Regulation of canonical Wnt signalling by the ciliopathy protein MKS1 and the E2 ubiquitin-conjugating enzyme UBE2E1
- University of Leeds, United Kingdom
- University of Tübingen, Germany
- University of Southampton, United Kingdom
Abstract
Primary ciliary defects cause a group of developmental conditions known as ciliopathies. Here, we provide mechanistic insight into ciliary ubiquitin processing in cells and for mouse model lacking the ciliary protein Mks1. In vivo loss of Mks1 sensitizes cells to proteasomal disruption, leading to abnormal accumulation of ubiquitinated proteins. We identified UBE2E1, an E2 ubiquitin-conjugating enzyme that polyubiquitinates β-catenin, and RNF34, an E3 ligase, as novel interactants of MKS1. UBE2E1 and MKS1 colocalized, and loss of UBE2E1 recapitulates the ciliary and Wnt signalling phenotypes observed during loss of MKS1. Levels of UBE2E1 and MKS1 are co-dependent and UBE2E1 mediates both regulatory and degradative ubiquitination of MKS1. We demonstrate that processing of phosphorylated β-catenin occurs at the ciliary base through the functional interaction between UBE2E1 and MKS1. These observations suggest that correct β-catenin levels are tightly regulated at the primary cilium by a ciliary-specific E2 (UBE2E1) and a regulatory substrate-adaptor (MKS1).
Data availability
Data generated or analysed during this study are included in the manuscript and supporting files. Source data is provided for Supplementary Dataset 1, Figure 4e-f, and for all gels and blots displayed in Figures 1-7 (apart from Figure 1e, Figure 1-figure supplement 1 panel e for beta-actin western, Figure 4-figure supplement 1 panel a). Imaging data for gels and blots is collated as both original files of the full unedited files, and figures with the uncropped gels or blots with the relevant bands highlighted.Data from Supplementary dataset 1 is also available from University of Leeds at https://doi.org/10.5518/814
Article and author information
Author details
Katarzyna Szymanska
Funding
Medical Research Council (MR/M000532/1)
- Colin A Johnson
FP7 Health (241955 SYSCILIA)
- Colin A Johnson
Wellcome Trust (204378/Z/16/Z)
- Gabrielle Wheway
Wellcome Trust (105615/Z/14/Z)
- Katarzyna Szymanska
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: The animal studies described in this paper were carried out under the guidance issued by the Medical Research Council in Responsibility in the Use of Animals for Medical Research (July 1993) in accordance with UK Home Office regulations under the Project Licence no. PPL40/3349.
Human subjects: Informed consent was obtained from all participating families or patients, with studies approved by the Leeds (East) Research Ethics Committee (REC ref. no. 08/H1306/85) on 4th July 2008.
Copyright
© 2022, Szymanska et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 1,067
- views
-
- 175
- downloads
-
- 6
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Regulation of canonical Wnt signalling by the ciliopathy protein MKS1 and the E2 ubiquitin-conjugating enzyme UBE2E1
eLife 11:e57593.
https://doi.org/10.7554/eLife.57593
Further reading
-
- Cell Biology
- Immunology and Inflammation
Arpin was discovered as an inhibitor of the Arp2/3 complex localized at the lamellipodial tip of fibroblasts, where it regulated migration steering. Recently, we showed that arpin stabilizes the epithelial barrier in an Arp2/3-dependent manner. However, the expression and functions of arpin in endothelial cells (EC) have not yet been described. Arpin mRNA and protein are expressed in EC and downregulated by pro-inflammatory cytokines. Arpin depletion in Human Umbilical Vein Endothelial Cells causes the formation of actomyosin stress fibers leading to increased permeability in an Arp2/3-independent manner. Instead, inhibitors of ROCK1 and ZIPK, kinases involved in the generation of stress fibers, normalize the loss-of-arpin effects on actin filaments and permeability. Arpin-deficient mice are viable but show a characteristic vascular phenotype in the lung including edema, microhemorrhage, and vascular congestion, increased F-actin levels, and vascular permeability. Our data show that, apart from being an Arp2/3 inhibitor, arpin is also a regulator of actomyosin contractility and endothelial barrier integrity.
-
- Cell Biology
The excessive cosolute densities in the intracellular fluid create a physicochemical condition called macromolecular crowding (MMC). Intracellular MMC entropically maintains the biochemical thermodynamic equilibria by favouring associative reactions while hindering transport processes. Rapid cell volume shrinkage during extracellular hypertonicity elevates the MMC and disrupts the equilibria, potentially ushering cell death. Consequently, cells actively counter the hypertonic stress through regulatory volume increase (RVI) and restore the MMC homeostasis. Here, we establish fluorescence anisotropy of EGFP as a reliable tool for studying cellular MMC and explore the spatiotemporal dynamics of MMC during cell volume instabilities under multiple conditions. Our studies reveal that the actin cytoskeleton enforces spatially varying MMC levels inside adhered cells. Within cell populations, MMC is uncorrelated with nuclear DNA content but anti-correlated with the cell spread area. Although different cell lines have statistically similar MMC distributions, their responses to extracellular hypertonicity vary. The intensity of the extracellular hypertonicity determines a cell's ability for RVI, which correlates with Nuclear Factor Kappa Beta (NFkB) activation. Pharmacological inhibition and knockdown experiments reveal that Tumour Necrosis Factor Receptor 1 (TNFR1) initiates the hypertonicity induced NFkB signalling and RVI. At severe hypertonicities, the elevated MMC amplifies cytoplasmic microviscosity and hinders Receptor Interacting Protein Kinase 1 (RIPK1) recruitment at the TNFR1 complex, incapacitating the TNFR1-NFkB signalling and consequently, RVI. Together, our studies unveil the involvement of TNFR1-NFkB signalling in modulating RVI and demonstrate the pivotal role of MMC in determining cellular osmoadaptability.
