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Real-time single-molecule co-immunoprecipitation analyses reveal cancer-specific Ras signalling dynamics

Co-immunoprecipitation (co-IP) has become a standard technique, but its protein-band output provides only static, qualitative information about protein–protein interactions. Here we demonstrate a real-time single-molecule co-IP technique that generates real-time videos of individual protein–protein interactions as they occur in unpurified cell extracts. By analysing single Ras–Raf interactions with a 50-ms time resolution, we have observed transient intermediates of the protein–protein interaction and determined all the essential kinetic rates. Using this technique, we have quantified the active fraction of native Ras proteins in xenograft tumours, normal tissue and cancer cell lines. We demonstrate that the oncogenic Ras mutations selectively increase the active-Ras fraction by one order of magnitude, without affecting total Ras levels or single-molecule signalling kinetics. Our approach allows us to probe the previously hidden, dynamic aspects of weak protein–protein interactions. It also suggests a path forward towards precision molecular diagnostics at the protein–protein interaction level.

To induce on-demand translocation of HRas to the plasma membrane, we used FKBP-FRB heterodimerization that can be triggered by rapamycin 13 . The FRB domain was fused to Lyn tyrosine kinase. Since the N-terminal 11 amino acids of Lyn contain sites for myristoylation and palmitoylation, Lyn-FRB is stably anchored to the plasma membrane.
The FKBP domain was fused to the CAAX-deleted HRas. When the rapamycin was added, the FKBP-conjugated HRas was captured by the Lyn protein through FKBP-FRB heterodimerization, which effectively translocated HRas to the plasma membrane. The protein-protein interaction between HRas and Ras binding domain of raf1 (cRaf RBD ) was assessed by seeing whether cRaf RBD was also translocated to the plasma membrane. (b) In order to analyze co-localization of HRas and cRaf RBD , we measured fluorescence intensity of a specific region of interest (ROI) inside cells. We designated ROI 1 and ROI 2, in which fluorescence intensity changes in the plasma membrane and cytoplasm were recorded, respectively. When constitutively active HRas (Q61L) was translocated to the plasma membrane, the fluorescence signal of HRas in ROI 1 increased by a factor of seven during 10 min imaging, which was accompanied by a decrease in fluorescence signal in ROI 2 to one sixth of the initial value. It is noted that the fluorescence signal of cRaf RBD showed concurrent increase in ROI 1 by a factor of six. When dominant negative HRas (S17N) was used for the same experiment, on the other hand, we observed similar level of HRas translocation to the plasma membrane. However, the cRaf RBD fluorescence levels in both ROI 1 and 2 largely remained unchanged, indicating a minimal interaction between cRaf RBD and dominant negative HRas. Figure S3. Conventional co-IP analyses of the HRas-cRaf RBD interaction. The conventional co-IP data in the right panel of Fig. 1b were reassessed by measuring the fluorescence signal of fluorescent protein tags. (a) For the co-IP analysis, the mCherry-HRas proteins, either in the constitutively active (CA) or dominant negative (DN) form, were immobilized on beads using the anti-mCherry antibodies. Fluorescence signals measured before and after incubation with beads and comparison with 150 nM recombinant mCherry proteins indicated that ~150 nM HRas proteins were immobilized on beads. A sample volume of 200 μl was used throughout the co-IP experiments. (b) The eGFP-cRaf RBD proteins were applied as the prey proteins. The fluorescence level of the original prey protein solution (200 nM eGFP-cRaf RBD ) was about 1.6×10 5 . After the 2 hour reaction at the prey protein concentration of 200 nM, the eGFP-cRaf RBD proteins that had been captured by the HRas proteins on beads were harvested by elution after thorough washing steps. The fluorescence signal of this eluted portion was higher for the reaction with CA HRas than that with DN HRas. However, even this elution portion produced by the CA HRas showed the fluorescence level of 4×10 3 , which corresponds to only 2.5% of the original prey proteins. Considering that a tight protein-protein binding would consume 75% of the prey proteins (150 nM baits/200 nM preys), this tiny portion of 2.5% captured during the co-IP process indicates a weak, transient interaction between HRas and cRaf RBD proteins. Figure S4. Controlling the mCherry-HRas immobilization number on surface. To assess the total immobilization number of bait proteins, we determined the slope for surface immobilization for different bait protein concentrations. The slope was determined by counting the numbers of immobilized single mCherry-HRas proteins while varying the mCherry-HRas concentration. For example, when an anti-mCherry antibody was used for capturing mCherry-HRas as in Figures 1 and 2, the slope was determined to be ~6 count per imaging area/[mCherry-HRas] when [mCherry-HRas] was given in pM.

Supplementary
Since one imaging area had an area of 4,050 μm 2 in our TIRF microscopy, we used 350 pM [mCherry-HRas] to locate on average 0.5 mCherry-HRas in each ROI (one ROI has an area of 1.1 μm 2 ). Since it is presumed that there are more than 10 5 antibodies in one imaging area, the first order approximation should hold well when we immobilize less than 10,000 baits on surface. Error bars denote S.D. (n=10). Figure S5. Analysis of real-time single-molecule co-IP traces. (a) To separate the diffuse background and fluorescence spikes in an objective way, we first obtained the fluctuation information using the reaction with dominant negative HRas. Since this negative control case showed few binding events during 1 min imaging, we were able to clearly separate fluctuations from the fluorescence spikes. The fluctuation was fitted to a Gaussian distribution, which yielded the standard deviation of fluctuation, σ. Moving average with a three point window was applied to real-time traces throughout analysis. (b) By increasing the prey protein concentration (eGFP-cRaf RBD ), we increased the background fluorescence to determine corresponding σ, which gave a calibration curve for σ as a function of the background fluorescence. Error bars denote S.D. (n>50). (c) To analyze the protein-protein interactions with constitutively active HRas, we first found the background fluorescence from each real-time trace and determined the relevant σ value using the calibration curve of (b). The 20 lowest fluorescence signals were averaged and used as the bottom limit of the individual trace. Six σ above the bottom limit was chosen as the confidence level, which should contain more than 99.9% of the fluctuation. Fluorescence signals that went past the confidence level were considered to be due to specific proteinprotein interactions.

Supplementary Figure S6. Co-localization study for the mCherry-HRas-eGFP-cRaf RBD interaction. (a)
We used the co-localization method for the HRas-cRaf RBD binding experiment described in Fig. 1. The positions of the pulled-down mCherry-HRas proteins were first identified using a 532 nm laser excitation, and the binding events of 20 nM eGFP-cRaf RBD were selectively recorded from the identified spots. To identify individual mCherry-HRas proteins with minimal overlap, we used a low surface HRas density of ~0.03 HRas per μm 2 , in which we had about 130 HRas proteins pulled down per our imaging area. When the mCherry-HRas proteins carried the active mutation (Q61L), approximately 70% of the detected mCherry spots showed any binding events of eGFP-cRafRBD. This percentage, on the other hand, dropped to 20% when the HRas proteins had the dominant negative mutation (S17N), meaning that 80% of the spots showed no binding events. We noted that even with the active mutation, the co-localization percentage was 70% and didn't reach 100%. These 30% spots seemed to be fluorescence junk. (b) We analyzed single-molecule kinetics from the identified co-localized spots. When we assessed the binding kinetics from the colocalized spots, the kinetic rates of k bind and k diss were measured to be 0.39 s -1 (τ off of 2.51 s) and 3.45 s -1 (τ on of 0.29 s), respectively, for the HRas with active mutation. But, the binding frequency, k bind , plunged to 0.063 s -1 (τ off of 15.87 s) when the dominant negative HRas proteins were used. All these kinetic rates obtained with the co-localization method are well comparable to those from (n>100).

Supplementary Figure S8. Distributions of the fluorescence spike intensity. (a)
Fluorescence spike intensity is quantitatively defined as the height of a fluorescence spike above the threshold. (b-d) All the histograms of fluorescence spike intensity, obtained at three different prey protein concentrations from 15 to 25 nM, display a unimodal peak at the same position of ~100, which is close to the single eGFP intensity. Nevertheless, we cannot completely rule out the possibility that there are multiple conformations for the final Ras-Raf complex because the histograms show relatively broad distributions around the main peak. However, in every histogram, there is no other appreciable peak that could be attributed to a distinct state. Moreover, both the dwell time kinetics for τ on and τ off can be fitted using single exponential functions ( Fig. 1 and 2). Thus, we have presumed one welldefined conformation state for the final Ras-Raf complex throughout our analysis.

Supplementary Figure S9. Numerical simulation on background fluorescence increase.
To investigate if multiple parallel binding events in a single ROI can produce a background fluorescence increase, we have constructed artificial real-time movies of co-IP imaging. In such a movie, a certain number of HRas molecules were distributed randomly on a virtual imaging area consisting of 256×512 pixels. On each HRas molecule, binding and unbinding events of single eGFP-cRaf RBD proteins were stochastically generated following single exponential probability distributions, time constants of which were τ off -1 of 1.77 s -1 and τ on -1 of 3.18 s -1 , respectively. A series of images were constructed by placing a two dimensional Gaussian point-spread-function (PSF) on each HRas that was then bound by an eGFP-cRaf RBD molecule. The intensity of a pixel was calculated by the equation, , where (i,j) is the position of the pixel under consideration, (x k , y k ) is the position of the kth PSF and σ is the standard deviation of the PSF. A is the peak intensity of the PSF that has been adjusted to match the experimental data from Figs. 1 and 2 (A=158.7 used). To simulate shot noise of imaging, a Poisson noise was added for each pixel. It should be noted that in this computer-generated movie, the fluorescence signals only come from the eGFP-cRaf RBD proteins in the final HRas-cRaf RBD complex, without any contribution from freely diffusing cRaf RBD s or encounter complexes.
The movies were generated as the total HRas numbers were increased up to 76,000, which corresponded to on average 20 HRas per μm 2 . In analyzing the movies, we used the same method described in Supplementary Figure  whereas 300% increase has been observed with 6.2 HRas per μm 2 in our experimental data in Fig. 2f. Thus, it is likely that the presence of multiple parallel binding events cannot be the main cause of the enhancement in background fluorescence, which has been experimentally observed during real-time co-IP. Error bars denotes S.D. (n>100). closely approximates 100%. This implies that the threshold for k bind inflation occurs when there are 2,400 active baits per imaging area, which corresponds to 0.6 active baits per μm 2 . It is noted that this threshold depends only on the geometric parameters of imaging apparatus, not on detailed molecular nature of surface baits. Different size of field of views and/or different algorithm for identifying binding events will shift the number of surface baits required for k bind inflation. But, once it is calibrated for a specific microscope, the threshold will be independent of the molecular nature of surface bait proteins. Error bars denote S.D. for total Ras immobilization (n=10) and S.E. for active binding site (n=2), respectively.

Supplementary
Supplementary Figure S14. Schematics of k bind inflation. Under a diluted condition where the average distance between active HRas is much larger than the diameter of ROI (upper left), the fluorescence spikes from each ROI faithfully reflect the binding events on a single HRas (lower left). The measured τ off,apparent should be identical to the genuine time constant of single HRas activity, τ off,single HRas . Since our imaging area is about 4,000 μm 2 while each ROI has an area of 1.1 μm 2 , the imaging area accommodates at least 1,000 active HRas molecules without significant overlapping of their ROIs (upper middle). The τ off,apparent is constant at the same value, continuously reflecting τ off,single HRas (lower middle).
This means that even when the total immobilization number of HRas is increased, the kinetic constant largely stays at the same value up to a certain point. However, if the total immobilization number is increased beyond a certain threshold, more than one active HRas molecules begin to be included in some of ROIs (upper right). These additional HRas molecules create additional fluorescence spikes (lower right, red spikes), which make τ off,apparent smaller than the genuine τ off,single HRas . As a result, the measured kinetic rate of k bind,apparent is expected show a biphasic behavior as a function of total HRas immobilization number. The k bind,apparent is initially maintained at a constant of k bind,single HRas , but begins to increase beyond the threshold. We note that this threshold for k bind,apparent corresponds to a specific surface density of active baits, which is independent of detailed molecular nature of bait proteins. Thus, comparing the thresholds can be directly used to assess the active portion of bait proteins on surface.
Supplementary Figure S15. k diss observed for native signaling Ras. Unlike k bind that shows inflation past the thresholds ( Fig. 3l and Fig. 4c), k diss is largely constant for every HRas and KRas sample we studied. We note that τ on values are in the range of hundreds of ms, showing that the rapid turnover in Figs. 1 and 2 is also observed for the native Ras pulled down from both tumor and control tissues. for MDA-MB-231 and 9.7 nM for MCF-7 in 1 mg/mL cell extracts, respectively. These values are little bit lower than those from our single-molecule western analysis from Fig. 4 (5.5 and 17.3 nM), which seems to be due to loss of proteins during transfer to PVDF membrane prior to immunoblotting. (c) We however note that the Ras-concentration ratio of MCF-7 to MDA-MB-231 is 3.03 (9.7 nM/3.2 nM), which is very close to the value of 3.14 (17.3 nM/5.5 nM) estimated by the single-molecule western analysis. This reconfirms our important finding that the mutant KRas in MDA-MB-231 cells has a smaller concentration than the wild-type KRas in MCF-7 cells.