αE-catenin-dependent mechanotransduction is essential for proper convergent extension in zebrafish

Han, Mitchell K. L.; Hoijman, Esteban; Noël, Emily S.; Garric, Laurence; Bakkers, Jeroen; Rooij, Johan de

doi:10.1242/bio.021378

Cited by 29 publications

(25 citation statements)

References 45 publications

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“…Similarly but delayed by 20 hours, in zGrad mRNA-injected Z ctnna-Citrine embryos, cells detached from the embryo by 24 hpf and about half of the embryos died (note, half the embryos are zygotically homozygous for ctnna:ctnna-Citrine since we crossed ctnna:ctnna-Citrine/ctnna:ctnna-Citrine males to ctnna:ctnna-Citrine /+ females, Figure 3H, I) while control injected embryos were unaffected (Figure 3H, I). The observed phenotype in Z ctnna-Citrine embryos injected with zGrad mRNA is similar to the defects reported for zygotic ctnna -/- embryos (18). Thus, zGrad depletes Ctnna-Citrine to levels too low to sustain cell-cell adhesion and embryonic development.…”

Section: Resultssupporting

confidence: 85%

zGrad: A nanobody-based degron system to inactivate proteins in zebrafish

Yamaguchi

Colak-Champollion

Knaut

2019

Preprint

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25The analysis of protein function is essential to modern biology. While protein function has mostly 26 been studied through gene or RNA interference, more recent approaches to degrade proteins 27 directly have been developed. Here, we adapted the anti-GFP nanobody-based system 28 deGradFP from flies to zebrafish. We named this system zGrad and show that zGrad efficiently 29 degrades transmembrane, cytosolic and nuclear GFP-tagged proteins in zebrafish in an 30 inducible and reversible manner. Using tissue-specific and inducible promoters in combination 31 with functional GFP-fusion proteins, we demonstrate that zGrad can inactivate transmembrane, 32 cytosolic and nuclear proteins globally, locally and temporally with different consequences. 33Global protein depletion results in phenotypes similar to loss of gene activity while local and 34 temporal protein inactivation yields more restricted and novel phenotypes. Thus, zGrad is a 35 versatile tool to study the spatial and temporal requirement of proteins in zebrafish. 36 37 109 degree of GFP degradation as the ratio of sfGFP to mScarlet fluorescence intensity in the 110 cytoplasm and the nucleus. Consistent with the initial description (4), Ab-SPOP efficiently 111 degraded nuclear sfGFP-ZF1 (70% reduction, Figure 1 -figure supplements 3A-C) but not 112 cytoplasmic sfGFP-ZF1 (13% reduction, Figure 1 -figure supplements 3A-C). Abmut-SPOP did 113 not cause any detectable sfGFP-ZF1 degradation (Figure 1 -figure supplements 3A-C). As 114 reported previously (4), this confirms that the Ab-SPOP/FP-tag degradation system degrades 115 nuclear proteins efficiently but is not suitable for the degradation of non-nuclear proteins in 116 zebrafish.117 118 Fourth, we tested the deGradFP degradation system. This system uses the F-box domain from 119 the Drosophila Slimb adaptor protein fused to the anti-GFP nanobody vhhGFP4 to target FP-120 tagged proteins for degradation ( Figure 1A, B and (7)). We co-injected one-cell stage embryos 121 with sfGFP-ZF1 mRNA, deGradFP mRNA and mScarlet-V5 mRNA and assessed sfGFP-ZF1 122 degradation 9 hours later at the epiboly stage. As above, mScarlet-V5 served as an internal 123 standard and GFP degradation was assessed as the ratio of GFP to mScarlet fluorescence 124 intensity. We found that deGradFP reduced sfGFP-ZF1 only slightly (19% reduction, Figure 1C, 125 D). Since the fusion of the anti-GFP nanobody to the Cullin-binding domain from SPOP resulted 126 in efficient albeit only nuclear degradation of GFP, we reasoned that the anti-GFP nanobody 127 recognizes GFP-tagged proteins but that the Slimb F-box domain is not efficiently recruited to 128 the E3 ligase complex in zebrafish. We therefore replaced the Slimb F-box domain in deGradFP 129 with the homologuous F-box domain from zebrafish, reasoning that this should result in more 130 efficient GFP degradation in zebrafish. Based on sequence homology we identified the 131 zebrafish fbxw11b gene as the Drosophila slmb orthologue. We then replaced the Drosophila F-132 box domain from Slimb ...

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Section: Resultssupporting

confidence: 85%

zGrad: A nanobody-based degron system to inactivate proteins in zebrafish

Yamaguchi

Colak-Champollion

Knaut

2019

Preprint

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“…Local contractions between E-cadherin adhesions provide a simple mechanism for testing the rigidity of neighboring cells that is analogous to local matrix contractions to test matrix rigidity even though the details are distinct. Many physiological processes are postulated to involve cadherin adhesion mechanosensing such as convergent extension 43 and epithelial tissue movements 44 . In those cases, monolayer morphology alteration is coupled with continued cell-cell sensing, and this indicates that there is a general mechanism of E-cadherin sensing in tissues 6 .…”

Section: Discussionmentioning

confidence: 99%

Local Contractions Regulate E-Cadherin Adhesions, Rigidity Sensing and Epithelial Cell Sorting

Yang

Nguyen

Narayana

et al. 2018

Preprint

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30 31 E-cadherin is a major cell-cell adhesion molecule involved in mechanotransduction at cell-32 cell contacts in tissues. Since epithelial cells respond to rigidity and tension in the tissue 33 through E-cadherin, there must be active processes that test and respond to the mechanical 34 properties of these adhesive contacts. Using sub-micrometer, E-cadherin-coated PDMS 35 pillars, we find that cells generate local contractions between E-cadherin adhesions and 36 pull to a constant distance for a constant duration, irrespective of pillar rigidity. These 37 cadherin contractions require non-muscle myosin IIB, tropomyosin 2.1, α-catenin and 38 binding of vinculin to α-catenin; plus, they are correlated with rigidity-dependent cell 39 spreading. Without contractions, cells fail to spread to different areas on soft and rigid 40 surfaces and to maintain monolayer integrity. We further observe that cadherin 41contractions enable cells to test myosin IIA-mediated tension of neighboring cells, and sort 42 out myosin IIA-depleted cells. Thus, we suggest that epithelial cells test and respond to the 43 mechanical characteristics of neighboring cells through cadherin contractions.For the proper organization of tissues, cells need to probe the mechanical properties of their 63 micro-environment including both extracellular matrix and neighboring cells through 64 adhesive contacts. These mechanical properties are then transduced into biochemical 65 information to regulate cell functions 1 , including single and collective cell motility 2, 3 , 66 proliferation 4 or differentiation 5 . Of the many mechanical properties that cells control, 67 stiffness appears to be an important parameter that is distinctive for a tissue and is reflected 68 in the cells that constitute the tissue 6 . It follows that cells should be able to measure the 69 stiffness of their neighbors to enable them to regulate their cell-cell contacts, cytoskeletal 70 rigidity and organize cell monolayers. Thus, it is important to understand how E-cadherin 71 rigidity might be sensed. Recent studies have indeed found that epithelial cells spread to 72 larger areas on rigid cadherin-coated surfaces than soft 7 . The testing of cadherin adhesion 73 rigidity 8 shares similarities with the testing of matrix rigidity described for fibroblasts 9 . In 74 the context of epithelial cell dynamics, this mechanism may allow cells to adapt to changes 75 in the local stiffness of their neighbors due to cytoskeleton remodeling and reinforcement 10-76 12 . 77 78 Cadherin rigidity is a complex mechanical parameter since it is defined as the force per 79 unit area needed to displace a cadherin adhesion by a given distance. In the case of matrix 80 rigidity sensing, cells pull matrix contacts to a constant deflection and measure the force 81 generated [13][14][15] . The local matrix rigidity sensor is a sarcomere-like contraction complex (2 82 micrometers in length) that contracts matrix adhesions by 120 nm and if the force exceeds 83 25 pN, then a rigid-matrix signal is activate...

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“…Not only do different cadherins regulate the magnitude and dynamics of the intercellular tension within the monolayer (Bazellieres et al, 2015), but the protein-protein interaction-induced clustering of these adhesion proteins on the membrane also plays an essential role in regulating the stability of cell-cell junctions, as well as their collective migration (Strale et al, 2015). The molecular mechanosensor of adherens junction α-catenin and its interaction with vinculin are also involved in the regulation of intercellular tension within monolayers and the collective migration dynamics both in vitro (Seddiki et al, 2018) and in vivo (Han et al, 2016). Suppression of α-catenin protein expressionor its forcedependent interaction with vinculin by decreasing the strength of cell-cell adhesions at the molecular scaledecreases collective cell migration correlation length in vitro and increases cell migration and the exchange of neighboring cells (Seddiki et al, 2018).…”

Section: Molecular Mechanosensing Mechanisms Contribute To Large-scalmentioning

confidence: 99%

“…Suppression of α-catenin protein expressionor its forcedependent interaction with vinculin by decreasing the strength of cell-cell adhesions at the molecular scaledecreases collective cell migration correlation length in vitro and increases cell migration and the exchange of neighboring cells (Seddiki et al, 2018). Furthermore, during embryonic development in zebrafish, the mechanosensing function of α-catenin is not required for epithelial barrier formation; it is, however, required for directional cell migration during convergent extension (Han et al, 2016).…”

Section: Molecular Mechanosensing Mechanisms Contribute To Large-scalmentioning

confidence: 99%

Mechanical forces in cell monolayers

Chen

Saw

Mège

et al. 2018

Journal of Cell Science

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In various physiological processes, the cell collective is organized in a monolayer, such as seen in a simple epithelium. The advances in the understanding of mechanical behavior of the monolayer and its underlying cellular and molecular mechanisms will help to elucidate the properties of cell collectives. In this Review, we discuss recent in vitro studies on monolayer mechanics and their implications on collective dynamics, regulation of monolayer mechanics by physical confinement and geometrical cues and the effect of tissue mechanics on biological processes, such as cell division and extrusion. In particular, we focus on the active nematic property of cell monolayers and the emerging approach to view biological systems in the light of liquid crystal theory. We also highlight the mechanosensing and mechanotransduction mechanisms at the sub-cellular and molecular level that are mediated by the contractile actomyosin cytoskeleton and cell-cell adhesion proteins, such as E-cadherin and α-catenin. To conclude, we argue that, in order to have a holistic understanding of the cellular response to biophysical environments, interdisciplinary approaches and multiple techniquesfrom large-scale traction force measurements to molecular force protein sensorsmust be employed.

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αE-catenin-dependent mechanotransduction is essential for proper convergent extension in zebrafish

Cited by 29 publications

References 45 publications

zGrad: A nanobody-based degron system to inactivate proteins in zebrafish

zGrad: A nanobody-based degron system to inactivate proteins in zebrafish

Local Contractions Regulate E-Cadherin Adhesions, Rigidity Sensing and Epithelial Cell Sorting

Mechanical forces in cell monolayers

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