Biology subjects

Guille, M.

Publications and source records attributed to Guille, M..

3 recordsLinked to original sources

bioRxiv2025

The Actin regulator Mena promotes Wnt signalosome endocytosis and Wnt signalling.

Wnt signaling controls embryonic development and tissue maintenance. Endocytosis of Wnt-receptors is required for signalling, yet uptake mechanisms remain poorly understood. Here, we identify the actin regulator and Ena/VASP protein, Mena, as a key mediator. Upon Wnt stimulation, Mena redistributes from focal adhesions to signalosomes, Wnt-receptor clusters. Mena directly binds Wnt-coreceptors LRP5/6 in a phosphorylation-dependent manner increasing Wnt signal transduction. Sequestration of Ena/VASP proteins impedes in vivo Wnt activation driving Xenopus embryonic development. We resolve previous controversies by showing that Wnt3a triggers rapid Clathrin-Mediated and Fast Endophilin-Mediated LRP6 endocytosis at low concentrations sufficient for Wnt activation. This efficient endocytosis requires Ena/VASP proteins and is specifically promoted by Mena. Our results suggest Mena as a crucial mediator of Wnt signalosome endocytosis thus promoting canonical Wnt signalling.

cell biology↗

bioRxiv2024

Cleland Immunoblotting Unmasks Unexpected Cysteine Redox Proteoforms

Cysteine redox proteoforms are virtually unstudied because they are extremely difficult to detect. They are difficult to detect by immunoblotting when the polyethylene glycol (PEG)-payloads used to mobility-shift proteoforms into distinct bands block antibody binding. Here, we synthesised a novel compound to reversibly crosslink the PEG-payloads to oxidised cysteines using disulfide bonds. To reductively release the PEG-payloads from mobility-shifted proteoforms, we soaked the gel in Clelands reagent: 1,4-dithiothreitol (DTT). Hence, Cleland immunoblotting. Cleland immunoblotting unmasked hitherto undetectable cysteine redox proteoforms. In Xenopus oocytes, we detected 2 cdc20-specific coordinates in an otherwise abstract space housing 1,024 theoretical proteoforms. The coordinates mapped to the fully, all 10 cysteine residues, 100%-reduced and 100%-oxidised proteoforms. The unexpected absence of partially oxidised molecules along the 10-reaction path to the 100%-oxidised proteoform implies novel biology. By providing the technological means to detect cysteine redox proteoforms, Cleland immunoblotting opens new avenues of discovery.

biochemistry↗

bioRxiv2021

An efficient miRNA knockout approach using CRISPR-Cas9 in Xenopus

In recent years CRISPR-Cas9 knockouts (KO) have become increasingly ultilised to study gene function. MicroRNAs (miRNAs) are short non-coding RNAs, 20-22 nucleotides long, which affect gene expression through post-transcriptional repression. We previously identified miRNAs-196a and -219 as implicated in the development of Xenopus neural crest (NC). The NC is a multipotent stem-cell population, specified during early neurulation. Following EMT NC cells migrate to various points in the developing embryo where they give rise to a number of tissues including parts of the peripheral nervous system and craniofacial skeleton. Dysregulation of NC development results in many diseases grouped under the term neurocristopathies. As miRNAs are so small it is difficult to design CRISPR sgRNAs that reproducibly lead to a KO. We have therefore designed a novel approach using two guide RNAs to effectively drop out a miRNA. We have knocked out miR-196a and miR-219 and compared the results to morpholino knockdowns (KD) of the same miRNAs. Validation of efficient CRISPR miRNA KO and phenotype analysis included use of whole-mount in situ hybridization of key NC and neural plate border markers such as Pax3, Xhe2, Sox10 and Snail2, q-RT-PCR and Sanger sequencing. miRNA-219 and miR-196a KOs both show loss of NC, altered neural plate and hatching gland phenotypes. Tadpoles show gross craniofacial and pigment phenotypes.

developmental biology↗