Département De Chimie, Ecole Normale Supérieure, UMR 8640 .

Engineering spatial gradients of signaling proteins using magnetic nanoparticlesL. Bonnemay, S. Hostachy, C. Hoffmann, J. Gautier and Z. GuerouiDépartement de Chimie, Ecole Normale Supérieure,UMR 8640 CNRS-ENS-UPMC Pasteur, 24, rue Lhomond, 75005 Paris, France.Corresponding Author, Zoher GuerouiSupplementary Experimental Procedures:1. Supplementary Methods and Discussion2. Supplementary Figures1

1. Supplementary Methods and DiscussionTransmission Electron MicroscopyTransmission Electron Microscopy was performed using a Jeol 2200FS microscope for 120nmdiameter nanoparticles and a Jeol JEM 100CX microscope for 30nm nanoparticles(Supplementary Figure S3).Characterization of Ran-NP complexesConjugation stoichiometry was determined by a semi-quantitative assay using SDS-PAGEelectrophoresis (Supplementary Figure S6). Samples were solubilized in 2% sodiumdodecylsulfate (SDS) with 0.3 M -mercaptoethanol. Electrophoresis was carried out with acurrent of 100mA for 30 minutes in a 4-12% polyacrylamide gel (Life technologies). Proteindetection was revealed by Coomassie Blue staining and molecular weight standard proteins(Biorad #161-0363). Protein density on nanoparticles was determined beside a BSA range usingImage J. Integrated colorimetric intensity of each band is expressed as a function of proteinquantity. The quantity of protein is reported to the known quantity of nanoparticles in thesamples, allowing us to determine the final Protein/NP ratio (Supplementary Figures S4a,b). Inorder to determine the stability of the complexes as a function of time (0, 30, and 90 min), thesolution was rinsed in XB1 buffer to remove unbound proteins. (Supplementary Figure S6c).Determination of the nanoparticle concentration using the intensity profile of fluorescentnanoparticles.The intensity of fluorescence is a readout of the number of fluorescently labeled nanoparticles.To determine the local concentration of nanoparticles, the geometry of the droplet need to be2

considered. In epifluorescence acquisition, the intensity profile of fluorescently homogeneousdroplets provides an estimation of the droplet curvature (Supplementary Figures S4a,b). Wecould thus obtain the local concentration in nanoparticles and consequently in Ran proteins alongthe droplets by extracting the droplet contour height. In Supplementary Figure S4a, we reportedthe intensity profile of fluorescent nanoparticles homogeneously distributed within 12 differentdroplets (red) compared to a theoretical profile of a semi-spherical droplet (blue). Next, weweighted each intensity by the droplet shape by computing are constituted droplet having ahomogeneous concentration using the average intensity profile determined in SupplementaryFigure S4b (Supplementary Figures S4c-g).The typical decay length L of the concentrationgradient was extract using exp(-c*L-1). We found a typical value of L of 20% of the dropletdiameter for the strong asymmetries; 40% of the droplet diameter for intermediate asymmetriesand 125% of the droplet diameter for weak asymmetries (Supplementary Figure S4h).Effect of the nanoparticle sizeThe size and composition of the nanoparticles matter to induce concentration gradient ofnanoparticles. In order to examine the rational of our observations we have compared the ratio(λ) between the magnetic energy of interaction between two particles to the thermal energyλ πµ 0 a 3 χ 2 H 29k BTwhere µ 0 is the magnetic permeability in vacuum, a the particle radius, χ its susceptibility, kB theBoltzmann constant, and T the temperature 1. If λ is larger than one, the particles are more likelyto aggregates. To the contrary if λ is smaller than one, the nanoparticles are under Brownianthermal diffusion control. Consequently, since the magnetic attraction scales with the third powerof the nanoparticle diameter, a, we expect a strong variation depending on the nanoparticle size3

for a given magnetic field. For instance, a fold change of 4 in nanoparticle size (30 to 120 nm)results in a fold change in magnetic energy of 64 (see figure above).Effect of the nanoparticle size: λ values for different nanoparticles radius for a magnetic field of 400mT(a) and 100mT (b).4

2. Supplementary FiguresSupplementary Figure S1: Experimental set-up. Two rectangular capillaries are positioned next to apermanent magnet.5

Supplementary Figure S2: 120 nm magnetic nanoparticles encapsulated in droplet and submitted to a50T.m-1 magnetic field gradient. We observed the formation of aggregates of nanoparticles.6

Supplementary Figure S3: Microscopic observations of different size nanoparticles. (a) Transmissionelectron microscopy of 120 nm nanoparticles, (b) Transmission electron microscopy of 30 nm diameternanoparticles.7

Supplementary Figure S4: Determination of the nanoparticle concentration using the fluorescenceintensity profile. (a) red: Intensity profile of fluorescent nanoparticles homogeneously dispersed within adroplet (12 droplets). Blue: profile of a semi-spherical droplet. (b) Mean shape of a homogenous droplet.(c) Fluorescence intensity profile of an asymmetric (blue) and a homogeneous (red) droplets. (d)Deconvoluated gradient of fluorescence intensity. The gradients are deconvoluated using the mean shapeof a homogeneous droplet. (e,f,g) Deconvolution of the droplet shape for each category of gradient. We8

obtained RanGTP concentration gradients within the droplets. (h) Average of the concentration profile foreach category.9

Supplementary Figure S5: Stability and reversibility of the nanoparticle gradient. (a) Temporalstability of the gradient of nanoparticle concentration upon magnetic induction. Left : observation offluorescent magnetic nanoparticles within Xenopus egg extracts encapsulated inside a droplet uponapplication of a magnetic field, Middle : fluorescence intensity profile along the droplet, Right : plot of thecorresponding nanoparticle concentration within the droplet (b) Temporal reversibility of the nanoparticlegradient upon removal of the magnetic field. Magnetic field is removed at t 0s Left : fluorescencemicroscopy observation of fluorescent magnetic nanoparticles within Xenopus egg extracts encapsulatedinside a droplet upon application of a magnetic field (t 0s), or after removing the magnetic field, Middle :fluorescence intensity profile along the droplet, Right : plot of the corresponding nanoparticleconcentration within the droplet.10

Supplementary Figure S6: Characterization of Ran-NP complexes. (a,b) Determination of conjugationstoichiometry by SDS-PAGE electrophoresis. (a) SDS-PAGE electrophoresis of various Ran-NPcomplexes. Lane 1: molecular weight protein standard, lane 2 to 5: unpurified complexes with variation ofRanQ69L/NP initial ratio: lane 2 0; lane 3 130; lane 4 600; lane 5 1300; lane 6 to 9 : purifiedcomplexes; lane 10to 15 : BSA RANGE : lane 10 0,1 µg BSA; lane 11 0,5 µg BSA; lane 12 1 µgBSA; lane 13 1,5 µg BSA; lane 14 2 µg BSA; lane 15 3 µg BSA. (b) RanQ69L/NP final molar ratioas a function of RanQ69L/NP initial molar ratio. (c) Determination of the stability of the Ran-NPcomplex. (d,e) Aster’s nucleation and assembly threshold of RanQ69L (d) or Ran-NP (e) concentration.Aster density was determined by fluorescence microscopy and normalized to 100% at the highest density.11

1.Liu, J.; Lawrence, E. M.; Wu, A.; Ivey, M. L.; Flores, G. A.; Javier, K.; Bibette, J.; Richard, J. Phys RevLett 1995, 74, (14), 2828-2831.12

Département de Chimie, Ecole Normale Supérieure, UMR 8640 CNRS-ENS-UPMC Pasteur, 24, rue Lhomond, 75005 Paris, France. Corresponding Author, Zoher Gueroui Supplementary Experimental Procedures: 1. Supplementary Methods and Discussion 2. Supplementary Figures