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 March 26, 2019

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: 10th June 2019

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