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1 Nanomechanical mapping of first binding steps of a virus to animal cells David Alsteens, Richard Newton, Rajib Schubert, David Martinez-Martin, Martin Delguste, Botond Roska and Daniel J. Müller This PDF file includes: Supplementary Figs. 1 to 9 Caption for Supplementary Movie 1 Other Supplementary Information for this manuscript includes the following: Supplementary Movie 1 NATURE NANOTECHNOLOGY 1

2 Supplementary Fig. 1 Validation of MDCK-TVA cells and EnvA-RABV(ΔG) virusbinding, transduction and AFM tip functionalisation. a-c, Confocal microscopy of MDCK cells expressing TVA-mCherry (MDCK-TVA cells, red), transduced with cytoplasmic egfp (green) and nuclei stained with DAPI (blue). a, Full projection, b, bottom slice, and c, top slice. d-f, Flow cytometry data of d, wild-type MDCK cells incubated with EnvA-RABV(ΔG::eGFP), of e, MDCK-TVA cells not incubated, and of f, MDCK-TVA cells incubated with the virus. The mcherry channel is plotted against the egfp channel. Insets show fluorescence images. Scale bars, 50 µm. g, Virus-binding assay. Confocal microscopy of co-cultured wild-type MDCK and MDCK-TVA cells (red). MDCK cells were incubated with EnvA-RABV(ΔG::eGFP), fixed washed and stained with anti-enva and egfp secondary antibodies (green). Nuclei were stained with Hoechst (blue). Confocal microscopy of an AFM tip h, functionalised with EnvA- RABV(ΔG) (see green spots in the inset) or i, with BSA. The functionalised tip was brought in contact with a confluent layer of mixed wild-type MDCK and MDCK-TVA (red) cells and stained with anti-enva and egfp secondary antibodies (green). Experiments were reproduced three times with similar results. 2 NATURE NANOTECHNOLOGY

3 SUPPLEMENTARY INFORMATION Supplementary Fig. 2 The combined confocal microscopy and AFM chamber keeps animal cells alive for several days. MDCK cells expressing egfp-labelled histones (H2B-eGFP, green) were plated on glass Petri dishes and grown for 3 days in the combined confocal microscopy and AFM chamber (see Methods). After day 3, time-lapse series of confocal images were taken for 14 hours. The images taken every 60 min show MDCK cells continuously dividing (arrows). The chip and AFM cantilever are visible in the darker upper rim of each of the images. MDCK cells NATURE NANOTECHNOLOGY 3

4 were kept at 37 C in DMEM medium and under humidified synthetic air supplemented with 5% CO 2 (see Methods). Experiments were reproduced three times. Figure relates to Supplementary Movie 1. 4 NATURE NANOTECHNOLOGY

5 SUPPLEMENTARY INFORMATION Supplementary Fig. 3 Scanning electron microscopy (SEM) of AFM tips functionalised with rabies viruses. a, SEM images of glass surfaces functionalised with EnvA-RABV( G) viruses. b, Imaged at higher resolution the characteristic bullet-shaped morphology of the rabies virus ( 75 nm 180 nm) is observed. c, Chip of an untreated AFM cantilever showing the pyramidal AFM tip being about 17 µm high. d, At higher resolution the SEM images show the small tip which has been added to top of the pyramidal tip to increase the lateral resolution of the AFM images. e and f, Side views of functionalised AFM tips showing either no virus attached (e) or a single virus attached close to the tip apex (f). Glass surfaces and AFM cantilevers (PFQNM- LC probes, Bruker) were functionalised and SEM was performed as described in the Methods. We found that critical point drying (CPD) could be applied to viruses attached to glass surfaces (a,b) but not to cantilevers (c-f). The reason was that the turbulent mixing of the liquid ethanol/co 2 during CPD introduced too large movements of the special cantilever design (c), which mechanically destroyed their tips. For this reason we dehydrated virus functionalized cantilevers before SEM imaging by placing them in a hexamethyldisilazane bath and evaporating the solvent at 37 C. The structural integrity of the virus might be slightly less preserved using this method compared to CPD. NATURE NANOTECHNOLOGY 5

6 Supplementary Fig. 4 Mapping EnvA-RABV( G) virus binding to MDCK-TVA cells using correlative confocal microscopy and FD-based AFM. Investigated is a mixed culture of wildtype MDCK cells and MDCK-TVA cells (red) grown for 3 days before data acquisition. The TVA-mCherry fusion construct allows MDCK-TVA cells to be distinguished from wild-type cells and serves as an internal control for evaluating the specificity of the virus binding experiments. Confocal fluorescence microscopy (1 st column) and FD-based AFM height images (2 nd column) recorded from the inset shown in the 1 st column. Adhesion images simultaneously obtained with FD-based AFM height images are shown in the 3 rd column. z-scales are given for AFM height images and adhesion maps. AFM images were acquired using an oscillation frequency of 0.25 khz and amplitude of 500 nm, under cell culture conditions (37 C, DMEM medium and humidified atmosphere supplemented with 5% CO 2, see Methods). 6 NATURE NANOTECHNOLOGY

7 SUPPLEMENTARY INFORMATION Supplementary Fig. 5 Consecutive mapping of EnvA-RABV( G) virus-binding to MDCK- TVA cells show similar results. FD-based AFM height images (1st column) and corresponding adhesion channels (2nd column) show similar results for two consecutive maps indicating that the virus was firmly attached to the tip and not degraded over time. AFM images were acquired using an oscillation frequency of 0.25 khz and amplitude of 500 nm, under cell culture conditions (see Methods). Experiments were reproduced ten times. Additional examples are given in Supplementary Fig. 4. NATURE NANOTECHNOLOGY 7

8 Supplementary Fig. 6 Extraction of average rupture forces for discrete loading rates. a, Dynamic force spectroscopy (DFS) plot showing the force required to separate the virus from the cell surface. The forces of single virus-receptor rupture events (circles) are plotted against the LR and are taken from Fig. 4g. b, Small LR ranges #1 #5 are binned and the distributions of the rupture forces are plotted as histograms. This classification reveals multiple force peaks with average values corresponding to single (red), double (green) or triple (blue) simultaneously established virus-receptor interactions. The average values of the force distributions are extracted and plotted on the DFS plot c, enabling their analysis using the Bell-Evans model (see Fig. 4g). Error bars denote S.D.. 8 NATURE NANOTECHNOLOGY

9 SUPPLEMENTARY INFORMATION Supplementary Fig. 7 Exploring the virus-binding free-energy landscape at close-toequilibrium. a, To explore the rupture force of the bond formed between virus and cell surface receptor at close-to-equilibrium the AFM tip was moved at very slow speeds of nm s 1. Therefore the AFM tip was oscillated at low frequencies of Hz and ramp size of 1,000 nm. b, The LR is extracted via the slope of the force-time curve and c, the effective spring constant is measured using the slope of the force vs piezo movement curve. Using this approach we determined the rupture forces of the virus-receptor bonds at lower LRs (Fig. 5a). The data is used to explore the close-to-equilibrium binding strengths between EnvA-RABV(ΔG) and TVA receptor. NATURE NANOTECHNOLOGY 9

10 Supplementary Fig. 8 Estimating the effective spring constant of the cantilever-peg 27 linker-virus-cell system. a, The effective spring constant k eff is the combination of the cantilever stiffness k C, the PEG 27 stiffness of the linker k L, the stiffness of the virus k virus and the stiffness of the cell surface k cell. b, Calculating the effective stiffness of the PEG 27 linker changing with the stretching force F in water. The stiffness was calculated using the PEG 27 elasticity model 1. Taking into account the stiffness of the cantilever (calibrated using thermal tune) gave the stiffness of the PEG 27 linker and cantilever k C-L (blue curve). Further considering the stiffness of cantilever, linker, and cell provided k C-L-cell (green curve). k virus was neglected from the estimation since we found no value in the literature. The effective spring constants k eff (circles) extracted from the experimentally recorded force curves (shown in Supplementary Fig. 6) lie below the values estimated for k C-L-cell (green curve). c, Zoom in of the gray region indicated in b. The calculations show that to model the effective spring constant of the cantilever-linker-virus-cell system, requires inclusion of the stiffness of the virus, if available. 10 NATURE NANOTECHNOLOGY

11 SUPPLEMENTARY INFORMATION Supplementary Fig. 9 Fitting the dynamic force spectrum of the rupture forces indicates the formation of one, two and three correlated bonds between virus and cell surface receptors. Solid lines fitting the data taken from Fig. 4g were calculated using the Bell-Evans model 2. The red line describes the rupture of a single bond, the green line the rupture of two correlated bonds and the blue line the rupture of three correlated bonds. Dashed lines were obtained by fitting the data using the Williams-Evans model, which describes the force-induced rupture of two (green) and three (blue) uncorrelated bonds. NATURE NANOTECHNOLOGY 11

12 Movie 1 The combined confocal microscopy and AFM chamber keeps animal cells alive for several days. MDCK cells expressing egfp-labelled histones (H2B-eGFP, green) were plated on glass Petri dishes and grown for 3 days in the combined confocal microscopy and AFM chamber. After day 3, time-lapse series of confocal images were taken for 14 hours. The movie is composed of images taken every 60 min and shows MDCK cells continuously dividing. The AFM chip and cantilever are visible in the darker upper rim of each of the images. MDCK cells were kept at 37 C in DMEM medium and under humidified synthetic air supplemented with in DMEM medium and under humidified synthetic air supplemented with 5% CO 2 as described in the Methods. Experiments were reproduced three times. Movie relates to Supplementary Fig. 2. References 1. Sulchek, T., Friddle, R.W. & Noy, A. Strength of multiple parallel biological bonds. Biophys. J. 90, (2006). 2. Evans, E. & Ritchie, K. Dynamic strength of molecular adhesion bonds. Biophys. J. 72, (1997). 12 NATURE NANOTECHNOLOGY