title: Low PIP(2) molar fractions induce nanometer size clustering in giant unilamellar vesicles.
abstract: Phosphatidylinositol (4,5) bisphosphate (PIP2) is an important signaling molecule located on the inner leaflet of the cell membrane. In order to perform its various signaling functions, it is suggested that PIP2 must be able to form localized clusters. In this study, we have used LAURDAN generalized polarization function (GP) with unlabeled PIP2 and single point fluorescence correlation spectroscopy and brightness analysis of various BODIPY labeled PIP2 to determine the presence of clusters in the membrane of giant unilamellar vesicles (GUVs) made of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) or a mixture of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), sphingomyelin and cholesterol. We determined the number of freely diffusing fluorescent BODIPY molecules in the membrane and found that in GUVs containing various amounts of labeled PIP2, this number was significantly lower than in GUVs made with the control BODIPY labeled hexadecyl phosphatidylcholine (BODIPY-HPC). Also, we noted an increase in brightness of the labeled PIP2 particles with increasing labeled PIP2 molar fraction. Together with the observed change in LAURDAN GP with increasing molar fraction of unlabeled PIP2, these results demonstrate the presence of PIP2 enriched clusters that are smaller than the resolution limit of the fluorescent microscope. In addition, we report the presence of a hypsochromic shift of the fluorescence for the BODIPY labeled lipids that we attributed to clustering. This clustering result in a change in the partitioning of the lipids with the BODIPY labeled PIP2 lipids able to move between the liquid ordered and liquid disordered phase.
authors.affiliation: Centre for Bioactive Discovery in Health and Ageing, School of Science & Technology, University of New England, Armidale, NSW 2351, Australia.
authors.forename: Iyrri L
authors.initials: IL
authors.lastname: Salvemini
authors.affiliation: Centre for Bioactive Discovery in Health and Ageing, School of Science & Technology, University of New England, Armidale, NSW 2351, Australia; Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA 15213, USA.
authors.forename: D M
authors.initials: DM
authors.lastname: Gau
authors.affiliation: Centre for Bioactive Discovery in Health and Ageing, School of Science & Technology, University of New England, Armidale, NSW 2351, Australia.
authors.forename: J
authors.initials: J
authors.lastname: Reid
authors.affiliation: Membrane Biophysics and Biophotonics Group/MEMPHYS-Center for Biomembrane Physics, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark.
authors.forename: L A
authors.initials: LA
authors.lastname: Bagatolli
authors.affiliation: Biomedical Imaging Facility, Mark Wainwright Analytical Centre, the University of New South Wales, Sydney, NSW, Australia.
authors.forename: A
authors.initials: A
authors.lastname: Macmillan
authors.affiliation: Centre for Bioactive Discovery in Health and Ageing, School of Science & Technology, University of New England, Armidale, NSW 2351, Australia. Electronic address: pmoens@une.edu.au.
authors.forename: P D J
authors.initials: PD
authors.lastname: Moens
mesh_terms: D001896:Boron Compounds / Q000737:chemistry; D010713:Phosphatidylcholines / Q000737:chemistry; D010714:Phosphatidylethanolamines / Q000737:chemistry; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000737:chemistry*; D053835:Unilamellar Liposomes / Q000737:chemistry*
chemical_list: C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001896:Boron Compounds; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D019269:Phosphatidylinositol 4,5-Diphosphate; D053835:Unilamellar Liposomes; C028694:1-palmitoyl-2-oleoylphosphatidylcholine
keywords: BODIPY; FCS; Number and brightness analysis; Phosphatidylinositol