Condensation of Sidekick at tricellular junctions organizes mechanical forces for cell-cell adhesion remodeling

Introduction:

Cells in an epithelial sheet are associated with each other through adhesion molecules at cellular junctions. Precise spatiotemporal control of these adhesion molecules is important for the regulation of tissue morphogenesis, however the mechanism through which this is achieved is not always clear.

Biomolecular condensation, known to regulate spatial compartmentalization in the cytoplasm and nucleoplasm, has recently been found to organise dynamic adhesive complexes at tight junctions and focal adhesions through membrane associated intracellular proteins like ZO-1 and Talin.

In this preprint, the authors focus on the transmembrane protein Sidekick (Sdk), which is known to localise at the tricellular junctions in Drosophila and plays an important role in junctional remodelling during germband extension. They explore how condensation of the intracellular domain of Sdk is involved in its spatial organisation and function.

 

Main findings:

Condensation of the intracellular domain localizes Sdk to tricellular junctions

The authors used CRISPR knock in to remove the intracellular domain of Sdk. They observed that without the intracellular domain, Sdk does not localise to the tricellular junctions and is instead distributed uniformly along the membranes. The intracellular domain of Sdk is comprised of intrinsically disordered regions, which promote phase transition into condensates. Constructs where these disordered regions of Sdk were replaced with the similarly disordered regions of Anakonda showed localisation at tricellular junctions, while replacing with less disordered regions of E-Cadherin resulted in uniform distribution along the membranes as seen with in Sdk without the intracellular domain.

Sdk intracellular domain can recapitulate Sdk’s localization patterns and physiological concentration

Purified Sdk intracellular domain forms condensates at high concentration on supported lipid bilayers in vitro, but it is unable to form these condensates in dilute conditions. The authors hypothesized that the bent lipid membrane surface at the tricellular junctions could be responsible for bringing Sdk intracellular domains in close proximity, and thus in high concentration, to facilitate condensate formation. This was tested in vitro by adding a giant unilamellar vesicle to the lipid bilayer, replicating a bent membrane. The results showed that purified Sdk intracellular domain was able to form condensates in the corner of the bent membrane in dilute conditions.

A conserved amino acid motif encodes the material property of Sdk intracellular domain condensates

The authors used a FRAP assay on bulk condensates, targeting one half of the condensate. They showed slow recovery at the bleached region and no change at the unbleached region, suggesting that the condensates are in a less dynamic, more gel like, state. However, when they mutated a conserved amino acid motif (ALEL) in the intracellular domain, the FRAP analysis showed faster recovery in the bleached region and decreased signal intensity at the unbleached region, suggesting that the ALEL motif was responsible for less dynamic, more liquid like, properties of Sdk condensate. Also, the in vivo mutant of ALEL was shown to phenocopy Sdk null mutants. 

The less dynamic state of Sdk confines its localization during cellular movement

During polarised cell intercalation, the ALEL mutants were more likely to disperse and mislocalise away from the lengthening junction compared to WT Sdk. The less dynamic properties of WT Sdk potentially allow stability.

The Sdk ALEL mutant alters dynamics of the downstream molecule myosin II

ALEL mutants decreased myosin II recruitment to tricellular junctions compared to WT Sdk and similar to Sdk RNAi, which suggests that the less dynamic properties of WT Sdk are also important for myosin dynamics.

 

Why I chose this preprint/What I like:

I chose this preprint as I am also working on Sidekick and came across it during my literature search. I think this is an interesting piece of work as it gives a unique physiological perspective on Sdk dynamics at the tricellular junctions.

I really like the FRAP experiment, I think it was such a clear way to show the difference in condensate dynamics with the ALEL mutants.

I also enjoyed the complementation of in-vivo and in-vitro experiments used to provide well rounded arguments in favour of the hypotheses put forward by the preprint authors.

 

Questions for the authors/future directions:

• Is there any way to calculate the critical proximity at which Sdk forms these condensates? For example, is there a way you could manipulate the GUVs contact angle to determine when condensates form?
• In your discussion section, you suggest that the ALEL motif could serve as a protein binding site, and that could potentially alter the condensate dynamics. Are there any particular proteins you are interested to explore?
• Do you think that the WT Sdk condensates remain in a less dynamic state consistently or is there a possibility of context-dependent altering of states?

 

doi: https://doi.org/10.1242/prelights.41698

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