title: Membrane binding properties of IRSp53-missing in metastasis domain (IMD) protein.
abstract: The 53-kDa insulin receptor substrate protein (IRSp53) organizes the actin cytoskeleton in response to stimulation of small GTPases, promoting the formation of cell protrusions such as filopodia and lamellipodia. IMD is the N-terminal 250 amino acid domain (IRSp53/MIM Homology Domain) of IRSp53 (also called I-BAR), which can bind to negatively charged lipid molecules. Overexpression of IMD induces filopodia formation in cells and purified IMD assembles finger-like protrusions in reconstituted lipid membranes. IMD was shown by several groups to bundle actin filaments, but other groups showed that it also binds to membranes. IMD binds to negatively charged lipid molecules with preference to clusters of PI(4,5)P2. Here, we performed a range of different in vitro fluorescence experiments to determine the binding properties of the IMD to phospholipids. We used different constructs of large unilamellar vesicles (LUVETs), containing neutral or negatively charged phospholipids. We found that IMD has a stronger binding interaction with negatively charged PI(4,5)P2 or PS lipids than PS/PC or neutral PC lipids. The equilibrium dissociation constant for the IMD-lipid interaction falls into the 78-170μM range for all the lipids tested. The solvent accessibility of the fluorescence labels on the IMD during its binding to lipids is also reduced as the lipids become more negatively charged. Actin affects the IMD-lipid interaction, depending on its polymerization state. Monomeric actin partially disrupts the binding, while filamentous actin can further stabilize the IMD-lipid interaction.
authors.affiliation: Department of Biophysics, Medical School, University of Pécs, Szigeti str. 12, Pécs H-7624, Hungary.
authors.forename: Kinga
authors.initials: K
authors.lastname: Futó
authors.affiliation: Department of Biophysics, Medical School, University of Pécs, Szigeti str. 12, Pécs H-7624, Hungary.
authors.forename: Emőke
authors.initials: E
authors.lastname: Bódis
authors.affiliation: Beatson Institute for Cancer Research, Garscube Estate, Switchback Road, Bearsden, Glasgow G61 1BD, UK.
authors.forename: Laura M
authors.initials: LM
authors.lastname: Machesky
authors.affiliation: Department of Biophysics, Medical School, University of Pécs, Szigeti str. 12, Pécs H-7624, Hungary; Szentágothai Research Center, Pécs, Ifjúság str. 34, H-7624, Hungary; Hungarian Academy of Sciences, Office for Subsidized Research Units, Budapest, Nádor str. 7, H-1051, Hungary.
authors.forename: Miklós
authors.initials: M
authors.lastname: Nyitrai
authors.affiliation: Department of Biophysics, Medical School, University of Pécs, Szigeti str. 12, Pécs H-7624, Hungary. Electronic address: balazs.visegrady@aok.pte.hu.
authors.forename: Balázs
authors.initials: B
authors.lastname: Visegrády
mesh_terms: D000818:Animals; D008840:Microfilament Proteins / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism; D011485:Protein Binding; D011817:Rabbits; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism
chemical_list: D008840:Microfilament Proteins; D009419:Nerve Tissue Proteins; D010743:Phospholipids; D020764:cdc42 GTP-Binding Protein
keywords: Actin; Fluorescence spectroscopy; I-BAR; IMD; IRSp53