title: [Modulation of growth factor receptors in membrane microdomains].
abstract: Membrane lateral heterogeneity is accepted as a requirement for the function of biological membranes, and the notion of the "raft/microdomain" gives specificity to this concept. Recently, fluorescence-based techniques such as fluorescence recovery after photobleaching (FRAP), single particle tracking (SPT), and fluorescence correlation spectroscopy (FCS) have shown promise for application to the dynamics of membrane molecules in microdomains. We previously revealed, by performing live-cell FRAP and SPT studies, a mechanism of insulin resistance in which dissociation of the insulin receptor (IR)-caveolin-1 (Cav1) complex was caused by an interaction between the IRβ subunit and the ganglioside GM3 cluster, a glycolipid-enriched membrane microdomain. We hoped to demonstrate that an alteration in the lipid component of microdomains affects lateral diffusion of membrane receptors. We therefore established an experimental system for monitoring the membrane organization of receptors by analyzing their lateral diffusion parameters in the plasma membranes of living cells using FRAP and SPT. In this study, measurement of the lateral diffusion of the IR was performed by fitting analysis to fluorescence recovery curves and trace analysis to individual fluorescent spots, which provided the diffusion constant. The results show how fast IR molecules diffuse before and after a change in membrane environment, such as stimulation by cholesterol depression or treatment with a glycosphingolipid (GSL) inhibitor. Using these techniques, we have established a method for determining the diffusion constant for the lateral movement of IR-EGFP, expressed in CHO-K1 cells. We will use these techniques for the lateral diffusion analysis of membrane receptors under other assay conditions, such as use of GSL-deficient cells or pathologic samples.
authors.affiliation: Institute of Glycoscience, Tokai University, Hiratsuka, Kanagawa, Japan. kaba@tokai-u.jp
authors.forename: Kazuya
authors.initials: K
authors.lastname: Kabayama
mesh_terms: D000818:Animals; D016466:CHO Cells; D051242:Caveolin 1 / Q000378:metabolism; D006224:Cricetinae; D005679:G(M3) Ganglioside; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D021962:Membrane Microdomains / Q000737:chemistry / Q000502:physiology*; D038761:Photobleaching; D011972:Receptor, Insulin / Q000378:metabolism*; D017978:Receptors, Growth Factor / Q000378:metabolism*; D013050:Spectrometry, Fluorescence / Q000379:methods
chemical_list: D051242:Caveolin 1; D005679:G(M3) Ganglioside; D017978:Receptors, Growth Factor; D049452:Green Fluorescent Proteins; D011972:Receptor, Insulin