Dynamic Re-organization of Individual Adhesion Nanoclusters in Living Cells by Ligand Patterned Surfaces**

Transcription

1 Supplementary information: SMALL Dynamic Re-organization of Individual Adhesion Nanoclusters in Living Cells by Ligand Patterned Surfaces** Ruth Diez-Ahedo, Davide Normanno, Olga Esteban, GertJan Bakker, Carl G. Figdor, Alessandra Cambi and Maria F. Garcia-Parajo*

2 Experimental Section: Pattern fabrication: Poly(dimethylsiloxane) (PDMS) elastomer stamps were fabricated by curing Sylgard 184 (Dow Corning) in a 10:1 proportion (base : crosslinker) in weight, on a silanized silicon patterned master generated in photoresist using standard photolithography techniques. The cured PDMS was peeled off the master and cut into 1 cm x 1 cm squares. PDMS stamps were sonicated in ethanol for 10 min. rinsed in milliq water and dried with a stream of nitrogen gas. Stamps were coated with 50 µg/ml goat anti human antibodies (GαHu IgG (Fab ) 2, Jackson Immuno Research) in TMS buffer (200mM Tris, 150mM NaCl, 1mM CaCl 2, 2mM MgCl 2, ph 8.0). After 45 min. of incubation, stamps were rinsed with milliq water, blown dry with nitrogen gas, and stamped into clean glass substrates (Menzel- Glaser Ø30mm #1). The glass coverslips were sonicated for 10 min in 1:1 ethanol: milliq water dried with nitrogen flow and then exposed to UV/Ozone (Bioforce nanosciences) plasma cleaner immediately prior to stamp application to increase the hydrophilicity of the glass surface. A circle with a DAKO pen was drawn to keep the solutions within the boundaries of the substrate. After stamping, the unexposed surface of the glass was blocked with 1% bovine serum albumin (BSA) in TMS buffer to minimize unspecific binding. The samples were then washed with TMS to remove the unbound protein. 5µg/ml chimera-fc-icam-1 in TMS buffer were adsorbed over the patterned area and incubated overnight at 4ºC. Unbound Chimera-Fc-ICAM-1 was washed off with TMS. Pattern identification: To examine the specific adsorption of ICAM-1 onto the GαHu patterned areas by fluorescence microscopy, 100 µl of 5µg/ml of Anti-human-ICAM-1 IgG 1 (BD Pharmingen) in TMS were incubated over the patterns for 1 hour at 37ºC. The unbounded antibody was washed with d I H 2 O. 100 µl of 5µg/ml of AlexaFluor-647 goat

3 anti-mouse IgG (H+L) (Molecular Probes) in TMS were incubated for 15 min at room temperature. Finally, samples were washed with MilliQ water and were ready for inspection. Cell culture and labeling experiments: THP-1 (Human acute monocytic leukemia) cells that inherently express LFA-1 were cultured in RPMI-1640 (Gibco) medium supplemented with fetal calf serum (FCS, 10%) and antibiotics/antimicotics (AA, 2%). The culture was kept 37ºC in a humidified atmosphere with CO 2 (5%). 200 µl of cells (10 5 cell/ml) were blocked with 1% human serum (HS) in RPMI serum-free, phenol red free and AA free for 10 min at room temperature and labeled with L16 antibody (home made) conjugated to ATTO-647N (Atto-tech) in a final concentration of µg/ml for 5 min at 37ºC. After incubation time, cells were centrifuged for 5 min at 1000 rpm and resuspended into 1 ml RPMI. 200 µl of labeled THP-1 cell were incubated over the patterned coverslips for 15 min before single-molecule fluorescence imaging. Before measurements, cells were washed and dissolved in serum-free, phenol red free, and AA free RPMI-1640 medium. Control experiments were carried out with the addition of APC conjugated mouse IgG2a isotype control (BD Pharmingen) and no fluorescence was observed. Control experiments using fibronectin coated glass coverslips and GαHu patterned surfaces: To confirm that the specific arrangement of LFA-1 on the ICAM-1 patterns shown in Figure 2B was indeed due to specific interaction with the ligand, we performed control experiments using fibronectin coated surfaces. For substrate preparation, 20µg/ml of fibronectin (Roche) in PBS were placed onto a clean coverslip (in the DAKO confined circle) and incubated for 60 min at 37ºC. Unbound protein was

4 washed with RPMI 1640 before cells seeding. Cell seeding was performed as described above. As control substrates for cell seeding we also used GαHu/BSA patterns (5x 5µm 2 ) as non-specific ligands for LFA-1. Single spot brightness analysis and simulations: Raw images were analyzed using a custom-made routine (written in LabVIEW v.6.1, National Instruments) that determines position and brightness of each individual fluorescence spot. In the analysis shown in Figure 3A, we only considered diffraction limited spots (i.e. with apparent dimension not larger than the diffraction limit of our set-up roughly 400nm or 3 pixels on the CCD). The brightness of each spot was defined as the (background-subtracted) sum of the intensities of all the pixels within the spot. To minimize the effect of photobleaching on the analysis, only the first frames from multiple movies were taken into account. Outcome of brightness analysis (over BSA and ICAM-1 regions) as shown in Figure 3A were compared using Wilcoxon-Mann-Whitney two-sample rank-sum test. Medians were 9.3a.u. and 16.5a.u. for BSA and ICAM-1 regions respectively. The distributions in the two groups differed significantly (normalized Mann-Whitney U = -8.32; n BSA = 128; n ICAM-1 = 176; two-sided P < for equal distribution hypothesis). To enquire on the apparent patchiness of the LFA-1 distribution on the ICAM-1 areas, we generated simulated images of randomly distributed nanoclusters at densities related to our experimental conditions using custom-made software (written in LabVIEW). The simulation algorithm places individual particles at random positions in a 2D area, similar in size to that typically imaged in our experiments. The adjustable parameters in the simulations are: image area (A) in µm 2, particle density (D) in particles/µm 2, particle brightness (I) in a.u., particle intensity standard deviation (I d ) in a.u., and imaging

5 resolution (R) in nm. During the simulation, A and D are used to calculate the total number of particles to be distributed in A. The (x,y) coordinates of each individual particle are obtained from a random number generator. Particles with the size of a single pixel are randomly distributed in the simulated image area. To include the effect of the diffraction limited resolution, the pixels are convoluted with a 2D Gaussian with a FWHM equal to the microscope resolution (R), 400 nm in our case. All the parameters of the simulation, i.e., I, I d, D and A were derived from experimental data. Finally, the pixel intensity distribution of the simulated images was plotted and compared to that of the pixel intensities obtained during experiments on the ICAM-1 areas (Figure 3B). Fluorescence trajectories analysis: Two-dimensional trajectories of individual LFA-1 fluorescent spots in the focal plane were obtained using custom-made single-particle tracking software (running in MatLab, The MathWorks). Only trajectories containing at least thirteen points were retained for further analysis. Mean Square Displacement (MSD) versus time lag, t lag, plots were generated for all possible t lag intervals throughout each trajectory. The slope of the linear fit through the first four points in the MSD plots was determined, and the relationship MSD=4Dt lag was used to derive the short range diffusion coefficient D. Localization accuracy on the determination of the (x,y) coordinates for generating single trajectories was 30-40nm in our experimental conditions. Single- particle motion analysis: Among all the trajectories observed over the ICAM-1 area, we selected those falling within regions proximal to ICAM-1 patterns. In particular, we considered rectangular regions 1µm wide and with a length similar to that of the patterns (5µm in the case of the results shown here). For the selected trajectories

6 we then defined an end-to-end vector R as the vector between the initial and the final position of each cluster along its trajectory. For easier analysis we translated all initial points into the origin of a virtual reference system (see Figure 5A,B). Each trajectory is represented by an effective distance travelled ( R ) and by the angle θ indicating the direction of diffusion. For global comparison of diffusion directions, opportune rotations have been imposed to the different lateral regions in order to locate the direction from the external region towards the pattern at an angle θ = 45. Thus, in case of an actively driven diffusion process towards the ligand rich areas, an accumulation of trajectories centered on this angular value is expected.

7 A 5 µm patterns B C 1 µm 2.5 µm 5 µm Intensity (a.u) Background (a.u) Intensity (a.u) Background (a.u) Intensity (a.u) Background (a.u) 982 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 46 Figure S1: MCP allows reproducible and homogenous fabrication of ICAM-1 patterns over large surface areas. (A) Circular arrays of ICAM-1 patterns (5 µm in diameter) as inspected by fluorescence microscopy. ICAM-1 patterned areas were coated with anti-human-icam-1 fluorescently labeled using goat anti-mouse Alexa Fluor 647. (B) Line profile over a distance of 100 µm. Variations in intensity account to 9% in this representative profile. (C) Average intensity of different fluorescent areas and chosen from different pattern sizes. Similar intensity values are obtained independent on the pattern size.

8 150 nm Figure S 2: LFA-1 forms nanoclusters on THP-1 cells, prior to ligand engagement. THP-1 cells were specifically labeled using the TS2/4 antibody and 10nm-gold particles. Cells were then treated for TEM inspection, f ollowing procedures described in Ref [3]. Clustering of LFA-1 (defined when gold particles are <50nm apart from a neighboring particle) are clearly observed on the TEM image.

9 A B C D height (nm) E 40 F 40 G Occurrences x scanning (µm) height (nm) Occurrences ,0 1,5 2,0 2,5 3,0 3,5 height (nm) Figure S3: ICAM-1 patterns are homogeneous down to the nm-scale. AFM inspection of the patterns used for cell seeding at difference spatial scales for (A) 40 x 40 µm 2 (256x256 pixels), (B) 20 x 20 µm 2 (256x256 pixels), (C) 8 x 8 µm 2 (512x512 pixels) and (D) 1 x 1 µm 2 (512x512 pixels). (E) Line profile along the ICAM-1 area shown in C. The difference is height between ICAM-1 and BSA regions is ~ 5nm. (F) Histogram of the different heights shown in E. Fitting with a two Gaussian function renders values of (4 ± 0.05) nm for the height of the BSA area and (8.8 ± 0.04) nm for the ICAM-1 area, while the width of the distributions reflect the heterogeneity of the layers which correspond to (1.2 ± 0.1)nm in BSA and ICAM-1 areas. (G) Histogram of the different heights obtained from image D. The width of the distribution reflects the heterogeneity of the region which is ~1nm.

10 A 5µm B 50pixel Figure S4: Comparison between experimental data (A) and simulated data (B) showing a preferential non-random distribution of LFA-1 nanoclusters on ligand rich areas. Simulated images, as the one shown in B, were generated using the experimental LFA-1 densities as measured on the patterns. Particles with the size of a single pixel were randomly distributed in the simulated image area. To include the effect of the diffraction limited resolution, the pixels were then convoluted with a 2D Gaussian with a FWHM of 400nm, equal to the microscope resolution (see also Supplementary information). A random distribution of LFA-1 is observed on the simulated images (B) in contrast to the punctuated (clustered) organization exhibited by LFA-1 on the experiments (A).

11 # trajectories 70 A D (µm 2 /s) MSD (µm 2 /s) B D = 0.084± µm 2 /s Timelag (s) Figure S5: (A) Diffusion histogram of LFA-1 nanocluster trajectories taking at the cell membrane side in contact with fibronectin coated surfaces. The histogram contains 186 individual trajectories from 5 representative cells. (B) Average MSD plot of the mobile population of LFA-1 for D values > 0.001µm 2 /s, indicating random diffusion of LFA-1 in the absence of its ligand. Fitting of the first four points of the MSD curve renders a D value of ± µm 2 /s.

12 Supplementary movie information: Movie 1: LFA-1 nanoclusters are selectively recruited to ICAM-1 patterned regions. THP-1 cells were seeded over ICAM-1/BSA patterns (5µm). Fluorescence spots correspond to LFA-1 labeled with L16-ATTO647N antibody. Movie composed out of 400 frames. Frame rate 10Hz. Imaged area: (27 x 27)µm 2. One individual cell covering multiple ICAM-1 patterns is shown in the movie. During the first few fames of the movie the focus is adjusted until the interface between the cell membrane and the patterns is localized. Movie 2: LFA-1 nanoclusters are randomly distributed and exhibit mostly random diffusion on THP-1 cells seeded over Fibronectin coated glass. Fluorescence spots correspond to LFA-1 labeled with L16-ATTO647N antibody. Movie composed out of 400 frames. Frame rate 10Hz. Imaged area: (27 x 27)µm 2. One individual cell is shown in the movie. During the first few fames of the movie the focus is adjusted until the interface between the cell membrane and the substrate is localized. Movie 3: LFA-1 nanoclusters are randomly distributed and exhibit mostly random diffusion on THP-1 cells seeded over GαHu/BSA patterns (5µm). Fluorescence spots correspond to LFA-1 labeled with L16-ATTO647N antibody. Movie composed out of 400 frames. Frame rate 10Hz. Imaged area: (27 x 27)µm 2. One individual cell is shown in the movie. During the first few fames of the movie the focus is adjusted until the interface between the cell membrane and the substrate is localized.