Supplementary information Membrane curvature enables N-Ras lipid anchor sorting to liquid-ordered membrane phases Jannik Bruun Larsen 1,2,3, Martin Borch Jensen 1,2,3,6, Vikram K. Bhatia 1,2,3,7, Søren L. Pedersen 2,3,8, Thomas Bjørnholm 1, Lars Iversen 1,2,3, Mark Uline 4, Igal Szleifer 5, Knud J. Jensen 2,3, Nikos S. Hatzakis 1,2,3 and Dimitrios Stamou 1,2,3, 1 Bio-Nanotechnology and Nanomedicine Laboratory, Nano-Science Center, 2 Department of Chemistry, 3 Lundbeck Foundation Center Biomembranes in Nanomedicine, University of Copenhagen, Denmark, 4 Department of Chemical Engineering, University of South Carolina, Columbia, SC 29208, USA 5 Department of Biomedical Engineering, Department of Chemistry and Chemistry of Life Processes Institute, Northwestern University, Evanston, IL 60208, USA. 6 Current address: The Buck Institute for Research on Aging, 8001 Redwood Boulevard, Novato, CA 94945, USA 7 Current address: Novozymes A/S, 2880 Bagsvaerd, Denmark 8 Current address: Gubra ApS, 2970 Hørsholm, Denmark email: stamou@nano.ku.dk 1
Supplementary Results Supplementary Fig. 1 Mass spectra and scheme of peptide (2). 2
Supplementary Fig. 2 The Single Liposome Curvature (SLiC) assay for measuring recruitment by membrane curvature. (a) Fluorescently labeled liposomes with a wide distribution of diameters, and thus curvatures, are tethered to a passivated glass surface through biotin-streptavidin coupling. A separately labeled protein-anchoring motif, here the minimal anchor of N-Ras (tn-ras), is added and binds to the liposomes from solution. Confocal fluorescence images of liposomes (b) and tn-ras (c) are compared to evaluate anchoring motif binding on individual liposomes. Zooms of each channel show 3
gaussian intensity profiles, from which liposome diameter and anchoring motif density on single liposomes are calculated. Scale bar is 5 µm. 4
Supplementary Fig. 3 Addition of tn-ras does not deform liposomes. In analogy with previous studies 1, 2 we believe that the ability to record membrane curvature dependent binding strongly suggests that under our experimental conditions we do not have liposome deformation. To evaluate this experimentally for tn-ras, we incubated micron sized GUVs with 2 µm tn-ras. After a 10 min incubation period we imaged the GUVs and saw no deformation. Since it has previously been theoretically predicted 3 and experimentally verified 4 that larger liposomes are more susceptible to deformation than smaller, we believe that we do not have significant deformation even for the smaller liposomes used in this work. 5
Supplementary Fig. 4 tn-ras is well solubilized and experiments are performed at binding equilibrium. (a) and (b) Micrographs of liposome and tn-ras channels respectively, revealing no significant precipitation, strongly 6
suggesting tn-ras to be well solubilized. Scale bars are 3 µm. (c) tn-ras intensity on a small (red) and large (blue) liposome as a function of time, showing tn-ras to fully equilibrate within minutes. (d) Micrograph zooms of liposomes used for analysis (red, diameter = 80 nm and blue, diameter = 255 nm) and of tn-ras bound to liposomes before bleaching (pre bleach) and at five time points after bleaching (post bleach). 7
Supplementary Fig. 5 Membrane phase-state modulate recruitment by membrane curvature of tn-ras. (a) and (b) Bound tn-ras density as a function of liposome diameter for l d (DOPC:PSM:Chol, 80:10:10) liposomes (red markers) and l o (DOPC:PSM:Chol, 20:20:60) liposomes (black markers). (a) l o data points omitted for clarity. (b) l d data points omitted for clarity. The power function fits reveal that l d and l o membrane systems show different increases in density on highly curved liposomes indicating that membrane phase-state can regulate recruitment by membrane curvature. Equivalent data and fit for a negative control (blue markers) shows efficient binding of streptavidin to biotinylated liposomes, but no change in density as a function of diameter. 8
Supplementary Fig. 6 Normalized density measurement of C 16 reveals more potent recruitment by l o than l d phase-state membranes. To further elucidate how membrane phase-state and curvature influence the recruitment of protein anchoring motifs, we simplified our model system to one of the most common protein anchoring motif, a palmitoyl acyl chain. We used a fluorescein headgroup with an attached palmitoyl anchor (C 16 ), which we have previously shown to be recruited by membrane curvature. 1 We employed the same assay and liposome compositions as in the tn-ras study and observed in full agreement that C 16 is also recruited more strongly by curvature on l o - than on l d membranes. 9
Supplementary Fig. 7 The relative recruitment ratio for tn-ras and C 16 quantified for different membrane compositions. 10
Comparison of the reported coefficient for l d - to l o domain partitioning of either N-Ras or single chained anchoring motifs with the membrane-curvature mediated density increase for various lipid compositions. The R value is presented as the average of at least three independent experiments, with the uncertainty calculated as the standard error of the mean. (a) The relative recruitment ratio for tn-ras on ternary mixtures of DOPC:PSM:Chol showing different bilayer phase-states. (b) The relative recruitment ratio for C 16 on different binary and ternary mixtures in a variety of pure and mixed phase-states (c-g) C 16 density versus liposome diameter for a number of membrane compositions. (h) Table summarizing the R values presented in (a) and (b). n represents the number of independent experiments. 11
Supplementary Fig. 8 Liposome size does not introduce a systematic change in the average lipid composition of liposomes. (a) Normalized ratio of Chol- NBD and DiD for individual liposomes plotted versus liposome diameter, reveal a constant average cholesterol concentration for all liposome sizes. (b) Normalized Chol-NBD and DiD intensity ratio histogram illustrates compositional heterogeneity. Gaussian fitting quantifies that the cholesterol concentration of 95 % of all liposomes will be within 40 % of average cholesterol concentration. The mean and standard deviation is calculated from two independent experiments. (c) Phase-diagram for ternary mixtures of DOPC:PSM:Chol with the l d and l o mixtures marked along with the 95 % cholesterol heterogeneity cutoff. Cholesterol heterogeneity cannot lead to a change in phase-state for essentially any of the liposomes within the ensemble. 12
Supplementary Fig. 9 Oval-C 16 control verifies that recruitment of C 16 by membrane curvature is not affected by flip-flop or attachment of organic dyes. Normalized density versus liposome diameter for C 16 and Oval-C 16 on either l d (red, n Oval-C16 = 3), l o (grey, n Oval-C16 = 3) or DOPC:PS (90:10) (blue, n Oval-C16 = 2) membrane systems. To ensure that C 16 flip-flop or organic dyes labeling were not affecting our measurement we compared the recruitment by membrane curvature of C 16 and a protein control Ovalbumin-C 16 (Oval-C 16 ). As previously described 1 we biochemically coupled a single palmitoyl to ovalbumin (Oval-C 16 ), producing a species anchored to the membrane by a C 16 chain and unable to flip-flop. Furthermore Oval-C 16 was labeled with Alexa488 as previously described 1 and represents a large protein, thus its membrane interaction and partitioning should not be influenced by the placement of organic dyes. We quantified non-significant differences in the recruitment by membrane curvature of Oval-C 16 and C 16 on the l d, l o or DOPC:PS (90:10) membrane systems (l d system: R C16 = 6.6 ± 0.4, R Oval-C16 = 6.36 ± 1.3, p = 0.44; l o system: R C16 = 36.8 ± 9.4, R Oval-C16 = 32.3 ± 3.8, p = 0.38; DOPC:PS system: R C16 = 6.2 ± 2.0, R Oval-C16 = 6.5 ± 0.6, p = 0.41). Thus we conclude that our measurements using C 16 are not significantly affected by flip-flop or the attachment of organic dyes. 13
Supplementary Fig. 10 Decreasing the lipid packing order in the headgroup area of the outer monolayer by inclusion of DOPE results in reduced recruitment by membrane curvature. Increasing amounts of DOPE were added to a DOPC membrane system, 5 mol% (black, n 5% = 3), 25 mol% (red, n 25% = 3) or 50 mol% (blue, n 50% = 3). To study how decreasing the lipid packing order of the headgroup area of the outer monolayer affects the recruitment by membrane curvature we included increasing amounts of DOPE lipids in a DOPC mixture. 5, 6 The incorporation of cone shaped lipids (like DOPE) is known to have opposite effects on the packing of the headgroup- and acyl chain regions of membranes. 7 Whereas the packing increases in the acyl-chain region, it decreases in the headgroup region, leading to increased binding and activity of peripheral membrane proteins. 6, 8 We examined the recruitment ability of C 16 on DOPC liposomes containing 5 mol%, 25 mol% or 50 mol% DOPE and as previously reported 6 an upward shift in the density versus size curves is observed for increasing DOPE content (grey arrow). Furthermore we recorded a small, but significant, decrease in the R values for increasing DOPE concentrations (R 5 mol% = 6.5 ± 0.6, R 50 mol% = 3.2 ± 0.2, p = 0.003). 14
Supplementary Fig. 11 Measurement of absolute density of tn-ras and C 16 on liposomes of different phase-state reveals that the partition coefficient depends on membrane curvature. Recording the density of tn-ras or C 16 on liposomes using identical microscope settings allowed us to compare the absolute densities on liposomes of different phase-states. To do this we plotted the non-normalized data of Supplementary Figure S5 (tn-ras) and Supplementary Figure S6 (C 16 ) and again fitted them with the offset power function a) tn-ras and b) C 16. For tn-ras it is evident that for larger liposomes the absolute density is significantly higher on liposomes in the l d phase than in the l o phase. For both tn-ras and C 16 the rate of the density increase with curvature however is higher for the l o versus the l d -membrane system, eventually leading to higher densities on l o membranes of high membrane curvatures (low diameters). 15
Supplementary Fig. 12 In GUVs having coexisting l o and l d domains tn-ras show preferential partitioning in the l d phase. a) Confocal micrographs and linescans of both fluorescent channels for two GUVs. Scale bar is 20 µm (left) and 5 µm (right) To study the partition coefficient of tn-ras between l d and l o domains on GUVs, we prepared GUVs from a 3:4:3 DOPC:PSM:Chol mixture showing coexisting l o and l d domains. Analogous to previous protocols 9, 10, we used line scans through the l o and l d domains on the same GUV to quantify the partition coefficients for tn-ras, K p (l o /l d ) GUV. We extracted the tn-ras density as the integrated intensity using a gaussian fit to the l o and l d phase-state intensity peaks (shaded areas) and 16
calculated K p (l o /l d ) GUV as the integrated intensity ratio. Only GUVs with clearly defined equatorial domains were used for partitioning calculations. b) We examined GUVs ranging in size from 6-67 µm (light red) and calculated the average K p (l o /l d ) GUV for three size bins (dark red) (8.3 ± 0.5 µm = 0.29 ± 0.05; 16.4 ± 1.5 µm = 0.21 ± 0.04; 40.4 ± 5.3 µm = 0.24 ± 0.06). For tn-ras we recorded a clear preference for the l d as opposed to the l o phase-state, in agreement with previous studies. 11, 12 Black markers represent the GUV shown in a). 17
Supplementary Fig. 13 The lateral pressure is dominated by the repulsive, not the attractive interactions. The total lateral pressure profile and the contribution of the repulsive interactions to the lateral pressure, π(z,c), as a function of position in the hydrophobic region of the l o membrane for (a) a planar bilayer and (b) the same bilayer curved to a diameter of 50 nm. 18
Supplementary Fig. 14 Illustration of areas in the trafficking pathway of N-Ras where increased membrane order and curvature coincide. Cellular membranes comprise areas of different order (i.e. composition) and geometrical curvature. Our prediction, based on in vitro experiments, is that N-Ras partitioning is maximized when the two coincide, as in the case for (1) budding transport vesicles from the Golgi apparatus, (2) vesicles trafficking between the Golgi and the plasma membrane or (5) caveolae. (3) illustrates a plasma membrane ordered domain and (4) an invagination in the plasma membrane. 19
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