Supporting Information

Transcription

1 Supporting Information I. Vesicle preparation protocol All lipids were purchased from Avanti Polar Lipids, USA. 1-Acyl-2-Acyl-sn- Glycero-3-Phosphocholine (POPC), L-a-Phosphatidylinositol-4,5-bisphosphate (Brain, PorcineTriammonium Salt) (PIP 2 ), 1-Stearoyl-2-Arachidonoyl-sn-Glycero-3- Phosphoinositol-3,4,5-trisphosphate (Tetra-ammonium Salt) (PIP 3 ) and the fluorescently labeled lipids 1-Oleoyl-2-[12-[[6-[(7-nitrobenz-2-oxa-1,3-diazol-4- yl)amino]hexanoyl]amino]-dode-canoyl]-sn-glycero-3-phosphoinositol-3,4,5-trisphosphate (Tetra-ammonium Salt) (PIP 3 -NBD),1-oleoyl-2-6-[4-(dipyrrometheneborondifluoride) butanoyl]amin] hexano-yl-sn-glycero-3-phosphoinositol-4,5- bisphosphate (ammonium salt) (TopFluorPIP 2 ) and 1-Palmitoyl-2-[12-[(7-nitro-2-1,3- benzoxadiazol-4-yl)amino]dodecanoyl]-sn-glycero-3-phosphocholine (16:0-12:0 NBD PC) (NBD-PC). The lipids were mixed to the desired composition in CHCl 3 to a total lipid content of 0.5 mg. All compositions in the text are given as weight percentages. After evaporation of the CHCl 3 under steady N 2 flow for 1 h the lipid film was rehydrated with Tris buffered saline (TBS, 10mM Tris(hydroxymethyl)aminomethane (Sigma-Aldrich), 150 mm NaCl (VWR BDH Prolabo), ph was adjusted to 7.4 by adding 2 M HCl (Sigma-Aldrich)) to a final lipid concentration of 0.5 mg/ml. After solubilisation in the buffer at room temperature the lipid mixture was extruded 31 times through two stacked polycarbonate membranes (pore size 100 nm, Avestin, Canada). Vesicle size was measured by dynamic light scattering at 90 in a Zetasizer (Zetasizer 3000 HAS (Malvern, USA)) and the average measured vesicle diameter was 109 ± 12 nm. II. QCM-D experiments A Q-Sense E4 quartz crystal microbalance with dissipation monitoring 1 (QCM-D) (Q-Sense AB, Sweden) 1 was used to monitor the formation of supported lipid bilayers with varied fractions of phosphoinositides on SiO 2 coated crystals (C). By measuring the change in resonance frequency, Δf, and the change in dissipation, ΔD, of the piezoelectric

2 quartz crystal adsorbed mass including structural water (roughly proportional to Δf ) and supramolecular conformation (sensed through changes in ΔD) can be deduced 2. The protocol used for formation of the SLBs was adapted from the method pioneered by McConnell et al. via lipid vesicle adsorption to the sensor surface, rupture and lateral association of the bilayer fragments to form a supported lipid bilayer (SLB) 3. The SiO 2 QCM-D crystals were sonicated in 2% SDS for 20 min, then in EtOH for another 20 min and subsequently exposed to UV-ozone for 30 min. QCM-D experiments were performed at 37 C. 357μl of lipid vesicles solution (50μg/ml lipid concentration in TBS, ph 7.4) were injected into the liquid chamber with 100ul/min and batch adsorbed until the saturated response from liposome adsorption or formation of an SLB was obtained. The bilayers were subsequently rinsed at 50 μl/min with TBS (ph 7.4) to remove excessive lipid material. Table 1. Frequency and dissipation shifts measured at the time point of maximum vesicle adsorption (1) and after rinsing of the SLB (2). The observation of a maximum in the frequency and dissipation shifts and a final low value in dissipation (< ) and frequency ( Hz). For pure POPC a frequency shift of ~-26 is expected 2. Increasing PIP 2 concentration in the SLB leads to lower frequencies as well as to slightly elevated dissipation shifts compared to pure POPC. SLB formation for PIP 3 containing SLB was only successful for 1% PIP % PIP 3 lead to only partial SLB formation whereas 5% resulted in association of a sub-monolayer of liposomes with the surface. PIP 2 Δf (1) Δf (2) ΔD * (10-6 ) (1) ΔD* (10-6 ) (2) 1%PIP ± ± ± ± %PIP ± ± ± ± %PIP ± ± ± ± 0.20 PIP 3 Δf (1) Δf (2) ΔD * (10-6 ) (1) ΔD* (10-6 ) (2) 1%PIP ± ± ± ± %PIP ± ± ± ± %PIP ± ±

3 Figure SI 1. QCM-D. Adsorption of 1% PIP 2 (POPC: PIP 2 [99:1]), 5% PIP 2 (POPC: PIP 2 [95:5]) and 10% PIP 2 (POPC: PIP 2 [90:10]) POPC vesicles on SiO 2 coated QCM-D crystals in TBS supplemented with 2mM Ca 2+. For illustration of a successful SLB formation a curve of POPC (pure) vesicles in TBS has been added showing the associated frequency and dissipation shifts. Δf solid symbols; ΔD open symbols.

4 Figure SI 2. QCM-D. ΔD vs. -Δf plots of QCM-D measurements for PIP 2 and PIP 3 SLBs. A 1% PIP 2 (POPC: PIP 2 [99:1]), 5% PIP 2 (POPC: PIP 2 [95:5]), 10% PIP 2 (POPC: PIP 2 [90:10]), POPC (pure). B. 1% PIP 3 (POPC: PIP 3 [99:1]), 2.5% PIP 3 (POPC: PIP 3 [97.5:2.5]), 5% PIP 3 (POPC: PIP 3 [95:5]), POPC (pure). III. FRAP experiments Fluorescence recovery after photobleaching (FRAP) experiments were performed on a confocal laser scanning microscope (CLSM, LSM 510, Zeiss Germany) equipped with an argon laser (30 mw, λ= 488 nm) and using a 63x (oil, 1.4 NA) objective. 0.1% of fluorescently labeled lipids were used in the bleaching experiments. The photobleaching experiments were performed to assess the fluidity of the lipids in the SLBs. The protocol for the FRAP experiments was adapted from Lopez et al. 4. In brief, SLBs were formed on a glass slide (Menzel Gläser, Braunschweig, Germany) in a confocal microscopy fluid cell cleaned by 20 min sonication in EtOH (sonication bath, ULTRAsonik 104H, NEY) and subsequently by 30 min exposure in a UV-Ozone cleaner (ProCleaner Plus TM, BioForce Nanosciences, Inc., Ames, IA, USA). After mounting the glass slide into the confocal microscopy fluid cell, 1 ml of 50 μg/ml vesicle solution was pipetted into the fluid cell and incubated at 37 C. The incubation time for formation of the SLBs was deduced from QCM-D experiments and the SLBs on the glass slides were rinsed after 2 h by exchanging the solution on top of the bilayers with Tris ph 7.4 for 10 times. Thereby, special care was taken to not ever let the surface dry out. Then the fluid cell was mounted

5 on the temperature chamber of the confocal microscope. The diffusion coefficient and recovered fractions were obtained by analysis of the bleached areas according to Jönsson et al. 5. Figure SI 3. FRAP experiments. Images before bleaching (left), immediately after bleaching (middle) and after 300s post-bleaching (right) are shown. (2A) FRAP experiments on POPC-NBD labeled supported POPC lipid bilayers containing increasing amounts of PIP 2 (a 1% PIP 2, b 5% PIP 2, c 10% PIP 2 ). After bleaching all bilayers recovered and diffusion coefficients were measured as listed in Table 1. (2B) FRAP experiments on supported POPC lipid bilayers containing increasing amounts of PIP 3 (a 1% PIP 3, b 2.5% PIP 3, c 5% PIP 3 ). In correspondence with the QCM-D experiments vesicles containing more than 1% PIP 3 were not able to form continuous SLBs and therefore did not recover completely after photo bleaching (Fig. b, c). IV. XPS The XPS experiments were performed with a Theta Probe (Thermo Electron Corporation, Waltham MA, USA) equipped with a radian lens and a two-dimensional detector. A monochromatic, microfocused AlK α source with a spot size of 300 μm was run at a power of 70 W. The pass energy was 25 ev for the detail and 100 ev for the survey spectra. Data were analyzed using the CasaXPS software (CasaXPS software

6 Version dev52, Software Ldt, UK). A Shirley background was subtracted before the peak areas were integrated and corrected for the cross section using the Scoffield factors 6, inelastic mean free path, attenuation length 7 and the energy dependent transmission function. The percentage of PIP2:POPC was assessed by quantifying the atomic ratio of the N1s:P2p peak. To account for the phosphorus contaminations in the SiO 2 wafers, the molar ratio of P:Si measured on reference wafers was subtracted before the N1s:P2p ratio was calculated. The resulting ratios of N:P were further normalized so that pure POPC bilayers resulted in a molar ratio of N:P = 1 (SI table 2). Statistics was done on 5-6 independent identical samples. SI table 2. N1s:P2p ratios measured by XPS and normalization values used for analysis of dried POPC SLBs with 1, 5 and 10 wt% PIP2 incorporated on SiO 2 wafers. ratio (N:P) average ratio (N:P) normalized amount PIP2 (mol%) amount PIP2 (wt%) POPC 0.30± % PIP2 0.29± % PIP2 0.25± % PIP2 0.21± The SLBs for XPS analysis were prepared on SiO 2 wafers (purchased from Silicon materials, Germany). The wafers were cleaned by 20 min sonication (ULTRAsonik 104H, NEY) first in toluol (Sigma-Aldrich) then in ethanol (Sigma- Aldrich) and subsequently exposed to UV-ozone for 30 min (ProCleaner PlusTM, BioForce Nanosciences, Inc., Ames, IA, USA). Lipid vesicles solutions (50 μg/ml in TBS) were adsorbed on the wafers for 3 h at RT. After exchanging the solution on top of the bilayers with water (Milli-Q grade) for 10 times the wafers were taken out of the water and the SLBs were dried under laminar flow. References 1. Rodahl, M.; Hook, F.; Krozer, A.; Brzezinski, P.; Kasemo, B., Quartz crystal microbalance setup for frequency and Q-factor measurements in gaseous and liquid environments. Review of Scientific Instruments 1995, 66, (7),

7 2. Keller, C. A.; Kasemo, B., Surface Specific Kinetics of Lipid Vesicle Adsorption Measured with a Quartz Crystal Microbalance. Biophysical Journal 1998, 75, (3), McConnell, H. M.; Watts, T. H.; Weis, R. M.; Brian, A. A., Supported planar membranes in studies of cell-cell recognition in the immune system. Biochimica et Biophysica Acta (BBA) - Reviews on Biomembranes 1986, 864, (1), Lopez, A.; Dupou, L.; Altibelli, A.; Trotard, J.; Tocanne, J. F., Fluorescence recovery after photobleaching (FRAP) experiments under conditions of uniform disk illumination. Critical comparison of analytical solutions, and a new mathematical method for calculation of diffusion coefficient D. Biophys. J. 1988, 53, (6), Jönsson, P.; Jonsson, M. P.; Tegenfeldt, J. O.; Höök, F., A Method Improving the Accuracy of Fluorescence Recovery after Photobleaching Analysis. Biophysical Journal 2008, 95, (11), Scofield, J. H., Hartree-Slater Subshell Photoionization Cross-Sections at 1254 and 1487ev. J Electron Spectrosc 1976, 8, (2), Seah, M. P.; Dench, W. A., Quantitative electron spectroscopy of surfaces: a standard data base for electron inelastic mean free paths in solids. Surface and Interface Analysis Surface and Interface Analysis 1979, 1, (1), 2-11.