Project description:In this set of experiments we identified the main scaffolding component of centriolar satellites, PCM1 or Cmb in Drosophila as a proximity interactor of the centriolar structural protein Sas4 by BioID in S2 cells and subsequently explored the Cmb proximity interactome by direct TurboID in S2 cells and indirect, GFP nanobody-targeted, TurboID in Drosophila testes.
Project description:Anaplastic lymphoma kinase (Alk) is an evolutionary conserved receptor tyrosine kinase of the insulin receptor family. In addition to its well-studied role in cancer, numerous studies on invertebrate and vertebrate models reveal that Alk-signaling is associated with a variety of complex traits such as: regulation of growth and metabolism, hibernation, regulation of neurotransmitters, synaptic coupling, axon targeting, decision making, memory formation and learning, alcohol use disorder, as well as steroid hormone metabolism. To shed light on ALK-driven signaling processes, we used BioID-based in vivo proximity labeling to reveal the molecules that interact with Alk in the Drosophila CNS. Therefore, we generated first and next generation BioID fusion proteins in the endogenous Alk locus by CRISPR/Cas9 induced homology-directed repair (HDR) resulting in viable and fertile flies. Our results show (i) that the next generation of BioID proteins (TurboID and miniTurbo) outperform the first generation BirA* fusion in terms of labeling efficiency, and (ii) allow identification of proximitomes without overexpression which are likely to be more physiologically relevant. LC-MS/MS-based BioID screening of the larval brain revealed an extensive neuronal Alk-proximitome identifying numerous novel potential components of Alk signaling complexes. Validation of Alk-proximitome candidates further revealed co-expression with Alk in the CNS.
Project description:SH-SY5Y cells were transfected with either BioID-GFP-NLS control or BioID-Flag-ERRg expressing adenoviruses and isolated with Trizol after 48 hours to compare gene expression and find potential ERRg targets specific to neuron-like cells.
Project description:ChIP-chip experiment for nuclear pore proteins Nup153 and Mtor in Drosophila S2 and Kc cells. This experiment is related to E-MEXP-2523.
Project description:In order to identify interaction partner of the Drosophila melanogaster TFIIA protein, we have immunoprecipitated an endogenously 3xFLAG-AID tagged TFIIA-L from Drosophila Schneider S2 cells