Phase separation of zonula occludens proteins drives formation of tight junctions

Oliver Beutel, Riccardo Maraspini, Karina Pombo-Garcia, Cécilie Martin-Lemaitre, Alf Honigmann

Preprint posted on 26 March 2019

Article now published in Cell at

Phase separation strikes again! Zonula occludens proteins concentrate tight junction machinery by forming non-membrane enclosed compartments.

Selected by Abagael Lasseigne

Categories: cell biology


Tight junctions are cell-cell adhesion complexes that prevent passage between epithelial or endothelial cells. Apically located, tight junctions consist of adhesion receptors, such as claudins, and a dense plaque of cytoplasmic proteins that interact with the cytoskeleton. It is known that the intracellular scaffolds zonula occludens 1 and 2 (ZO1 and ZO2, respectively) are required for tight junction formation, but how they organize these structures on a mechanistic level is unknown. ZO proteins have conserved protein-protein binding domains and are homologs of PSD95 which was recently found to organize the chemical synapse via phase separation. Within a cell, non-membrane enclosed organelles can be formed when condensates of particular proteins create liquid droplets within the surrounding liquid (for an in depth review of phase separation, see Alberti, 2017). At the chemical synapse, PSD95 oligomerization facilitates phase separation therefore creating protein nanodomains that sequester other postsynaptic machinery to the sight of synaptogenesis. Similarly, ZO protein oligomerization is required for effective tight junction formation, thus the authors hypothesize that 1) ZO proteins phase separate, and 2) this phase separation is required for tight junction formation and integrity.  

Key Findings:

  • Both ZO1 and ZO2 localize to tight junctions between cultured epithelial cells, although ZO1 is more abundant.
  • When overexpressed in cell culture, ZO1 and ZO2 proteins form large condensates at the membrane. Utilizing live imaging techniques and fluorescence recovery after photobleaching (FRAP) assays, the authors showed that these enriched protein domains could fuse in a liquid-like manner and rapidly recover after photobleaching. These dynamics are indicative of phase separation.   
  • ZO protein phosphorylation inhibits phase separating abilities.
  • Intramolecular and intermolecular interactions between ZO proteins facilitate phase separation. Most notably, the PDZ3-SH3-GUK domain (PSG), necessary for oligomerization, is required for phase separation of each ZO protein in vitro. Without the PSG, proteins homogeneously diffuse instead of forming droplets.  
  • ZO1 selectively phase separates with tight junction proteins (including afadin, cingulin, and claudin-1), and FRAP experiments suggest that this is a dynamic and ongoing process.
  • ZO1 phase separation is required for tight junction formation in cell culture. Without phase separating, ZO1 disperses in the cytoplasm. If this occurs, ZO1 does not localize well to the tight junctions between cultured epithelial cells and junctional integrity is lost.

Future directions:

To further understand how phase separation of ZO proteins organizes tight junctions, it is important to next understand how interactions with transmembrane adherens receptors and the cytoskeleton influence the condensation dynamics of ZO proteins. In other words, how does phase separation physically influence other tight junction proteins, and how are ZO protein dynamics regulated?

Why I like this preprint:

The findings in this preprint personally inform my research. I study ZO1 at the electrical synapse in zebrafish. We think it may play an organizational role similar to PSD95 at the chemical synapse. It is likely that phase separation is a part of this role and functions in electrical synapse formation. This contributes to the overall model we are trying to develop of how an electrical synapse is formed, maintained, and regulated.

Questions for the author:

  • How would you test this hypothesis in an organism?
  • How should readers, including myself, think about the process of phase separation in an organism, and how might that influence the standard ways in which we think about proteins acting in a cell?

Tags: epithelial cells, intracellular scaffolds, phase separation, tight junctions, zo1, zo2, zo3, zonula occludens

Posted on: 10 June 2019


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 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.


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


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


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


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

Biophysical Society Annual Meeting 2019

Few of the preprints that were discussed in the recent BPS annual meeting at Baltimore, USA


List by Joseph Jose Thottacherry

ASCB/EMBO Annual Meeting 2018

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


List by Dey Lab, Amanda Haage