Juha Saarikangas, Hongxia Zhao, Anette Pykäläinen, Pasi Laurinmäki, Pieta K. Mattila, Paavo K.J. Kinnunen, Sarah J. Butcher, and Pekka Lappalainen
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1 Supplemental Data Molecular Mechanisms of Membrane Deformation by I-BAR Domain Proteins Juha Saarikangas, Hongxia Zhao, Anette Pykäläinen, Pasi Laurinmäki, Pieta K. Mattila, Paavo K.J. Kinnunen, Sarah J. Butcher, and Pekka Lappalainen Supplemental Experimental Procedures Plasmid construction and protein purification RNA from mouse tissues was extracted using Trizol reagent and transcribed into cdna with Superscript II enzyme (Invitrogen) using the manufacturers instructions. IRTKS bp and FLJ bp constructs were cloned into phat1 [1][1] and pgfp-n1 vectors from mouse E17 and liver cdnas, respectively. IRSp bp was cloned from mouse E17 cdna to pgfp-n1 and pcherry-n1 vectors (Clonetech). MIM 1-750bp was subcloned from ppl 329 plasmid [2] into pcherry-n1 vector. IRSp53 I-BAR E.coli protein expression construct and MIM and ABBA constructs have been previously characterized in [2,3]. C.elegans M04F bp was cloned from YK1363a03 cdna clone (Yuri Kohara) into phat1 and pgfp-n1 and pcherry-n1 vectors. Amphiphysin was subcloned into phat1 vector from human kidney cdna. Site-directed mutagenesis was performed as described in [2]. Following plasmids were received as gifts: Fascin EGFP (Daniela Vignjevic, Institut Curie, France), Myosin-10 EGFP (Johanna Ivaska, VTT Medical Biotechnology, Finland) and VASP-EGFP (Frank Gertler, MIT, USA). All protein constructs were expressed as his-tagged fusion proteins, enriched with Ni-NTA Superflow beads (Sigma-Aldrich), and further purified with ion exchange chromatography columns, either Q-, or S-sepharose (GE Healthcare) using FPLC (Pharmacia). Preparation of vesicles Vesicles were prepared as in [2]. Briefly, lipids in desired concentrations were mixed, dried under a stream of nitrogen and hydrated in 20 mm Hepes, ph 7.5, 100 mm 1.5 M 1
2 NaCl to yield multilamellar vesicles in a lipid concentration of 1 mm. To obtain unilamellar vesicles, vesicles were extruded through a polycarbonate filter (100-nm pore size) using a mini extruder (Avanti Polar Lipids). Giant unilamellar vesicles (GUV) were prepared as described elsewhere [4]. Briefly, 2 4 µl of the desired lipids dissolved in diethylether: methanol (9:1, v/v, final total lipid concentration 1 mm) were spread on the surface of two Pt electrodes and subsequently dried under a stream of nitrogen. Possible residues of organic solvent were removed by evacuation in vacuum for 1 h. An AC field (sinusoidal wave function with a frequency of 8 Hz and amplitude of 0.2 V) was applied before adding 0.6 ml of 200 mm sucrose dissolved in 0.5 mm Hepes buffer, ph 7.5. During the first minute of hydration the voltage was increased to 2V. The AC field was turned off after 2 h. For the experiments, GUVs were transferred to 200 mm glucose in 0.5 mm Hepes buffer, ph 7.5. The GUVs had NBD-PC (2 %) and Bodipy-TMR- PI(4,5)P 2 (1-2 %) fluorescent labels and were imaged every 2 seconds as described in the light microscopy section. Total volume of the samples during imaging was 300 μl and a final protein concentration of μm was added to the vesicles. Fluorescence spectroscopy experiments All fluorescence measurements were performed in quartz cuvettes with 3 mm path length. In quenching experiments, fluorescence spectra were measured with a Perkin- Elmer LS 55 spectrometer with both emission and excitation band passes set at 10 nm. Bodipy-TMR-PI(4,5)P 2 fluorescence was excited at 547 nm and the emission spectra were recorded from 555 to 600 nm in the presence of different concentrations of proteins. The percentage of quenching was calculated using the following equation: % quenching=(1-f/f 0 ) 100 where F is the fluorescence intensity of Bodipy-TMR-PI(4,5)P2 in the presence of protein, and F 0 is the fluorescence intensity of Bodipy-TMR-PI(4,5)P2 in the absence of protein. When quenching of Trp emission by acrylamide was measured, the excitation of Trp at 295 nm instead of 280 nm was used to reduce absorbance by acrylamide [5]. Aliquots of the 10 M solution of this water-soluble quencher were added in the absence or presence of liposomes at a protein/lipid molar ratio of 1:100. The values obtained were 2
3 corrected for dilution, and the scatter contribution was derived from acrylamide titration of a vesicle blank. The data were analyzed according to the Stern-Volmer equation [6], F 0 /F=1+K sv [Q], where F 0 and F represent the fluorescence intensities in the absence and the presence of the quencher (Q), respectively, and K sv is the Stern-Volmer quenching constant, which is a measure of the accessibility of Trp to acrylamide. On the premise that acrylamide does not significantly partition into the membrane bilayer [5], the value for K sv can be considered to be a reliable reflection of the bimolecular rate constant for collisional quenching of the Trp residue present in the aqueous phase. Accordingly, K sv is determined by the amount of non-vesicle-associated free protein as well as the fraction of the protein residing on the surface of the bilayer. When quenching of Trp by brominated phosphatidylcholines was measured, the tryptophan residue was excited at 280 nm, and emission spectra were recorded from 300 to 450 nm, averaging five scans. Spectra were corrected for the contribution of light scattering in the presence of vesicles. Collisional quenching of Trp by brominated phospholipids (Br 2 -PCs) was introduced to assess the localization of this residue in bilayers [5,7]. Br 2 -PCs are considered to be well suited for this purpose, since they should introduce insignificant perturbation into the membrane [7,8]. The indicated liposomes were added to the protein solution (final concentration of 0.5 µm in 20 mm Hepes, 100 mm Nacl, ph 7.5) and the emission spectra were recorded by averaging five spectra. The differences in the quenching of Trp fluorescence by (6,7)-, (9,10)-, and (11,12)-Br 2 -PC were used to calculate the probability for location of the fluorophore in the membrane using the parallax method [9]. The depth of the Trp residue is calculated as follows, Z cf =L cl +[(-ln(f 1 /F 2 )/πc-l 2l 2 ]/2L 2l where Z cf represents the distance of the fluorophore from the center of the bilayer, L c1 is the distance of the shallow quencher from the center of the bilayer, L 21 is the distance between the shallow and deep quencher, F 1 is the fluorescence intensity in the presence of the shallow quencher, F 2 is the fluorescence intensity in the presence of the deep 3
4 quencher, and C is the concentration of quencher in molecules/å 2. The average bromine distances from the bilayer center, based on x-ray diffraction are 10.8, 8.3, and 6.3 Å for (6,7)-, (9,10)-, and (11,12)-Br 2 -PC, respectively [8]. When equal concentrations of the Br-lipids are used, the value for c(h) is unity [10]. Fluorescence anisotropy of DPH was measured by including DPH into liposomes at X= The lipid concentration used was 40 µm. Fluorescence anisotropy for DPH was measured with a Perkin-Elmer LS 55 spectrometer, with excitation at 360 nm and emission at 450 nm, using 10nm bandwidths. Salt sensitivity assay Protein (final concentration of 4 μm) was incubated with 160 μm multilamellar vesicles (PC:PS:PE:PIP 2 = 45:20:5:30) in reaction buffer (20 mm Hepes ph 7.5, mm Nacl) for 30 min at RT and subsequently centrifuged 360,000 g for 30 min. Equal amounts of supernatants and pellets were separated on a 13.5 % SDS-PAGE gel. 4
5 SUPPLEMENTAL REFERENCES 1. Peranen, J., Rikkonen, M., Hyvonen, M., and Kaariainen, L. (1996). T7 vectors with modified T7lac promoter for expression of proteins in Escherichia coli. Anal. Biochem. 2, Mattila, P.K., Pykalainen, A., Saarikangas, J., Paavilainen, V.O., Vihinen, H., Jokitalo, E., and Lappalainen, P. (2007). Missing-in-metastasis and IRSp53 deform PI(4,5)P2-rich membranes by an inverse BAR domain-like mechanism. J. Cell Biol. 7, Saarikangas, J., Hakanen, J., Mattila, P.K., Grumet, M., Salminen, M., and Lappalainen, P. (2008). ABBA regulates plasma-membrane and actin dynamics to promote radial glia extension. J. Cell. Sci. Pt 9, Zhao, H., Bose, S., Tuominen, E.K., and Kinnunen, P.K. (2004). Interactions of histone H1 with phospholipids and comparison of its binding to giant liposomes and human leukemic T cells. Biochemistry 31, De Kroon, A.I., Soekarjo, M.W., De Gier, J., and De Kruijff, B. (1990). The role of charge and hydrophobicity in peptide-lipid interaction: a comparative study based on tryptophan fluorescence measurements combined with the use of aqueous and hydrophobic quenchers. Biochemistry 36, Eftink, M.R., and Ghiron, C.A. (1976). Exposure of tryptophanyl residues in proteins. Quantitative determination by fluorescence quenching studies. Biochemistry 3, Bolen, E.J., and Holloway, P.W. (1990). Quenching of tryptophan fluorescence by brominated phospholipid. Biochemistry 41, McIntosh, T.J., and Holloway, P.W. (1987). Determination of the depth of bromine atoms in bilayers formed from bromolipid probes. Biochemistry 6, Chattopadhyay, A., and London, E. (1987). Parallax method for direct measurement of membrane penetration depth utilizing fluorescence quenching by spin-labeled phospholipids. Biochemistry 1, Ladokhin, A.S. (1999) Analysis of protein and peptide penetration into membranes by depth-dependent fluorescence quenching: theoretical considerations. Biophys. J. 76,
6 Figure S1. (A) Structure of mouse MIM I-BAR domain (Protein Data Bank ID: 2D1L). Mutations that were used in this study are listed in the table and indicated with different colors in the structure. (B) ClustalX sequence alignment of different I-BAR domains. Amino acid sequences are retrieved from the Ensembl ( and Wormbase ( databases with the following peptide IDs: (Hs MIM ENSP ); (Mm ENSMUSP ); (Hs ABBA ENSP ); (Mm ABBA ENSMUSP ); (Hs IRSp53 ENSP ); (Mm IRSp53 ENSMUSP ); ( Hs IRTKS ENSP ); (Mm IRTKS 6
7 ENSMUSP ); (Mm FLJ22582 ENSP ); (Mm FLJ22582 ENSMUSP ); (Ce M04f3.5 WP:CE31974). 7
8 8
9 Figure S2. (A) The tubulation efficiency of MIM I-BAR is decreased when electrostatic interactions are compromised. In 0.5 M NaCl, there are still many tubules found of similar diameter to those in the physiological salt concentration. MIM I-BAR domain was not efficient in tubulating membranes in high (1.0 and 1.5 M) NaCl concentrations. (B) There was no significant difference in the diameter of MIM and IRSp53 I-BAR domain induced membrane tubules on unilamellar vesicles compared to the multilamellar vesicles (Fig. 3B-C). 9
10 Figure S3. I-BAR domains segregate in filopodia when co-expressed in cells. (A) MIM I-BAR-GFP and MIM I-BAR-Cherry were used as a control. The measured 10
11 intensity profiles from both channels were similar to each other. (B) Nearly identical intensity profiles were detected in cells transfected with ABBA I-BAR-GFP and MIM I- BAR-Cherry. (C) IRSp53-GFP and MIM-cherry show different distribution in cells. Typically, IRSp53 signal was stronger in the cytoplasm, whereas MIM displayed stronger fluorescence intensity in filopodia. (D) MIMΔN I-BAR displayed similar intensity in filopodia as IRSp53 I-BAR. (E) IRTKS and IRSp53 I-BARs displayed similar fluorescence intensity throughout the cell. (F) IRSp53 I-BAR segregated from C.elegans I-BAR in filopodia. In the cytoplasm and at the root of the filopodia the IRSp53 I-BAR signal is stronger but at the distal ends of the filopodia the IRSp53 I-BAR signal is lacking and the C.elegans I-BAR signal is dominant. 11
12 Figure S4. (A) Helical wheel representations reveal the amphiphatic properties in the N- terminal region of MIM and ABBA I-BAR domains. Hydrophobic residues (in green) are clustered on one side of the helix flanked by charged residues. (B) DPH anisotropy 12
13 curves derived from MIM I-BAR mixed with vesicle samples with or without negatively charged lipids demonstrate that weak electrostatic interactions are necessary for the membrane insertion of MIM I-BAR. (C) The tryptophans introduced at positions 3 and 31 of the N-terminal α-helix of IRSp53 I-BAR are protected from aqueous quencher as demonstrated by Stern-Volmer plots for the quenching of IRSp53 I-BAR by acrylamide in an aqueous buffer (open symbol) and in the presence of liposomes (filled symbols). (D and E) Lack of quenching of tryptophan emission by brominated lipids indicates that Trp3 and Trp31 do not insert into the hydrophobic core of the bilayer. 13
14 Figure S5. I-BAR domain induced membrane protrusions in cells contain filopodia markers. (A) Immunofluorescence stainings from U2OS cells that express MIM I-BAR domain, full length MIM, IRSp53 I-BAR domain and full length IRSp53. Cells were 14
15 stained with anti-myosin-10 antibody. In many, but not all cases, myosin-10 was detected at the tips of the filopodia (white arrowheads) induced by I-BARs or the full-length proteins. (B) U2OS cells simultaneously expressing MIM I-BAR domain and fascin, VASP or myosin-10. Fascin was seen in the shaft of the protrusions of the double transfected cells and was typically surrounded by I-BAR. VASP was detected both at the tips and in the shafts and, myosin-10 at the tip or in the shaft of filopodia, indicating intrafilopodial motility (see also Supplementary video 2). Scale bar: 2 μm. 15
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