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PLCγ1 promotes phase separation of the T cell signaling clusters

Longhui Zeng, Ivan Palaia, Anđela Šarić, Xiaolei Su

Preprint posted on July 01, 2020 https://www.biorxiv.org/content/10.1101/2020.06.30.179630v1.article-info

Not just a signalling protein - PLCɣ1 can regulate T cell receptor signalling clusters by controlling cluster size and formation

Selected by Sruthi S Balakrishnan

Introduction

Activation of T cell receptors (TCR) at the immunological synapse is known to result in the formation of signalling microclusters. These clusters comprise of the TCR, along with a host of adaptor and effector proteins such as ZAP70, Grb2, and LAT. The clusters show a key characteristic of liquid-liquid phase separation, a feature that marks them as distinct microdomains within the plasma membrane [1].

The formation and maintenance of these clusters has been well-studied over the years [2]. Scientists have identified several protein players involved in driving downstream signalling cascades as well. While we know a lot about the process of cluster formation, there are still open questions about its regulation. The specific functions of many cluster proteins are not clear [3]. This preprint answers one of these questions.

An important member of TCR microclusters is the enzyme phospholipase C gamma 1 (PLCɣ1), that drives downstream signalling through its enzymatic activity. However, this is not its only function. Using a combination of experiments and theoretical modelling, this preprint shows that PLCɣ1 regulates cluster formation through interactions with LAT, a key component of the TCR microcluster.

Key Results

Given that PLCɣ1 is known to bind to the adaptor protein LAT, the focus of the experiments was on the interactions of these two proteins and consequent effects on cluster formation.

The researchers did their first set of experiments in vitro, using supported lipid bilayers. Adaptor proteins Grb2 and Sos1 were used to induce clustering of LAT. When Grb2 was replaced by PLCɣ1, cluster formation still happened. However, the size, number, and dynamics of PLCɣ1-induced LAT clusters differed from the Grb2-induced clusters in that they were smaller, more numerous, and less dynamic.

They next probed the domain-specific interactions of PLCɣ1 and LAT using PLCɣ1 truncation mutants lacking either the nSH2, cSH2, or SH3 domains. While the SH3 domain was found to be dispensable for clustering, the SH2 domains were found to be required as they each interact with specific phospho-tyrosines on LAT.

The scientists then looked at how PLCɣ1 influences clustering efficiency. They found that seeding a lipid bilayer already containing Grb2, Sos1, and LAT with very small amounts of PLCɣ1 could significantly speed up cluster formation. By acting as a cross-linker of LAT, PLCɣ1 enhanced clustering induced by Grb2 and Sos1.

A curious observation was made during the above experiments – the addition of PLCɣ1 influenced cluster formation in a non-monotonic manner. When increasing concentrations of PLCɣ1 were added to the bilayer, the enzyme only enhanced clustering until a certain point. Beyond this, at higher concentrations, both cluster formation and size were reduced by PLCɣ1 addition.

To try and explain this, the researchers teamed up with theoretical scientists. Using a minimal coarse-grained model, they simulated LAT, Sos1, Grb2, and PLCɣ1 as two-dimensional particles. When they recreated clustering in silico, they were able to replicate the same non-monotonic effects seen in experiments. By accounting for the influence of PLCɣ1 on the likelihood of clusters merging and the compactness of a cluster, they were able to offer an explanation for the non-monotonic effect.

At low concentrations, the cross linking effect of PLCɣ1 with LAT and Sos1 opens up the latter two molecules to merge with other clusters, allowing the formation of bigger clusters. At higher concentrations, however, PLCɣ1 saturates the binding sites on LAT and Sos1, reducing the merging potential and hence, cluster size. This saturation effect also makes clusters containing PLCɣ1 more compact and stable, possibly explaining the slow dynamics of PLCɣ1-induced clusters.

The scientists then recreated their experiments in Jurkat T cells. They found that cells lacking PLCɣ1 still formed clusters, albeit at far lower rates than wild-type cells. They also observed similar deficiencies in cluster formation in PLCɣ1-null cells reconstituted with constructs lacking either the nSH2 or SH3 domains. This nicely recapitulated their observations from the lipid bilayer experiments.

Lastly, the authors examined whether PLCɣ1 regulates cluster stability by protecting LAT from dephosphorylation. Phosphorylation of LAT by ZAP70 is a key event in the initiation of cluster formation. Previous reports have also shown that LAT microclusters are conspicuously devoid of CD45, an abundant plasma membrane phosphatase [1]. In cells lacking PLCɣ1, one of the phospho-tyrosines on LAT, which exclusively binds PLCɣ1, was found to be less phosphorylated. Overexpression of a PLCɣ1 fragment containing the nSH2 and SH3 domains increased said phosphorylation levels. It appeared that PLCɣ1 regulates phosphorylation of LAT in a bidirectional manner.

Thus, the scientists were able to identify a role for PLCɣ1 in enhancing cluster formation by binding to LAT and potentially protecting it from dephosphorylation.

Why I Chose This Preprint

This preprint uses a nice mix of experimental and theoretical methods to answer a nuanced question. PLCɣ1 has traditionally been thought to be a relatively passive member of the TCR signalling cluster, present mostly to activate downstream signalling via DAG and IP3 production.

This paper uses lipid bilayers to confirm initial hypotheses, and then further validates them using cell lines. They also use theoretical models to try and explain unusual observations. This represents a more holistic approach to answering scientific questions, harnessing the strengths of different methodologies and using the outputs in a complementary manner.

Questions

  • Your final model for PLCɣ1 involvement proposes that eventually, activation of its lipase function leads to the enzyme falling off of the cluster. What is the timescale for such activation and falling off? Does it roughly correspond to the lifespan of a cluster?
  • Given that LAT phosphorylation is one of the first steps in cluster formation, isn’t it curious that PLCɣ1 appears to increase LAT phosphorylation? Does it mean that PLCɣ1 is involved even before the cluster initiation? Or does the increased phosphorylation happen after cluster initiation?
  • In Fig. S1A, it looks like the truncated PLCɣ1, with only the SH2 and SH3 domains, is better at forming clusters than the full-length PLCɣ1. Is this because the cSH2 can stay bound to LAT without interference from the PLCɣ1 core? Or is the truncated protein just better at cross-linking because of lower steric hindrances?
  • In Fig. S2C, the truncated PLCɣ1 (with just SH2 and SH3 domains), does not seem to have the non-monotonic effect on cluster formation that the full-length PLCɣ1 has. Is this because of different concentrations/ratios of adaptor proteins?
  • The theoretical model offers plausible explanations for the experimental observations. Does it also make some testable predictions about the nature of interactions between the four proteins?
  • One of the current models for LAT cluster formation proposes that plasma membrane LAT is used for initiation of clusters, whereas vesicular LAT is used in later stages [4]. How does PLCɣ1 factor into this model? Has it fallen off the cluster by the time the vesicular LAT has entered the picture?

References

  1. Su, X., Ditlev, J. A., Hui, E., Xing, W., Banjade, S., Okrut, J., … & Vale, R. D. (2016). Phase separation of signaling molecules promotes T cell receptor signal transduction. Science, 352(6285), 595-599.
  2. Balagopalan, L., Kortum, R. L., Coussens, N. P., Barr, V. A., & Samelson, L. E. (2015). The linker for activation of T cells (LAT) signaling hub: from signaling complexes to microclusters. Journal of Biological Chemistry, 290(44), 26422-26429.
  3. Gaud, G., Lesourne, R., & Love, P. E. (2018). Regulatory mechanisms in T cell receptor signalling. Nature Reviews Immunology, 18(8), 485-497.
  4. Balagopalan, L., Yi, J., Nguyen, T., McIntire, K. M., Harned, A. S., Narayan, K., & Samelson, L. E. (2018). Plasma membrane LAT activation precedes vesicular recruitment defining two phases of early T-cell activation. Nature communications, 9(1), 1-17.

Tags: immunological synapse, microdomain, rafts, t cell

Posted on: 7th September 2020

doi: https://doi.org/10.1242/prelights.24386

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