamine B into bead-supported lipid bilayer membranes revealing quantitative differences in saturable binding sites in DOPC and 1:1:1 DOPC/SM/cholesterol membranesTione Buranda
Department of Pathology and Cancer Center, University of New Mexico School of Medicine, Albuquerque, New Mexico 87131, USA
J Phys Chem B 114:1336-49. 2010..This approach represents a first step toward a nanoscale probing of membrane heterogeneity in living cells by analyzing differential local FRET among sites of unique receptor expression in living cells...
- Chapter 11. Subsecond analyses of G-protein coupled-receptor ternary complex dynamics by rapid mix flow cytometryTione Buranda
Department of Pathology and Cancer Center, University of New Mexico Health Science Center, Albuquerque, New Mexico, USA
Methods Enzymol 461:227-47. 2009..These results are compatible with a cell activation model involving G-protein conformational changes rather than disassembly of Galphabetagamma heterotrimer...
- Flow cytometry for real-time measurement of guanine nucleotide binding and exchange by Ras-like GTPasesSamantha L Schwartz
Department of Pathology and Cancer Research and Treatment Center, University of New Mexico, Albuquerque, NM 87131, USA
Anal Biochem 381:258-66. 2008..In sum, the flow cytometric assay can be used to measure nucleotide binding properties of GTPases in real time and to quantitatively assess differences between GTPases...
- Spectroscopic characterization of streptavidin functionalized quantum dotsYang Wu
Department of Pathology and Cancer Center, University of New Mexico School of Medicine, Albuquerque, NM 87131, USA
Anal Biochem 364:193-203. 2007..The results are discussed in the context of bridging the gap between what is currently known in the physical chemistry literature of quantum dots and the quantitative needs of assay development in biological applications...
- The development of quantum dot calibration beads and quantitative multicolor bioassays in flow cytometry and microscopyYang Wu
Department of Pathology and Cancer Center, University of New Mexico, Albuquerque, NM 87131, USA
Anal Biochem 364:180-92. 2007..The utility of the calibration beads is also extended to the characterization surface densities of dot-labeled epidermal growth factor ligands as well as quantitative indicators of the binding of dot-labeled virus particles to cells...
- Regulation of cell adhesion by affinity and conformational unbending of alpha4beta1 integrinAlexandre Chigaev
Department of Pathology and Cancer Center, University of New Mexico Health Sciences Center, Albuquerque, NM 87131, USA
J Immunol 178:6828-39. 2007....
- Activated epidermal growth factor receptor induces integrin alpha2 internalization via caveolae/raft-dependent endocytic pathwayYan Ning
College of Pharmacy, University of New Mexico, Albuquerque, New Mexico 87131, USA
J Biol Chem 282:6380-7. 2007....
- Rapid-mix flow cytometry measurements of subsecond regulation of G protein-coupled receptor ternary complex dynamics by guanine nucleotidesYang Wu
Department of Pathology and Cancer Research Facility, University of New Mexico Health Sciences Center, University of New Mexico, Albuquerque, NM 87131, USA
Anal Biochem 371:10-20. 2007..These results are compatible with a cell activation model involving G protein conformational changes rather than disassembly of Galphabetagamma heterotrimer...
- Quantum dots for quantitative flow cytometryTione Buranda
Department of Pathology and Cancer Center, University of New Mexico School of Medicine, Albuquerque, NM, USA
Methods Mol Biol 699:67-84. 2011....