Browsing by Subject "oligomerization"

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    Ligand Mediated Sequestering of Integrins in Raft-Mimicking Lipid Mixtures: The Role of Bilayer Asymmetry and Cholesterol Content
    (Office of the Vice Chancellor for Research, 2013-04-05) Hussain, Noor F.; Gao, Jiayun; Siegel, Amanda P.; Jordan, Rainer; Naumann, Christoph A.
    Lipid microdomains play an important functional role in plasma membranes. However, the small size and transient nature of lipid/membrane heterogeneities in the plasma membrane make characterization of microdomains and microdomain-related membrane processes quite challenging. To address this issue, we recently introduced a powerful model membrane system that allows the investigation of membrane protein sequestering and oligomerization in raft-mimicking lipid mixtures using combined confocal fluorescence spectroscopy, photon counting histogram (PCH), and epifluorescence microscopy. Our experiments on bilayer-spanning domains showed that αvβ3 and α5β1 integrins predominantly exist as monomers and sequester preferentially to the liquid-disordered (ld) phase in the absence of ligands. Notably, addition of vitronectin (αvβ3) and fibronectin (α5β1) caused substantial translocations of integrins into the liquid-ordered (lo) phase without altering receptor oligomerization state. Here we expand our previous studies and report on the sequestering and oligomerization state of αvβ3 and α5β1 in asymmetric bilayer compositions containing coexisting lo and ld phases located exclusively in the top leaflet of the bilayer (bottom leaflet shows only ld phase). Remarkably, in such a membrane environment, both integrins show a higher affinity for the top leaflet-restricted lo domains in the absence of their respective ligands. A slight change in the integrin sequestration was observed after addition of their respective ligands. We also present experimental findings, which show that cholesterol content has a substantial influence on integrin sequestering and oligomerization in raft-mimicking lipid mixtures. The described experimental results highlight the potential importance of membrane asymmetry and lipid composition in the sequestering of membrane proteins in biological membranes.
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    Monitoring peripheral protein oligomerization on biological membranes
    (Elsevier B.V., 2013) Stahelin, Robert V.; Department of Biochemistry & Molecular Biology, IU School of Medicine
    Peripheral proteins transiently interact with cellular membranes where they regulate important cellular events such as signal transduction. A number of peripheral proteins harbor lipid-binding modules that not only bind selectively with nanomolar affinity to biological membranes but also oligomerize on the membrane surface. In some cases specific lipid binding or specific lipid compositions can induce peripheral protein oligomerization on cellular membranes. These oligomers serve different roles in biological signaling such as regulating protein-protein interactions, induction of membrane bending, or facilitating membrane scission. A number of technologies have been employed to study protein oligomerization with fluctuation analysis of fluorescently labeled molecules recently developed for use with commercial laser scanning microscopes. In this chapter the approach of Raster Image Correlation Spectroscopy coupled with Number and Brightness (N&B) analysis to investigate protein oligomerization on cellular membranes in live cells is presented. Important considerations are discussed for designing experiments, collecting data, and performing analysis. N&B analysis provides a robust method for assessing membrane binding and assembly properties of peripheral proteins and lipid-binding modules.