Biology subjects

Basler, K.

Publications and source records attributed to Basler, K..

4 recordsLinked to original sources

bioRxiv2018

Generation of a versatile BiFC ORFeome library for analyzing protein-protein interactions in live Drosophila

Transcription factors achieve specificity by establishing intricate interaction networks that will change depending on the cell context. Capturing these interactions in live condition is however a challenging issue that requires sensitive and non-invasive methods. We present a set of fly lines, called \"multicolor BiFC library\", which covers most of the Drosophila transcription factors for performing Bimolecular Fluorescence Complementation (BiFC). The multicolor BiFC library can be used to probe binary or tripartite interactions and is compatible for large-scale interaction screens. The library can also be coupled with established Drosophila genetic resources to analyze interactions in the developmentally relevant expression domain of each protein partner. We provide proof of principle experiments of these various applications, using Hox proteins in the live Drosophila embryo as a case study. Overall this novel collection of ready-to-use fly lines constitutes an unprecedented genetic toolbox for the identification and analysis of protein-protein interactions in vivo.

genomics↗

bioRxiv2018

Pharmacophore-guided discovery of CDC25 inhibitors causing cell cycle arrest and cell death

CDC25 phosphatases have a key role in cell cycle transitions and are important targets for cancer therapy. Here, we set out to discover novel CDC25 inhibitors. Using a combination of computational approaches we defined a minimal common pharmacophore in established CDC25 inhibitors and performed a virtual screening of a proprietary library. Taking advantage of the availability of crystal structures for CDC25A and CDC25B and using a molecular docking strategy, we carried out hit expansion/optimization. Enzymatic assays revealed that naphthoquinone scaffolds were the most promising CDC25 inhibitors among selected hits. At the molecular level, the compounds acted through a mixed-type mechanism of inhibition of phosphatase activity, involving reversible oxidation of cysteine residues. In 2D cell cultures, the compounds caused arrest of the cell cycle at the G1/S or at the G2/M transition. Mitotic markers analysis and time-lapse microscopy confirmed that CDK1 activity was impaired and that mitotic arrest was followed by death. Finally, studies on 3D organoids derived from intestinal crypt stem cells of Apc/K-Ras mice revealed that the compounds caused arrest of proliferation.

cancer biology↗

bioRxiv2018

Mutations in Bcl9 and Pygo genes cause congenital heart defects by tissue-specific perturbation of Wnt/β-catenin signaling

Genetic alterations in human BCL9 genes have repeatedly been found in congenital heart disease (CHD) with as-of-yet unclear causality. BCL9 proteins and their Pygopus (Pygo) co-factors can participate in canonical Wnt signaling via binding to {beta}-catenin. Nonetheless, their contributions to vertebrate heart development remain uncharted. Here, combining zebrafish and mouse genetics, we document tissue-specific functions in canonical Wnt signaling for BCL9 and Pygo proteins during heart development. In a CRISPR-Cas9-based genotype-phenotype association screen, we uncovered that zebrafish mutants for bcl9 and pygo genes largely retain {beta}-catenin activity, yet develop cardiac malformations. In mouse, both systemic and lineage-specific loss of the Pygo-BCL9-{beta}-catenin complex caused heart defects with outflow tract malformations, aberrant cardiac septation and valve formation, and compact myocardium hypoplasia. Mechanistically, these phenotypes coincide with transcriptional deregulation during heart patterning, and Pygo2 associates with {beta}-catenin at cis-regulatory regions of cardiac genes. Taken together, our results establish BCL9 and Pygo as tissue-specific {beta}-catenin co-factors during vertebrate heart development. Our results further implicate alterations in BCL9 and BCL9L in human CHDs as possibly causative.

genetics↗

bioRxiv2017

Systematic transcriptomics reveals a biphasic mode of sarcomere morphogenesis in flight muscles regulated by Spalt

Muscles organise pseudo-crystalline arrays of actin, myosin and titin filaments to build force-producing sarcomeres. To study how sarcomeres are built, we performed transcriptome sequencing of developing Drosophila flight muscles and identified 40 distinct expression profile clusters. Strikingly, two clusters are strongly enriched for sarcomeric components. Temporal gene expression together with detailed morphological analysis enabled us to define two distinct phases of sarcomere development, which both require the transcriptional regulator Spalt major. During the sarcomere formation phase, 1.8 m long immature sarcomeres assemble myofibrils that spontaneously contract. During the sarcomere maturation phase, these sarcomeres grow to their final 3.2 m length and 1.5 m diameter and acquire stretch-sensitivity. Interestingly, the final number of myofibrils per flight muscle fiber is determined at the onset of the first phase. Together, this defines a biphasic mode of sarcomere and myofibril morphogenesis - a new concept that may also apply to vertebrate muscle or heart development.

developmental biology↗