Wnt Signaling in 3D: Recent Advances in the Applications of Intestinal Organoids

Intestinal organoids grown from adult stem cells have emerged as prototype 3D organotypic models for studying tissue renewal and homeostasis. Owing to their strict dependence on Wnt signaling, intestinal organoids offer an unprecedented opportunity to examine Wnt pathway regulation in normal physiology and cancer. We review how alterations in growth factor dependency and organoid morphology can be exploited to identify Wnt signaling mechanisms, characterize mutated pathway components, and predict responses of patient-derived tumors to targeted therapy. We discuss current deficits in the understanding of genotype–phenotype relationships that are to be considered when interpreting mutation-induced changes in organoid morphology

Organoid-based modeling of intestinal development, regeneration, and repair

The intestinal epithelium harbors a remarkable adaptability to undergo injury-induced repair. A key part of the regenerative response is the transient reprogramming of epithelial cells into a fetal-like state, which drives uniform proliferation, tissue remodeling, and subsequent restoration of the homeostatic state. In this review, we discuss how Wnt and YAP signaling pathways control the intestinal repair response and the transitioning of cell states, in comparison with the process of intestinal development. Furthermore, we highlight how organoid-based applications have contributed to the characterization of the mechanistic principles and key players that guide these developmental and regenerative events

Determining biophysical protein stability in lysates by a fast proteolysis assay, FASTpp.

The biophysical stability is an important parameter for protein activity both in vivo and in vitro. Here we propose a method to analyse thermal melting of protein domains in lysates: Fast parallel proteolysis (FASTpp). Combining unfolding by a temperature gradient in a thermal cycler with simultaneous proteolytic cleavage of the unfolded state, we probed stability of single domains in lysates. We validated FASTpp on proteins from 10 kDa to 240 kDa and monitored stabilisation and coupled folding and binding upon interaction with small-molecule ligands. Within a total reaction time of approximately 1 min, we probed subtle stability differences of point mutations with high sensitivity and in agreement with data obtained by intrinsic protein fluorescence. We anticipate a wide range of applications of FASTpp in biomedicine and protein engineering as it requires only standard laboratory equipment

Large extent of disorder in Adenomatous Polyposis Coli offers a strategy to guard Wnt signalling against point mutations.

Mutations in the central region of the signalling hub Adenomatous Polyposis Coli (APC) cause colorectal tumourigenesis. The structure of this region remained unknown. Here, we characterise the Mutation Cluster Region in APC (APC-MCR) as intrinsically disordered and propose a model how this structural feature may contribute to regulation of Wnt signalling by phosphorylation. APC-MCR was susceptible to proteolysis, lacked α-helical secondary structure and did not display thermal unfolding transition. It displayed an extended conformation in size exclusion chromatography and was accessible for phosphorylation by CK1ε in vitro. The length of disordered regions in APC increases with species complexity, from C. elegans to H. sapiens. We speculate that the large disordered region harbouring phosphorylation sites could be a successful strategy to stabilise tight regulation of Wnt signalling against single missense mutations

mRNA spindle localization and mitotic translational regulation by CPEB1 and CPEB4

Transition through cell cycle phases requires temporal and spatial regulation of gene expression to ensure accurate chromosome duplication and segregation. This regulation involves dynamic reprogramming of gene expression at multiple transcriptional and posttranscriptional levels. In transcriptionally silent oocytes, the CPEB-family of RNA-binding proteins coordinates temporal and spatial translation regulation of stored maternal mRNAs to drive meiotic progression. CPEB1 mediates mRNA localization to the meiotic spindle, which is required to ensure proper chromosome segregation. Temporal translational regulation also takes place in mitosis, where a large repertoire of transcripts is activated or repressed in specific cell cycle phases. However, whether control of localized translation at the spindle is required for mitosis is unclear, as mitotic and acentriolar-meiotic spindles are functionally and structurally different. Furthermore, the large differences in scale-ratio between cell volume and spindle size in oocytes compared to somatic mitotic cells may generate distinct requirements for gene expression compartmentalization in meiosis and mitosis. Here we show that mitotic spindles contain CPE-localized mRNAs and translating ribosomes. Moreover, CPEB1 and CPEB4 localize in the spindles and they may function sequentially in promoting mitotic stage transitions and correct chromosome segregation. Thus, CPEB1 and CPEB4 bind to specific spindle-associated transcripts controlling the expression and/or localization of their encoded factors that, respectively, drive metaphase and anaphase/cytokinesis.Fil: Pascual, Rosa. Barcelona Institute Of Science And Technology.; EspañaFil: Segura Morales, Carolina. Barcelona Institute Of Science And Technology.; EspañaFil: Omerzu, Manja. University of Utrecht; Países BajosFil: Bellora, Nicolás. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte. Instituto Andino Patagónico de Tecnologías Biológicas y Geoambientales. Universidad Nacional del Comahue. Instituto Andino Patagónico de Tecnologías Biológicas y Geoambientales; ArgentinaFil: Belloc, Eulàlia. Barcelona Institute Of Science And Technology.; EspañaFil: Castellazzi, Chiara Lara. Barcelona Institute Of Science And Technology.; EspañaFil: Reina, Oscar. Barcelona Institute Of Science And Technology.; EspañaFil: Eyras, Eduardo. Universitat Pompeu Fabra; España. Institució Catalana de Recerca i Estudis Avançats; EspañaFil: Maurice, Madelon M.. University of Utrecht; Países BajosFil: Millanes Romero, Alba. Barcelona Institute Of Science And Technology.; EspañaFil: Méndez, Raúl. Barcelona Institute Of Science And Technology.; Españ

E3 ligases RNF43 and ZNRF3 display differential specificity for endocytosis of Frizzled receptors

The transmembrane E3 ligases RNF43 and ZNRF3 perform key tumour suppressor roles by inducing endocytosis of members of the Frizzled (FZD) family, the primary receptors for WNT. Loss-of-function mutations in RNF43 and ZNRF3 mediate FZD stabilisation and a WNT-hypersensitive growth state in various cancer types. Strikingly, RNF43 and ZNRF3 mutations are differentially distributed across cancer types, raising questions about their functional redundancy. Here, we compare the efficacy of RNF43 and ZNRF3 of targeting different FZDs for endocytosis. We find that RNF43 preferentially down-regulates FZD1/FZD5/FZD7, whereas ZNRF3 displays a preference towards FZD6. We show that the RNF43 transmembrane domain (TMD) is a key molecular determinant for inducing FZD5 endocytosis. Furthermore, a TMD swap between RNF43 and ZNRF3 re-directs their preference for FZD5 down-regulation. We conclude that RNF43 and ZNRF3 preferentially down-regulate specific FZDs, in part by a TMD-dependent mechanism. In accordance, tissue-specific expression patterns of FZD homologues correlate with the incidence of RNF43 or ZNRF3 cancer mutations in those tissues. Consequently, our data point to druggable vulnerabilities of specific FZD receptors in RNF43- or ZNRF3-mutant human cancers

Intestinal Paneth cell differentiation relies on asymmetric regulation of Wnt signaling by Daam1/2

The mammalian intestine is one of the most rapidly self-renewing tissues, driven by stem cells residing at the crypt bottom. Paneth cells form a major element of the niche microenvironment providing various growth factors to orchestrate intestinal stem cell homeostasis, such as Wnt3. Different Wnt ligands can selectively activate β-catenin-dependent (canonical) or -independent (noncanonical) signaling. Here, we report that the Dishevelled-associated activator of morphogenesis 1 (Daam1) and its paralogue Daam2 asymmetrically regulate canonical and noncanonical Wnt (Wnt/PCP) signaling. Daam1/2 interacts with the Wnt inhibitor RNF43, and Daam1/2 double knockout stimulates canonical Wnt signaling by preventing RNF43-dependent degradation of the Wnt receptor, Frizzled (Fzd). Single-cell RNA sequencing analysis revealed that Paneth cell differentiation is impaired by Daam1/2 depletion because of defective Wnt/PCP signaling. Together, we identified Daam1/2 as an unexpected hub molecule coordinating both canonical and noncanonical Wnt, which is fundamental for specifying an adequate number of Paneth cells

NEDD4 and NEDD4L regulate Wnt signalling and intestinal stem cell priming by degrading LGR5 receptor

The intestinal stem cell (ISC) marker LGR5 is a receptor for R-spondin (RSPO) that functions to potentiate Wnt signalling in the proliferating crypt. It has been recently shown that Wnt plays a priming role for ISC self-renewal by inducing RSPO receptor LGR5 expression. Despite its pivotal role in homeostasis, regeneration and cancer, little is known about the post-translational regulation of LGR5. Here, we show that the HECT-domain E3 ligases NEDD4 and NEDD4L are expressed in the crypt stem cell regions and regulate ISC priming by degrading LGR receptors. Loss of Nedd4 and Nedd4l enhances ISC proliferation, increases sensitivity to RSPO stimulation and accelerates tumour development in Apcmin mice with increased numbers of high-grade adenomas. Mechanistically, we find that both NEDD4 and NEDD4L negatively regulate Wnt/β-catenin signalling by targeting LGR5 receptor and DVL2 for proteasomal and lysosomal degradation. Our findings unveil the previously unreported post-translational control of LGR receptors via NEDD4/NEDD4L to regulate ISC priming. Inactivation of NEDD4 and NEDD4L increases Wnt activation and ISC numbers, which subsequently enhances tumour predisposition and progression

RSPO3 Furin domain-conjugated liposomes for selective drug delivery to LGR5-high cells

The transmembrane receptor LGR5 potentiates Wnt/β-catenin signaling by binding both secreted R-spondin (RSPOs) and the Wnt tumor suppressors RNF43/ZNRF3, directing clearance of RNF43/ZNRF3 from the cell surface. Besides being widely used as a stem cell marker in various tissues, LGR5 is overexpressed in many types of malignancies, including colorectal cancer. Its expression characterizes a subpopulation of cancer cells that play a crucial role in tumor initiation, progression and cancer relapse, known as cancer stem cells (CSCs). For this reason, ongoing efforts are aimed at eradicating LGR5-positive CSCs. Here, we engineered liposomes decorated with different RSPO proteins to specifically detect and target LGR5-positive cells. Using fluorescence-loaded liposomes, we show that conjugation of full-length RSPO1 to the liposomal surface mediates aspecific, LGR5-independent cellular uptake, largely mediated by heparan sulfate proteoglycan binding. By contrast, liposomes decorated only with the Furin (FuFu) domains of RSPO3 are taken up by cells in a highly specific, LGR5-dependent manner. Moreover, encapsulating doxorubicin in FuFuRSPO3 liposomes allowed us to selectively inhibit the growth of LGR5-high cells. Thus, FuFuRSPO3-coated liposomes allow for the selective detection and ablation of LGR5-high cells, providing a potential drug delivery system for LGR5-targeted anti-cancer strategies