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

  1. Zhongwen Chen
  2. Dongmyung Oh
  3. Kabir Hassan Biswas
  4. Ronen Zaidel-Bar
  5. Jay T Groves

  • Curated by eLife

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    Evaluation Summary:

    These data provide in vivo evidence for a previously described kinetic-proofreading mechanism in phase-separated condensates. The strength is being able to compare the impacts of clustering of signaling molecules with a non-clustered control in the same cell. The limitations are that there is not necessarily new biological insight gained and the effects reported are surprisingly modest compared with expectations from reconstituted systems. This paper will be of broad interest to scientists who study membrane-associated cell signaling.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

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Abstract

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 1 µm, 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 both receptor phosphorylation and downstream signaling activity. Single-molecule imaging clearly resolves increased molecular binding dwell times at EphA2 clusters for both Grb2:SOS and NCK:N-WASP 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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  1. Version published to 10.7554/elife.67379 on eLife
  2. eLife

    Reviewer #3 (Public Review):

    In the manuscript entitled "Probe the effect of clustering on EphA2 receptor signaling efficiency by subcellular control of ligand-receptor mobility" by Chen and colleagues, the authors develop an innovative method to directly evaluate the effect of membrane receptor clustering on signaling. Using a fabrication system in which they are able to produce neighboring mobile and immobile substrates, the authors studied the effects of EphA2 receptor mobility in Grb2:SOS and Nck:N-WASP signaling pathways. The authors found that EphA2 clustering enhances signal transduction and results in increased dwell-time of signaling molecules on membranes, analogous to what has been observed in vitro with LAT and nephrin signaling clusters.

    This manuscript is well-constructed and provides the reader with an innovative tool to directly evaluate clustered vs. non-clustered receptor in a cellular context. The images present are well-analyzed and provide clear data that support many of the authors conclusions. Importantly, the data presented here directly shows the importance of Eph2A receptor clustering in a cellular context. However, this work and the conclusions regarding distinct physiochemical properties of clusters would be strengthened by direct comparisons of substrate:receptor densities and signaling molecules. This work offers new insight into Eph2A signaling mechanisms as well as new techniques that can be used to study numerous receptor tyrosine kinase signaling pathways. As such, this study will be of interest to a wide variety of readers who study membrane-associated signaling and phase separation.

  3. eLife

    Reviewer #2 (Public Review):

    The authors describe a system using lithographically patterned substrates that contain small patches or corrals, consisting of either supported lipid bilayer allowing free diffusion of a specific ligand ("mobile"), or PEG-derived regions in which the ligand is fixed ("immobile"). Areas around the patches are functionalized with RGD peptides to facilitate cell adhesion via integrins. Cultured cells are incubated on the patterned substrate, allowing direct comparison within the same cells of signaling responses (receptor phosphorylation, adaptor and effector engagement) to patches in which receptors cluster, vs. those in which clustering is not possible. An important feature is that the same amount of ligand (ephrin-1 in this case) is found in the mobile and immobile patches, allowing direct quantitative comparison between the two.

    The strength of this manuscript is in the experimental approach, which brings methodologies and precision more associated with in vitro reconstitutions, to studies of living cells. However the advantage of assaying clustered vs. unclustered patches in the same cell are also to some extent a disadvantage, in that it is not really possible to assess the impact on downstream signaling. In this sense it is somewhat disappointing that the investigators did not compare downstream effects in cells plated on patches where only immobile ligands are available vs. those where only mobile ligands are available (for example, Erk nuclear localization could serve as a downstream readout of Grb2/Sos engagement). If the relatively modest differences in receptor phosphorylation and in adaptor/effector recruitment seen in clustered vs. unclustered patches are really biologically meaningful, then we would expect to see significantly more nuclear Erk (on average) in cells where ephrins are mobile and allow clustering.

    Related to the former point, the authors suggest that there is so much cell-to-cell variability that they only were able to see an effect of clustering where mobile and immobile patches are clustered in the same cell. However, there appears also to be a great deal of variation in the behavior of patches within the same cell as well. It would be informative to see a quantitative analysis of the variation from patch to patch in the same cell vs. the variation in overall signals for different cells. If, as appears from the figures, there is indeed great variation from patch to patch in individual cells, that would be quite interesting and lead to future experiments to find the source of intracellular variability and its impact on downstream outputs.

    A second major question regarding these studies is the effect of time on both clustering and on signal output. Typically, tyrosine phosphorylation reaches a maximum very quickly (within a minute or two) upon stimulation of RTKs. Most of the data in these studies are recorded after much longer times, e.g. 30-60 minutes. I understand some of this is likely due to the time needed for cells to adhere to the patterned substrate, but downstream outputs such as Erk activation also tend to be relatively rapid, so inability to monitor differences between mobile/clustered and immobile ligands means the investigators may be missing the most important and relevant window for downstream signaling (and may actually be measuring cells at a time when feedback inhibition and receptor internalization dominate).

    It also would be quite useful in the spt-PALM experiments to see whether the apparent diffusion rate of tracked particles is different between the mobile and immobile patches. It has been suggested that SH2-containing proteins repeatedly rebind to membtanes with high local concentrations of phosphorylated receptors, so counterintuitively one might expect lower apparent diffusion for SH2 domains when receptors are mobile and clustered (where local concentrations of phosphorylated receptor are high) vs. when receptors are fixed, and rebinding is relatively inefficient. This data should already be available to the investigators, since all particles are tracked for the duration of observation.

    In conclusion, the strength of this manuscript is the nanofabrication approach allowing direct comparison in cells of signal outputs from clustered vs. unclustered receptors. The rigor of this approach provides new capabilities for understanding the role of clustering in signal processing. The weaknesses are in my view a lack of attention to downstream signal outputs (to assess how small differences in dwell times to clustered vs. unclustered receptors actually impact outputs), and a failure to take into account the dynamics of the response to receptor binding. These factors diminish the overall impact of these studies on our understanding of the precise role of clustering in information processing by RTKs.

  4. eLife

    Reviewer #1 (Public Review):

    One proposed function of biomolecular condensates is in controlling the membrane dwell time to modulate activity of signaling factors. This has been well quantified in vitro but is challenging to quantitatively demonstrate in cells because of substantial cell-to-cell variability in behavior. This is important because cell-to-cell variations can obscure quantitatively measuring the functional impacts of clustering.

    In this manuscript, Chen et al. describe the application and adaptation of a micro-patterned substrate that addresses this intrinsic heterogeneity problem by analyzing the activity from clustered (condensed) and non-clustered (diffuse) in the same cell. This was achieved by creation of discrete zones of mobile (membrane-bound) and immobile (polymer-bound) ligands. Using this substrate, the authors examine receptor clustering upon binding to mobile ligands. This clustering drives receptor phosphorylation, increases molecular dwell time of key downstream signaling effectors, and drives local actin polymerization. Importantly, this patterning of substrate allows the authors to de-couple the effects of receptor binding from receptor clustering within individual cells. While there is not a major new conceptual insight in the study, the technical platform allows for a critical quantitative analysis of kinetic proof-reading in signaling pathways that appear phase-separated in cells.

    The results are convincing and conclusions are well supported by the data. The impact of this work include providing quantitative, in vivo evidence for functions of membrane-associated condensates and may extends to other questions related to signaling where it is critical to assess diffusive versus non-diffusive ligands within the same cell.

  5. eLife

    Evaluation Summary:

    These data provide in vivo evidence for a previously described kinetic-proofreading mechanism in phase-separated condensates. The strength is being able to compare the impacts of clustering of signaling molecules with a non-clustered control in the same cell. The limitations are that there is not necessarily new biological insight gained and the effects reported are surprisingly modest compared with expectations from reconstituted systems. This paper will be of broad interest to scientists who study membrane-associated cell signaling.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

  6. Version published to 10.1101/2021.02.16.431455v1 on bioRxiv