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Mechanical heterogeneity along single cell-cell junctions is driven by lateral clustering of cadherins during vertebrate axis elongation

Robert J. Huebner, Abdul Naseer Malmi-Kakkada, Sena Sarikaya, Shinuo Weng, D. Thirumalai, John B. Wallingford

Preprint posted on November 16, 2020 https://www.biorxiv.org/content/10.1101/2020.02.11.944033v2

Cadherin clusters locally and asymmetrically along shortening junctions to facilitate convergence-extension movements in vertebrate embryos.

Selected by Sundar Naganathan

Background

Convergence-extension (CE) is an evolutionarily conserved process that elongates the body axis during embryonic development. At the cellular scale, this process occurs through a combination of mediolateral intercalation and directed cell migration1. This polarized behaviour is set up by establishment of polarity at the molecular scale through the Wnt-PCP pathway, first discovered in Drosophila embryos2. In addition to these descriptions at cellular and molecular scales, a coarse-grained approach has also been undertaken to understand large-scale tissue movements, which has enabled understanding the hydrodynamics of tissue flows3 and macroscopic tissue stiffening4. Despite these diverse approaches and advances, it remains unknown how the polarized molecular patterns enable the observed cellular behaviours during CE. Huebner RJ et al. tackle this problem and bridge the missing link by performing high-resolution imaging of cell junctions and cadherin clustering during CE.

Key results

The authors analysed CE in Xenopus embryos, which can be predominantly considered in terms of four-cell neighbour exchanges, where mediolaterally (ML)-aligned cell-cell junctions shorten with concomitant elongation of perpendicularly-aligned junctions in anteroposterior (AP) direction. The authors first observed that junction shortening in the ML direction was asymmetric, with one vertex (termed ‘active’) shortening significantly more than the other (termed ‘passive’) as previously observed in Drosophila embryos5. Comparisons with a mechanical model revealed that this asymmetric shortening is essential for CE and that the active vertex displayed a fluid-like motion, whereas the passive vertex exhibited a glass-like dynamics. Furthermore, the increased local stiffness along an active vertex, as required by the model for CE, was consistent with in vivo observations where active vertices exhibited directed motion in the ML direction with significantly lesser transverse fluctuations. Taken together, mechanical heterogeneity at sub-cellular scales at the level of individual vertices is critical for CE in embryos.

The authors next addressed the molecular mechanisms that underlie this heterogeneous mechanical signature by imaging Cdh3, a cell-cell adhesion protein. Cdh3 is known to undergo both cis– (i.e within the same cell) and trans-clustering (i.e across neighbouring cells). Strikingly, the Cdh3 cluster size was larger along active vertices compared to passive vertices and this asymmetry was not observed in non-shortening junctions. When cis-clustering was specifically targeted through point mutations in the hydrophobic pocket of Cdh3, cluster formation was affected. Importantly, under these mutant conditions, axis elongation was specifically perturbed. The authors then showed that this effect on axis elongation is due to the elimination of mechanical heterogeneity along shortening junctions, thus providing the first functional significance for Cdh3 cis-clustering in vivo. Finally, the PCP pathway was shown to act upstream of Cdh3 cis-clustering, thus providing a mechanistic link between polarity establishment and the observed heterogeneous junctional dynamics.

Why I chose this preprint

  1. This is the first demonstration of asymmetric junctional shortening in vertebrate embryos. Moreover, the authors perform in-depth quantifications and compare their observations with a mechanical model to describe mechanistically the importance of mechanical heterogeneities along shortening junctions.
  2. While cadherin adhesion proteins have long been known to undergo cis-clustering, this preprint provides the first functional significance of such widely observed clustering events.
  3. The preprint discusses interesting observations of fluid-like and glass-like dynamics occurring concurrently in shortening junctions at sub-cellular scales. The transition between these contrasting motions have so far only been observed and described at macroscopic tissue-scales.

Open questions

  1. Given a 4-cell configuration, what determines whether the left junction or the right junction exhibits active shortening? On a larger scale, how are these active and passive junctions spatially distributed in a tissue?
  2. What percentage of cells in a converging tissue exhibit this heterogeneous active-passive shortening?
  3. It is not clear if the quantification of Cdh3 cluster size reported in Fig. 4 exclusively represents cis-clustering or it represents a combination of cis- and trans-clustering? If it does represent cis-clustering, how were the contributions from trans-clustering removed from this analysis? Does cis-clustering also simultaneously result in an increase in trans-interactions?
  4. In the model, junctions failed to shorten if the viscoelastic parameter was equal and small for both vertices. How did previous models in the CE field achieve CE even without explicitly considering heterogeneous shortening?
  5. Can you explain in simple terms how despite an increased local stiffness along an active vertex compared to a passive vertex, the active vertex still exhibits more fluid-like motion?
  6. Any hypothesis on how does the PCP pathway enables asymmetric junctional cis-clustering?

References

  1. Keller R. et al., Mechanisms of convergence and extension by cell intercalation, Phil. Trans. Royal Society London. Series B: Biological Sciences, 2000
  2. Vinson C. R. and Adler P. N., Directional non-cell autonomy and the transmission of polarity information by the frizzled gene of Drosophila, Nature, 1987
  3. Popović M. et al., Active dynamics of tissue shear flow, New J. Phys., 2017
  4. Mongera A. et al., A fluid-to-solid jamming transition underlies vertebrate body axis elongation, Nature, 2018
  5. Vanderleest T. E. et al., Vertex sliding drives intercalation by radial coupling of adhesion and actomyosin networks during Drosophila germband extension, eLife, 2018

Tags: adherens junctions, frog, morphogenesis, tissue flows

Posted on: 1st December 2020

doi: https://doi.org/10.1242/prelights.26039

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Author's response

John B. Wallingford and Abdul Naseer Malmi-Kakkada shared

1. This is the big question, clearly, and we don’t yet have an answer. Our working hypothesis is that active and passive junction behaviors are an emergent property of the larger-scale mechanical environment. That is, the mechanics of all neighboring cells determine the behavior at each vertex.

2. Another good question, and one we are trying to answer in order to test the idea mentioned in point #1. We are examining wider-field movies now for this purpose.

3. It’s generally thought that cis-clustering occurs predominantly among trans-ligated dimers. So, most work quantifying clusters of cadherins tacitly assume that clustering is a function of cis-interaction among already trans-ligated cadherin dimers. We made the same assumption. As a practical matter, if you block trans-ligation, the cells dissociate, so clustering becomes irrelevant since you can’t study CE! Interestingly, there is some very cool work, mainly from Ronen Zaidel-Bar’s lab, showing clustering and even functions for “extra-junctional” cadherin monomers, so there’s lots more to do.

4. Our observation of heterogenous shortening contributions from active and passive vertices came about due to us being able to observe vertex dynamics at a high frequency – in this case every 2 seconds over a 400 sec+ time interval. We consider it plausible that local vertex viscoelasticity could be a time dependent property with a persistence time in the order of 100s of seconds. This would imply that a previously active vertex could become a passive vertex and vice versa when observed over a time interval longer than the persistence time of the local vertex viscoelasticity. In this scenario, earlier models of CE accounting for a more time averaged cellular behaviors would not have observed such heterogeneity in the dynamics.

5. It is counter-intuitive, but quite simple really: We argue that the stiffer environment near active vertices acts mainly to prevent transversemotion of the vertex, thus vertex movement in-line (i.e. to shorten the junction) is more persistent and thus more fluid-like.

6. Of course, this is my favorite part! Many studies of cadherin clustering suggest a key role fo actomyosin in clustering. PCP clearly regulates actomyosin contractility, which in turn likely impacts clustering. We are trying to sort this out now.

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