Close

PLCγ1 promotes phase separation of the T cell signaling clusters

Longhui Zeng, Ivan Palaia, Anđela Šarić, Xiaolei Su

Preprint posted on 1 July 2020 https://www.biorxiv.org/content/10.1101/2020.06.30.179630v1.article-info

Article now published in Journal of Cell Biology at http://dx.doi.org/10.1083/jcb.202009154

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: 7 September 2020

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

Read preprint (No Ratings Yet)

Have your say

Your email address will not be published. Required fields are marked *

This site uses Akismet to reduce spam. Learn how your comment data is processed.

Sign up to customise the site to your preferences and to receive alerts

Register here

preLists in the biochemistry category:

Preprint Peer Review – Biochemistry Course at UFRJ, Brazil

Communication of scientific knowledge has changed dramatically in recent decades and the public perception of scientific discoveries depends on the peer review process of articles published in scientific journals. Preprints are key vehicles for the dissemination of scientific discoveries, but they are still not properly recognized by the scientific community since peer review is very limited. On the other hand, peer review is very heterogeneous and a fundamental aspect to improve it is to train young scientists on how to think critically and how to evaluate scientific knowledge in a professional way. Thus, this course aims to: i) train students on how to perform peer review of scientific manuscripts in a professional manner; ii) develop students' critical thinking; iii) contribute to the appreciation of preprints as important vehicles for the dissemination of scientific knowledge without restrictions; iv) contribute to the development of students' curricula, as their opinions will be published and indexed on the preLights platform. The evaluations will be based on qualitative analyses of the oral presentations of preprints in the field of biochemistry deposited in the bioRxiv server, of the critical reports written by the students, as well as of the participation of the students during the preprints discussions.

 



List by Marcus Oliveira

CellBio 2022 – An ASCB/EMBO Meeting

This preLists features preprints that were discussed and presented during the CellBio 2022 meeting in Washington, DC in December 2022.

 



List by Nadja Hümpfer et al.

20th “Genetics Workshops in Hungary”, Szeged (25th, September)

In this annual conference, Hungarian geneticists, biochemists and biotechnologists presented their works. Link: http://group.szbk.u-szeged.hu/minikonf/archive/prg2021.pdf

 



List by Nándor Lipták

Fibroblasts

The advances in fibroblast biology preList explores the recent discoveries and preprints of the fibroblast world. Get ready to immerse yourself with this list created for fibroblasts aficionados and lovers, and beyond. Here, my goal is to include preprints of fibroblast biology, heterogeneity, fate, extracellular matrix, behavior, topography, single-cell atlases, spatial transcriptomics, and their matrix!

 



List by Osvaldo Contreras

ASCB EMBO Annual Meeting 2019

A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)

 



List by Madhuja Samaddar et al.

EMBL Seeing is Believing – Imaging the Molecular Processes of Life

Preprints discussed at the 2019 edition of Seeing is Believing, at EMBL Heidelberg from the 9th-12th October 2019

 



List by Dey Lab

Cellular metabolism

A curated list of preprints related to cellular metabolism at Biorxiv by Pablo Ranea Robles from the Prelights community. Special interest on lipid metabolism, peroxisomes and mitochondria.

 



List by Pablo Ranea Robles

MitoList

This list of preprints is focused on work expanding our knowledge on mitochondria in any organism, tissue or cell type, from the normal biology to the pathology.

 



List by Sandra Franco Iborra

Also in the cell biology category:

‘In preprints’ from Development 2022-2023

A list of the preprints featured in Development's 'In preprints' articles between 2022-2023

 



List by Alex Eve, Katherine Brown

preLights peer support – preprints of interest

This is a preprint repository to organise the preprints and preLights covered through the 'preLights peer support' initiative.

 



List by preLights peer support

The Society for Developmental Biology 82nd Annual Meeting

This preList is made up of the preprints discussed during the Society for Developmental Biology 82nd Annual Meeting that took place in Chicago in July 2023.

 



List by Joyce Yu, Katherine Brown

CSHL 87th Symposium: Stem Cells

Preprints mentioned by speakers at the #CSHLsymp23

 



List by Alex Eve

Journal of Cell Science meeting ‘Imaging Cell Dynamics’

This preList highlights the preprints discussed at the JCS meeting 'Imaging Cell Dynamics'. The meeting was held from 14 - 17 May 2023 in Lisbon, Portugal and was organised by Erika Holzbaur, Jennifer Lippincott-Schwartz, Rob Parton and Michael Way.

 



List by Helen Zenner

9th International Symposium on the Biology of Vertebrate Sex Determination

This preList contains preprints discussed during the 9th International Symposium on the Biology of Vertebrate Sex Determination. This conference was held in Kona, Hawaii from April 17th to 21st 2023.

 



List by Martin Estermann

Alumni picks – preLights 5th Birthday

This preList contains preprints that were picked and highlighted by preLights Alumni - an initiative that was set up to mark preLights 5th birthday. More entries will follow throughout February and March 2023.

 



List by Sergio Menchero et al.

CellBio 2022 – An ASCB/EMBO Meeting

This preLists features preprints that were discussed and presented during the CellBio 2022 meeting in Washington, DC in December 2022.

 



List by Nadja Hümpfer et al.

Fibroblasts

The advances in fibroblast biology preList explores the recent discoveries and preprints of the fibroblast world. Get ready to immerse yourself with this list created for fibroblasts aficionados and lovers, and beyond. Here, my goal is to include preprints of fibroblast biology, heterogeneity, fate, extracellular matrix, behavior, topography, single-cell atlases, spatial transcriptomics, and their matrix!

 



List by Osvaldo Contreras

EMBL Synthetic Morphogenesis: From Gene Circuits to Tissue Architecture (2021)

A list of preprints mentioned at the #EESmorphoG virtual meeting in 2021.

 



List by Alex Eve

FENS 2020

A collection of preprints presented during the virtual meeting of the Federation of European Neuroscience Societies (FENS) in 2020

 



List by Ana Dorrego-Rivas

Planar Cell Polarity – PCP

This preList contains preprints about the latest findings on Planar Cell Polarity (PCP) in various model organisms at the molecular, cellular and tissue levels.

 



List by Ana Dorrego-Rivas

BioMalPar XVI: Biology and Pathology of the Malaria Parasite

[under construction] Preprints presented at the (fully virtual) EMBL BioMalPar XVI, 17-18 May 2020 #emblmalaria

 



List by Dey Lab, Samantha Seah

1

Cell Polarity

Recent research from the field of cell polarity is summarized in this list of preprints. It comprises of studies focusing on various forms of cell polarity ranging from epithelial polarity, planar cell polarity to front-to-rear polarity.

 



List by Yamini Ravichandran

TAGC 2020

Preprints recently presented at the virtual Allied Genetics Conference, April 22-26, 2020. #TAGC20

 



List by Maiko Kitaoka et al.

3D Gastruloids

A curated list of preprints related to Gastruloids (in vitro models of early development obtained by 3D aggregation of embryonic cells). Updated until July 2021.

 



List by Paul Gerald L. Sanchez and Stefano Vianello

ECFG15 – Fungal biology

Preprints presented at 15th European Conference on Fungal Genetics 17-20 February 2020 Rome

 



List by Hiral Shah

ASCB EMBO Annual Meeting 2019

A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)

 



List by Madhuja Samaddar et al.

EMBL Seeing is Believing – Imaging the Molecular Processes of Life

Preprints discussed at the 2019 edition of Seeing is Believing, at EMBL Heidelberg from the 9th-12th October 2019

 



List by Dey Lab

Autophagy

Preprints on autophagy and lysosomal degradation and its role in neurodegeneration and disease. Includes molecular mechanisms, upstream signalling and regulation as well as studies on pharmaceutical interventions to upregulate the process.

 



List by Sandra Malmgren Hill

Lung Disease and Regeneration

This preprint list compiles highlights from the field of lung biology.

 



List by Rob Hynds

Cellular metabolism

A curated list of preprints related to cellular metabolism at Biorxiv by Pablo Ranea Robles from the Prelights community. Special interest on lipid metabolism, peroxisomes and mitochondria.

 



List by Pablo Ranea Robles

BSCB/BSDB Annual Meeting 2019

Preprints presented at the BSCB/BSDB Annual Meeting 2019

 



List by Dey Lab

MitoList

This list of preprints is focused on work expanding our knowledge on mitochondria in any organism, tissue or cell type, from the normal biology to the pathology.

 



List by Sandra Franco Iborra

ASCB/EMBO Annual Meeting 2018

This list relates to preprints that were discussed at the recent ASCB conference.

 



List by Dey Lab, Amanda Haage

Also in the immunology category:

Journal of Cell Science meeting ‘Imaging Cell Dynamics’

This preList highlights the preprints discussed at the JCS meeting 'Imaging Cell Dynamics'. The meeting was held from 14 - 17 May 2023 in Lisbon, Portugal and was organised by Erika Holzbaur, Jennifer Lippincott-Schwartz, Rob Parton and Michael Way.

 



List by Helen Zenner

Fibroblasts

The advances in fibroblast biology preList explores the recent discoveries and preprints of the fibroblast world. Get ready to immerse yourself with this list created for fibroblasts aficionados and lovers, and beyond. Here, my goal is to include preprints of fibroblast biology, heterogeneity, fate, extracellular matrix, behavior, topography, single-cell atlases, spatial transcriptomics, and their matrix!

 



List by Osvaldo Contreras

Single Cell Biology 2020

A list of preprints mentioned at the Wellcome Genome Campus Single Cell Biology 2020 meeting.

 



List by Alex Eve

Autophagy

Preprints on autophagy and lysosomal degradation and its role in neurodegeneration and disease. Includes molecular mechanisms, upstream signalling and regulation as well as studies on pharmaceutical interventions to upregulate the process.

 



List by Sandra Malmgren Hill

Antimicrobials: Discovery, clinical use, and development of resistance

Preprints that describe the discovery of new antimicrobials and any improvements made regarding their clinical use. Includes preprints that detail the factors affecting antimicrobial selection and the development of antimicrobial resistance.

 



List by Zhang-He Goh

Zebrafish immunology

A compilation of cutting-edge research that uses the zebrafish as a model system to elucidate novel immunological mechanisms in health and disease.

 



List by Shikha Nayar

Also in the molecular biology category:

‘In preprints’ from Development 2022-2023

A list of the preprints featured in Development's 'In preprints' articles between 2022-2023

 



List by Alex Eve, Katherine Brown

CSHL 87th Symposium: Stem Cells

Preprints mentioned by speakers at the #CSHLsymp23

 



List by Alex Eve

9th International Symposium on the Biology of Vertebrate Sex Determination

This preList contains preprints discussed during the 9th International Symposium on the Biology of Vertebrate Sex Determination. This conference was held in Kona, Hawaii from April 17th to 21st 2023.

 



List by Martin Estermann

Alumni picks – preLights 5th Birthday

This preList contains preprints that were picked and highlighted by preLights Alumni - an initiative that was set up to mark preLights 5th birthday. More entries will follow throughout February and March 2023.

 



List by Sergio Menchero et al.

CellBio 2022 – An ASCB/EMBO Meeting

This preLists features preprints that were discussed and presented during the CellBio 2022 meeting in Washington, DC in December 2022.

 



List by Nadja Hümpfer et al.

EMBL Synthetic Morphogenesis: From Gene Circuits to Tissue Architecture (2021)

A list of preprints mentioned at the #EESmorphoG virtual meeting in 2021.

 



List by Alex Eve

FENS 2020

A collection of preprints presented during the virtual meeting of the Federation of European Neuroscience Societies (FENS) in 2020

 



List by Ana Dorrego-Rivas

ECFG15 – Fungal biology

Preprints presented at 15th European Conference on Fungal Genetics 17-20 February 2020 Rome

 



List by Hiral Shah

ASCB EMBO Annual Meeting 2019

A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)

 



List by Madhuja Samaddar et al.

Lung Disease and Regeneration

This preprint list compiles highlights from the field of lung biology.

 



List by Rob Hynds

MitoList

This list of preprints is focused on work expanding our knowledge on mitochondria in any organism, tissue or cell type, from the normal biology to the pathology.

 



List by Sandra Franco Iborra
Close