Probe the effect of clustering on EphA2 receptor signaling efficiency by subcellular control of ligand-receptor mobility

Clustering of ligand:receptor complexes on the cell membrane is widely presumed to have functional consequences for subsequent signal transduction. However, it is experimentally challenging to selectively manipulate receptor clustering without altering other biochemical aspects of the cellular system. Here, we develop a microfabrication strategy to produce substrates displaying mobile and immobile ligands that are separated by roughly one micron and thus experience an identical cytoplasmic signaling state, enabling precision comparison of downstream signaling reactions. Applying this approach to characterize the ephrinA1:EphA2 signaling system reveals that EphA2 clustering enhances receptor phosphorylation. Single molecule imaging clearly resolves increased molecular binding dwell time at EphA2 clusters for both Grb2:SOS and NCK:NWASP signaling modules. This type of intracellular comparison enables a substantially higher degree of quantitative analysis than is possible when comparisons must be made between different cells and essentially eliminates the effects of cellular response to ligand manipulation.


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The cell membrane surface is studded with a broad array of receptor proteins that interact with 33 numerous ligands, which can be soluble, membrane-bound on an apposed cell surface, or 34 associated with the extracellular matrix ( factor for modulating signaling activity (Bray et al., 1998;Cebecauer et al., 2010). 46 More recently, receptor clustering in some cases has also been found to involve downstream 47 signaling molecules that appear to undergo phase transitions (  We then monitored the interaction of live cells with ephrinA1 on the supported membrane corrals.  135   MDA-MB-231 breast cancer cell line was used as a model cell here, since it expresses EphA2 at a high  136 level and has been characterized previously on ephrinA1 functionalized supported membranes (Chen et  surface density on supported membranes was calibrated with quantitative fluorescence measurement 139 (Galush et al., 2008) (Fig. S2), and was controlled to be around 100 molecules /μm 2 in 4 μm-diameter 140 membrane corrals for these experiments. Cells seeded on the micropatterned substrate spread 141 presumably through engagement of RGD ligands on the substrate with integrins on the cell membrane. 142 This dynamic spreading enabled cell membrane protrusions to physically interact with multiple ephrinA1 143 membrane corrals on the substrate, resulting in the clustering of diffusive ephrinA1 ligands through 144 binding of cellular EphA2 receptors (Salaita et al., 2010) ( Fig. 1C and Movie 1 ). The clustering of 145 ephrinA1 is relatively fast: almost all the ligands inside each corral were clustered within 6 min of 146 physical contact by the cell membrane, leading to the formation of typically a micro-cluster in each of 147 the corrals (Fig. 1C). Therefore, this system allows for spatiotemporally resolved observation of 148 ephrinA1 interaction with EphA2, as well as its downstream signaling events. The supported membranes 149 in the micropatterned substrates could also be functionalized with other ligands, for example, E-150 cadherin to form cadherin junctions (Fig. S3), demonstrating versatility of this technology to study 151 different receptors. 152 Spatially segregated display of mobile and immobile ephrinA1 for single cells 153 Next, the substrates were extended to contain both mobile and immobile ephrinA1 to compare the 154 effects of ligand clustering on receptor signaling (Fig. 1D). For this, PLL-(g)-PEG-biotin coated glass 155 substrate was selectively UV-etched to coat another polymer PLL-(g)-PEG-NTA. This hybrid 156 substrate then underwent a second UV etching for supported membranes, which led to a three-157 component substrate (Fig. S4A). The sequential UV etching processes can be overlaid randomly for 158 regular circular arrays, because it is able to generate sufficiently large areas with clearly separated 159 mobile and immobile regions in a centimeter-size pattern. However, the glass coverslip and the 160 photomask can also be aligned under microscope before each UV exposure for accurate layouts of 161 multiple components as needed, at a cost of extra time and alignment instrument (Fig. S4B). 162 EphrinA1 was functionalized on both PLL-(g)-PEG-NTA polymers and supported membranes 163 through the same Ni-NTA-poly-histidine interactions, while RGD was functionalized on PLL-(g)-PEG-164 Biotin to allow cells to spread. Fig. 1E shows an example of cells spreading on the substrate with 165 alternating mobile ephrinA1 membrane corrals and immobile ephrinA1 polymers. Clearly, only 166 mobile ephrinA1 became clustered after contact with cells. Importantly, the molecular density of 167 ephrinA1 on the polymer could be titrated by mixing PLL-(g)-PEG-NTA with non-reactive PLL-(g)-168 PEG (Fig. 1F). We controlled ephrinA1 density to be similar in both mobile and immobile regions at 169 a range of 50-100 molecules /μm 2 for all cell experiments. 170

Mobile ephrinA1 increases EphA2 phosphorylation through clustering 171
Display of mobile and immobile ephrinA1 on micron-scale corrals (membrane and polymer, respectively) 172 allowed for comparison of clustered and non-clustered EphA2 receptor signaling in a spatially resolved 173 manner in individual cells. Immunostaining of cells using an antibody against the intracellular domain of 174 EphA2 verified engagement of the receptors to both mobile and immobile ephrinA1 ( Fig. 2A). Although 175 EphA2 cluster formation is dependent on the ligand mobility, the total amount of EphA2 receptors 176 recruited to mobile or immobile ephrinA1 were very similar, with a slight decrease in mobile ephrinA1 177 area possibly due to endocytosis (Greene et al., 2014;Sugiyama et al., 2013), suggesting a conservation 178 of binding between the receptors and ligands at the cell: substrate interface (Fig. 2B). EphA2 is known to 179 undergo a ligand binding-induced autophosphorylation at tyrosine 588, and this phosphorylation site is 180 key to the recruitment of downstream signaling molecules (Parri et al., 2005). Immunostaining of cells 181 using an anti-pY588-EphA2 specific antibody showed that EphA2 phosphorylation increased by an 182 average of 60% in response to mobile ephrinA1 stimulation compared to immobile stimulation ( Fig. 2A  183 and 2C). These results suggest that clustering of ephrinA1:EphA2 complexes enabled by the mobile 184 ligands resulted in a change in their physicochemical properties and the level of receptor activations.  (A) Representative immunofluorescent images of EphA2 or pY588-EphA2 in MDA-MB-231 cells fixed after 45 mins spread on the substrate. Quantification of (B) EphA2 / EphrinA1 intensity ratio or (C) pY588-EphA2 / EphrinA1 intensity ratio in mobile and immobile ephrinA1 corrals. Each data point represents an averaged ratio from multiple corrals from a single cell. The two grouped data from the same cell are paired for comparison. Significance is analyzed by paired-group student's t test. N = 22 or 26 cells respectively.

EphA2 clustering enhances Grb2:SOS signaling transductions by increasing on-rate and molecular 186 dwell time 187
Increased phosphorylation of EphA2 observed on mobile ephrinA1 membrane corrals is expected to lead 188 to increased recruitment of intracellular effector proteins. Therefore, we monitored the local signaling 189 transmission events in clustered or non-clustered EphA2 receptors in single living cells. Grb2 is an 190 important cytosolic adaptor protein that is known to be recruited to EphA2 receptors after ligand 191 binding-induced phosphorylation (Pratt & Kinch, 2002). Grb2 further recruits SOS, which catalyzes Ras-192 GDP to Ras-GTP exchange, and activates the MAPK pathway (Fig. 3A). For this, MDA-MB-231 cells 193 transfected with Grb2-tdEos were seeded on the substrates to allow live imaging of Grb2 recruitment by 194 total internal reflection fluorescence (TIRF) microscopy (Movie 2). A remarkable difference was 195 observed in the local recruitment of Grb2 to mobile or immobile ephrinA1:EphA2 complexes as cells 196 spread ( Fig. 3B and 3C). For the same number of ephrinA1 molecules, the mobile ligands increased Grb2 197 recruitment by about 80% compared to immobile ones both in terms of maximal ( Fig. 3D) and 30 mins 198 cumulative signals (Fig. 3E), which is more prominent than the enhancement in receptor 199 phosphorylation described above. confirmed to be single molecules as shown by single-step photobleaching, and the fluorescence 208 intensities of individual particles also exhibited a unimodal distribution (Fig. S5). The coordinates of 209 every Grb2 molecule in a continuous movie were then assembled to generate a high-resolution 210 localization image (also termed single particle tracking Photo-Activation Localization Microscopy;  (A) Schematic illustration of Grb2 or SOS recruitment to mobile or immobile EphA2 receptors under the same cell. (B) Representative live cell images of a Grb2-tdEos transfected cell spreading on the substrate after 45 mins, with white circles indicating mobile ephrinA1 corrals and cyan circles indicating immobile ephrinA1. A yellow square marked region is enlarged to highlight the differential Grb2 recruitment to a mobile ephrinA1 corral and an immobile one. (C) Heat map of temporal Grb2-tdEos intensities in 3 cells. Each block represents the normalized intensity of Grb2 in an ephrinA1 corral at a given time, starting from cell contact to a total period of 30 mins, at 30 sec/frame acquisition speed. The intensity of Grb2 is normalized according to its highest intensity of all corrals through the whole time period for each cell, and color-coded for visualization. Quantification of (D) maximum Grb2 intensity or (E) 30 mins cumulative intensity in mobile or immobile ephrinA1 corrals, normalized with ephrinA1 ligand intensity in each corral. Significance is analyzed by student's t test. (F) Single molecule imaging of Grb2-tdEos or SOS-tdEos. The coordinates of Grb2 or SOS single molecules when they first appear in a continuous movie are assembled to generate a localization image. (G) Distribution of Grb2-tdEos single molecule dwell time. Membrane located CAAX-tdEos is applied as a photobleaching control measured from another cell. Quantification of (H) Grb2-tdEos or (I) single molecule dwell time. The dwell time distribution is fitted by a 2-order exponential decay function and the slower time constant τ2 is used to represent characteristic dwell time for pairwise comparison in a group of cells. N = 5 cells. Significance is analyzed by paired-group student's t test.
sptPALM (Manley et al., 2008)), which clearly showed clustering of Grb2 in mobile ephrinA1 corrals 212 while relatively uniformly distributed in immobile corrals (Fig. 3F). By mask-separating these two 213 regions, we found that Grb2 has a longer dwell time distribution on clustered ephrinA1-EphA2 214 complexes, in comparison to the non-clustered ones (Fig. 3G). A membrane localized CAAX-tdEos 215 control was used to measure the photobleaching rate under the same experimental condition, which 216 was significantly slower than the apparent Grb2 binding dynamics. The dwell time difference is 217 consistent in all measured cells as shown by pairwise comparison of mobile or immobile regions in each 218 individual cell (Fig. 3H). Notably the cell-to-cell variations of dwell time are in the similar level to the 219 mobile-to-immobile differences; and therefore, only side-by-side comparison of each individual cell 220 made it possible to detect the increase of Grb2 membrane dwell time induced by clustering. 221 Similar to the observations with Grb2, EphA2 clustering also consistently increases SOS-tdEos single 222 molecule dwell time on the membrane ( Fig. 3F and 3I). Taken together, these data indicate that Here we sought to test if such molecular timing mechanism applies to EphA2 234 signaling in living cells (Fig. 4A). 235  Significance is analyzed by paired-group student's t test.
Similar to Grb2, NCK-mEOS3.2 and NWASP-mEOS3.2 were clearly found to be enriched at 236 ephrinA1:EphA2 clusters in comparison to immobile ephrinA1 region (Fig. 4B). Live imaging of F-tractin-237 EGFP showed local actin polymerization in each mobile ephrinA1 corrals shortly after cluster formation; 238 however, no enrichment of F-actin can be resolved in regions of immobile ephrinA1 (Fig. 4C and movie  239   4). The average maximal intensity of F-tractin-EGFP increased by about 80% in EphA2 clusters compared 240 with immobile corrals (Fig. 4D), showing that only clustered EphA2 are effective to trigger signaling 241 transduction towards actin polymerizations. Single molecule imaging confirmed clustering of NCK-242 mEos3.2 and NWASP-mEos3.2 in mobile ephrinA1 corrals (Fig. 4E). The molecular dwell time also 243 increased consistently in all measured cells in EphA2 clusters ( Fig. 4F and 4G). Therefore the live cell 244 measurement is in agreement with in vitro reconstitution experiments (Case et al., 2019), that the 245 increased dwell time of NWASP by crosslinking is important for its activation. 246

EphA2 clustering increases Grb2 and NWASP dwell time in COS7 cells 247
The model cell line MDA-MB-231 expresses a very high level of EphA2 receptors, which may raise 248 concern whether the observed dwell time difference is due to EphA2 pre-clustering. We validated these 249 results by testing another cell line COS7, which expresses EphA2 at a low to moderate level (Sabet et al., 250 2015). EphrinA1 clustering and subsequent cytosolic Grb2 and NWASP recruitment upon cell contact is 251 readily observed in mobile membrane corrals (Fig. 5A). The clustering consistently increases Grb2-tdEos 252 dwell time in all measured cells (Fig. 5B), and increases NWASP-mEOS3.2 dwell time in most cells only 253 with a rare exception (Fig. 5C).  An Eclipse Ti inverted microscope (Nikon) with a TIRF system and Evolve EMCCD camera (Photometrics) 347 was used for live cell imaging. TIRF microscopy was performed with a 100x TIRF objective with a 348 numerical aperture of 1.49 (Nikon) and an iChrome MLE-L multilaser engine as a laser source (Toptica 349 Photonics). Immunofluorescent imaging was also acquired in an Eclipse Ti inverted microscope (Nikon) 350 with CSU-X1 confocal spinning disk unit (Yokogawa). 351 Time-lapse single molecule imaging of Grb2-tdEos, SOS-tdEos, NCK-mEos3.2 and NWASP-mEos3.2 were 352 performed by TIRF microscopy, in a way such as to optimize signal-to-noise and temporal resolution by 353 coupling minimizing laser power and maximizing video rate. To increase tracking accuracy, the density of 354 individual molecules was controlled by 405 nm laser illumination to be about ~0.5 / µm 2 . Far-red 355 channel (ex =647 nm, em > 655 nm) were acquired before single molecule recording to localize mobile 356 and immobile ephrinA1 corrals. The autofluorescence on the red channel was completely 357 photobleached before photo-switching Eos by a 405 nm beam. After photo-switching, a small amount of 358 Eos molecules were visualized and recorded by EMCCD with 20 frame per second video rate. Each movie 359 contains 1000 frames for further analysis. Membrane localized CAAX-tdEos movies were used to 360 calculate photobleaching rate, acquired at the same microscopic set up. 361

Image analysis 362
Live cell and immunofluorescence images were analyzed to quantify Grb2-tdEos, F-tractin-EGFP, anti-363 EphA2, and anti-pY588-EphA2 intensities in mobile and immobile ephrinA1 regions. The regions of 364 mobile and immobile ephrinA1 were outlined to generate masks, so the average intensity of different 365 channels can be measured in the same corral and the ratios were calculated after subtraction of noises. 366 For live cell single particle tracking, a cross-correlation single particle tracking method was used to 367 determine the centroid positions of tdEos or mEos3.2 single molecules (Oh et al., 2012;Oh et al., 2014). 368 A trajectory was created by connecting the subsequent xy coordinates through the frames using the 369 nearest neighbor method. The first positions of each trajectory were assembled to generate a 370 localization image. By using pre-acquired ephrinA1 image as a mask, single molecule signals coming 371 from mobile or immobile ephrinA1 regions were separated to calculate spatially resolved binding 372 kinetics. The dwell time distributions of molecules in the two different regions were fitted with a 2-order 373 exponential decay function y=y0+A1e -t/τ1 + A2e -t/τ2 , providing two characteristic time constants τ1 and τ2. 374 To be consistent τ2 were used for pairwise comparison of each cells.    (B) Versa�le layouts of the three-component substrate by microscopy assisted alignment. Before each UV etch process, the maker on the polymer coated coverslip and the one on the photomask are aligned at the same posi�on so that the three components layout can be controlled as design.