Divalent cation-induced cluster formation by polyphosphoinositides in model membranes.

Lipids and Reagents Natural PIP2 (porcine brain L-α-phosphatidylinositol-4,5-bisphosphate), synthetic PIP2 analogs (dioleoyl phosphatidylinositol-(x,y)- bisphosphate) and neutral phospholipids such as SOPC (1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine), DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) and Rho-DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl)) were from Avanti (Alabaster, AL). Fluorescently-labeled PIP2 analogs GloPIPs BODIPY-TMR PI(4,5)P2 (C16) and GloPIPs BODIPY-FL PI(4,5)P2 (C16) are purchased from Echelon Biosciences (Salt Lake City, UT). Lipids were dissolved in chloroform/methanol 2:1 mixed solvent, and the concentrations of unlabeled lipid stock solutions were routinely monitored by a phosphorus assay as described elsewhere. 21 The concentrations of fluorescently labeled lipids were calibrated by their fluorescence intensity. Subphase reagents HEPES, EDTA, CaCl 2 , MgCl 2 , NaCl, sucrose and glucose were purchased from Fisher Scientific (Hampton, NH); other subphase reagents such as ethylenediamine (EDA), diethyl-enetriamine (DETA), triethylenetetramine (TETA), ZnCl 2 and cholestanol were purchased from Sigma-Aldrich (St. Louis, MO) and dithiothreitol (DTT) was purchased from Research Product Int. Corp. (Mt. Prospect, IL).

Binding Affinity Measurements

Similar to the cation binding affinity studies performed by Ohki et. al. 22 - 24 , a simplified Ca 2+ -binding affinity assay was carried out as described previously. 14 Limited by the complexity in the binding stoichiometry of highly charged PIP2 molecules, which have net charges that can vary from -3 to -5 under most experimental conditions 25 , 26 , only a global binding constant is reported using a Langmuir adsorption model. The measurement is performed on a MicroTroughX Langmuir trough (Kibron Inc. Helsinki, Finland) controlled by the FilmWare 3.57 software package (Kibron). Monolayer subphases were prepared with 10 mM HEPES, 1 μM EDTA, and 5 mM DTT at pH 7.4 dissolved in 18.2 MΩ ddH 2 O. For each measurement, 7 nmol of pre-mixed lipid was deposited on 30 mL buffered solution, and the monolayer surface pressure was monitored with a surface probe using the Wilhelmy method. 27 When the surface pressure reached equilibrium at 20 mN/m, concentrated cation stock solution (less than 0.3% of subphase volume fraction) was injected into the subphase and gently mixed without perturbing the monolayer. The surface pressure change was then recorded until the surface pressure again reached equilibrium. For other cations, such as Mg 2+ and polyamines, a competitive Ca 2+ -binding assay was carried out by titrating Ca 2+ in the presence of the other cations at various concentrations. The surface pressure measurement was analyzed using the Langmuir competitive adsorption model. 28 , 29 Imaging Supported Lipid Monolayers Supported lipid monolayers were prepared by transferring monolayers from the Langmuir trough onto glass coverslips using the Langmuir-Schaeffer method. For fluorescence microscopy, lipid monolayers were doped with 0.1 mol% BODIPY-FL PIP2 and Rho-DOPE and examined under an inverted microscope (Leica, DM IRBE) with a 100X oil objective. AFM images of air-dried supported lipid monolayers were taken using tapping mode AFM (Digital Instruments, Santa Barbara, CA) and processed by Nanoscope® IIIa software (v. 5.12; Digital Instruments). For fluid phase AFM, the transferred lipid sample was again immersed in its subphase solution and imaged by the Bioscope AFM (Digital Instruments, Santa Barbara, CA). Air-dried supported lipid monolayer samples were further processed for EM imaging. Samples were unilaterally coated with a thin layer of platinum (1 nm) from a 20° angle and carbon (5 nm) from an ~80° angle with an Auto306 vacuum evaporator (Edwards, UK). The coated sample was floated on a diluted hydrofluoric acid solution to separate from the coverslip, and transferred onto formvar-coated EM grids. Samples were analyzed using a JEM-1011 transmission electron microscope (JEOL USA, Peabody, MA) at an accelerating voltage of 100 kV. Images were captured by an ORIUS 835.10W CCD camera (Gatan, Warrendale, PA) Infrared Spectroscopy of Supported Lipid Monolayers Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectra were collected using a Magna-IR 860 spectrometer with a Harrick’s Horizon attachment and equipped with Opus software. Pure PIP2 monolayers were transferred onto a germanium internal reflection element (IRE) and immersed in their subphase buffer solution. The infrared spectra of supported PIP2 monolayers with different divalent cation concentrations were monitored at room temperature in the range of 650-4000 cm -1 , using a resolution of 1 cm -1 . PIP2 Phase-Partitioning on LUVs Steady-state probe-partitioning Förster resonance energy transfer (SP-FRET) 30 was used to probe PIP2 phase demixing within a bilayer membrane. For this purpose, two different fluorescent PIP2 analogs were used: BODIPY-FL and BODIPY-TMR PI(4,5)P2. 5.2 mol% PIP2-containing LUVs (including 0.3 mol% each fluorescent PIP2 analog) were prepared using a mini-extruder (Avanti, Alabaster, AL). While the probe-probe distance is expected to be 120 Å and the Förster distance for the selected FRET pair is about 57 Å 31 , an increase in FRET efficiency is expected as titrated cations induce the formation of PIP2-rich clusters. Holding the PIP2 mole fraction and overall PIP2 concentration in the LUV as constants, the fluorescence spectra for D-A (donor with acceptor), D (donor only) and A (acceptor only) were collected independently using a LS-50B Luminescence Spectrometer (Perkin-Elmer, Beaconsfield, UK). The spectra were combined linearly to calculate the concentration-dependent fluorescence intensity change and FRET efficiency change, and to rule out potential artifacts of fluorescence decay due to environmental changes. For polyamine titrations, the pH of each polyamine stock solution was adjusted to 7.4, and 5 mM DTT was added to stock solutions to keep polyamines from oxidizing. Nanocluster Formation and Diffusion Retardation on GUVs 5 mol% PIP2-containing GUVs were prepared by electroswelling. 32 0.1 nmol of lipid mixture solution was spread and dried on indium-doped tin oxide (ITO) glasses (Delta Technologies, Loveland, CO) on a hotplate at 60 °C. Following vacuum drying for 2 hrs, the ITO was assembled with another clean ITO with a Fastwell silicon spacer (Grace Bio-Labs, Bend, OR) and filled with a 300 mM sucrose solution. The samples were then left in a homemade heating block, heated at 60 °C and applied with a 1V AC field at 5 Hz for two hours using a function generator (B & K Precision, Yorba Linda, CA) and monitored by an oscilloscope (B & K Precision). GUVs were asymmetrically labeled with approximately 0.5 and 0.01 mol% BODIPY-TMR PIP2 for imaging and FCS, respectively. GUVs, diluted in isotonic glucose solutions containing 10 mM HEPES and various multivalent cations, were added to vacuum grease-sealed chambers covered with clean coverslips. The samples were kept in the dark for at least 30 mins to allow the GUVs to settle down to the glass surface. The diffusion of PIP2 in model membranes was studied by fluorescence correlation spectroscopy (FCS). The experimental setup, sample preparation, data acquisition, and analysis protocols are described elsewhere. 33 GUVs sealed in the chamber were allowed to sit for 30 min prior to fluorescence intensity fluctuation measurements. A 514 nm laser was focused near the top center of the GUVs to avoid lipid-solid support interaction. Each FCS curve was obtained by correlating the fluorescence signal for a duration of about 30 s and fit by a multi-component two dimensional diffusion equation to yield its characteristic diffusion times (τ D ). For each condition, 11 to 25 auto-correlation curves were collected from multiple vesicles.

Supplementary Material 1_si_001