Biology subjects

Radaszkiewicz, T.

Publications and source records attributed to Radaszkiewicz, T..

6 recordsLinked to original sources

bioRxiv2025

Armed with PRICKLE(3)s: Stabilizing WNT/PCP complexes against RNF43-mediated ubiquitination

The human Prickle protein family, consisting of PRICKLE1, PRICKLE2, PRICKLE3, and PRICKLE4, is an integral component of the WNT/planar cell polarity (WNT/PCP) pathway and is essential for various cellular and developmental processes. Despite their significance, the detailed roles and involvement in molecular mechanisms of these proteins in cells remain not fully understood. In this study, we used enhanced proximity biotinylation (miniTurboID) combined with mass spectrometry to characterize the microenvironment of PRICKLE1-3. Our results reveal that PRICKLE3 is directly linked to the WNT/PCP pathway, primarily localizing at the plasma membrane and forming complexes with VANGL proteins. This observation prompted us to examine its role in the non-canonical WNT signalling pathway in more detail. Using an inducible expression system to achieve protein levels closer to physiological conditions, we found that PRICKLE3 enhances the stability of VANGL1 and VANGL2 by shielding them from Casein kinase 1 {varepsilon}-mediated phosphorylation. Furthermore, our results indicate that PRICKLE3 modulates WNT receptor complexes by negatively regulating the interaction between Casein kinase 1 {varepsilon} and ubiquitin ligase RNF43, resulting in decreased ubiquitination and increased stabilization of VANGL1/2 at the plasma membrane. Notably, these effects were specific to PRICKLE3, with PRICKLE1 showing no comparable activity. Contrary to previous findings based mainly on standard overexpression studies, neither PRICKLE3 nor PRICKLE1 influenced the levels or phosphorylation status of WNT proteins DISHEVELLED2 and DISHEVELLED3, which are the PRICKLE proteins binding partners. In summary, we have identified a key mechanism specific to PRICKLE3 that positively regulates WNT/PCP complexes by suppressing RNF43. Additionally, we present a comprehensive interactome and new tools for the functional specification of Prickle isoforms to support further research.

cell biology↗

bioRxiv2025

Proximity interactomics identifies RAI14, EPHA2 and PHACTR4 as essential components of Wnt/planar cell polarity pathway in vertebrates

Wnt/planar cell polarity (Wnt/PCP) pathway is an evolutionarily conserved signaling cascade playing an inevitable role in cell biology. Deregulation of Wnt/PCP leads to severe developmental defects or cancer progression. Here, we applied proximity-dependent biotinylation (BioID) to capture the intracellular interactome of key Wnt/PCP components: transmembrane ROR1, ROR2 and VANGL2, and cytoplasmic DVL3 and PRICKLE1. Mapping of individual preys across the baits and subcellular compartments identified a group of 30 proteins that we tested by loss-of-function in zebrafish. Among those rai14, epha2 and phactr4 were essential for several Wnt/PCP-dependent processes such as zebrafish convergent extension, orientation of lateral organ cells, or migration of melanoma cells. Mechanistically, RAI14, EPHA2 and PHACTR4 connect the receptor complex to effector actomyosin. In summary, this study identified novel essential components of vertebrate WNT/PCP pathway and provides a detailed characterization of PCP complex composition that can serve as comprehensive resource for further analysis of Wnt/PCP function.

cell biology↗

bioRxiv2023

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 actively cycling stem cells residing at the crypt bottom1,2. Together with stromal cells, Paneth cells form a major element of the niche microenvironment that provides various growth factors to orchestrate intestinal stem cell homeostasis, such as Wnt33. With 19 family members, different Wnt ligands can selectively activate {beta}-catenin dependent (canonical) or independent (non-canonical) signaling4,5. Here, we report that Dishevelled-associated activator of morphogenesis 1 (Daam1) and its paralogue Daam2 asymmetrically regulate canonical and non-canonical Wnt (Wnt/PCP) signaling, and their function is required for Paneth cell progenitor differentiation. We found that Daam1/2 interacts with the Wnt antagonist Rnf43, and Daam1/2 double knockout stimulates canonical Wnt signaling by preventing Rnf43-dependent endo-lysosomal degradation of the ubiquitinated Wnt receptor, Frizzled (Fzd). Moreover, single-cell RNA sequencing analysis revealed that Paneth cell differentiation is impaired by Daam1/2 depletion, as a result of defective Wnt/PCP signaling. Taken together, we identified Daam1/2 as an unexpected hub molecule coordinating both canonical and non-canonical Wnt signaling, the regulation of which is fundamental for specifying an adequate number of Paneth cells while maintaining intestinal stem cell homeostasis.

cell biology↗

bioRxiv2022

LuminoCell: a versatile and affordable luminometer platform for monitoring in-cell luciferase-based reporters

Luciferase reporter assays represent a simple and sensitive experimental system in cell and molecular biology to study multiple biological processes. However, the application of these assays is often limited by the costs of conventional luminometer instruments and the versatility of their use in different experimental conditions. Therefore, we aimed to develop a small, affordable luminometer allowing continuous measurement of luciferase activity, designed for inclusion into various kinds of tissue culture incubators. Here we introduce LuminoCell - an open-source platform for the construction of an affordable, sensitive, and portable luminometer capable of realtime monitoring in-cell luciferase activity. The LuminoCell costs $40, requires less than 1 hour to assemble, and it is capable of performing real-time sensitive detection of both magnitude and duration of the activity of major signalling pathways in cell cultures, including receptor tyrosine kinases (EGF, FGF), WNT/{beta}-catenin, and NF-{kappa}B. Additionally, we show that the LuminoCell is suitable to be used in cytotoxicity assays as well as for monitoring periodic circadian gene expression.

molecular biology↗

bioRxiv2021

RNF43 inhibits WNT5A driven signaling and suppresses melanoma invasion

RNF43 is a E3 ubiquitin ligase and known negative regulator of WNT/{beta}-catenin signaling. We demonstrate that RNF43 is also regulator of noncanonical WNT5A-induced signaling in human cells. Analysis of the RNF43 interactome using BioID and immunoprecipitation showed that RNF43 can interact with the core receptor complex components dedicated to the noncanonical Wnt pathway such as ROR1, ROR2, VANGL1 and VANGL2. RNF43 triggers VANGL2 ubiquitination and proteasomal degradation and clathrin-dependent internalization of ROR1 receptor. This activity of RNF43 is physiologically relevant and blocks pro-metastatic WNT5A signaling in melanoma. RNF43 inhibits responses to WNT5A, which results in the suppression of invasive properties of melanoma cells. Furthermore, RNF43 prevented WNT5A-assisted development of resistance to BRAF V600E inhibitor. In line with these findings, RNF43 expression decreases during melanoma progression and RNF43-low patients have worse prognosis. We conclude that RNF43 is a newly discovered negative regulator of WNT5A-mediated biological responses that desensitizes cells to WNT5A.

cancer biology↗

bioRxiv2019

RNF43 truncating mutations mediate a tumour suppressor-to-oncogene switch to drive niche-independent self-renewal in cancer

Wnt/{beta}-catenin signalling is a primary pathway for stem cell maintenance during tissue renewal and a frequent target for mutations in cancer. Impaired Wnt receptor endocytosis due to loss of the ubiquitin ligase RNF43 gives rise to Wnt-hypersensitive tumours that are susceptible to anti-Wnt-based therapy. Contrary to this paradigm, we identify a class of RNF43 truncating cancer mutations that strongly induce {beta}-catenin-mediated transcription, despite exhibiting retained Wnt receptor downregulating activity. Mechanistically, these RNF43 mutants trap Casein Kinase (CK)1 at the plasma membrane, which prevents {beta}-catenin turnover and propels ligand-independent Wnt target gene transcription. When introduced in human colon stem cells, these oncogenic RNF43 mutants cooperate with p53 loss to drive a niche-independent program for self-renewal and proliferation. Importantly, onco-RNF43 mutations, unlike conventional LOF RNF43 mutations, confer resistance to anti-Wnt-based therapy. Our data demonstrate the relevance of studying patient-derived mutations for understanding disease mechanisms and improved applications of precision medicine.

cancer biology↗