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Updated: Oct 17, 2025
Ex vivo Culturing of Whole, Developing Drosophila Brains
Published on: July 27, 2012
In vivo Profiling of the Alk Proximitome in the Developing Drosophila Brain
Ezgi Uçkun1, Georg Wolfstetter1, Vimala Anthonydhason1
1Department of Medical Biochemistry and Cell Biology, Instititute of Biomedicine at the Sahlgrenska Academy, University of Gothenburg, SE-40530 Gothenburg, Sweden.
Abstract:
Anaplastic lymphoma kinase (Alk) is an evolutionary conserved receptor tyrosine kinase belonging to the insulin receptor superfamily. In addition to its well-studied role in cancer, numerous studies have revealed 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. In this study, we used BioID-based in vivo proximity labeling to identify molecules that interact with Alk in the Drosophila central nervous system (CNS). To do this, we used CRISPR/Cas9 induced homology-directed repair (HDR) to modify the endogenous Alk locus to produce first and next generation Alk::BioID chimeras. This approach allowed identification of Alk proximitomes under physiological conditions and without overexpression. Our results show that the next generation of BioID proteins (TurboID and miniTurbo) outperform the first generation BirA* fusion in terms of labeling speed and efficiency. LC-MS3-based BioID screening of AlkTurboID and AlkminiTurbo larval brains revealed an extensive neuronal Alk proximitome identifying numerous potential components of Alk signaling complexes. Validation of Alk proximitome candidates further revealed co-expression of Stardust (Sdt), Discs large 1 (Dlg1), Syntaxin (Syx) and Rugose (Rg) with Alk in the CNS and identified the protein-tyrosine-phosphatase Corkscrew (Csw) as a modulator of Alk signaling.
Insights
Anaplastic lymphoma kinase (Alk) signaling impacts complex traits. Using BioID proximity labeling in Drosophila, researchers identified novel Alk interacting proteins in the central nervous system, revealing new signaling pathways.
Area of Science:
- Neuroscience
- Molecular Biology
- Genetics
Background:
- Anaplastic lymphoma kinase (Alk) is a conserved receptor tyrosine kinase implicated in cancer and various complex biological processes.
- Alk signaling is involved in growth, metabolism, neurotransmission, learning, and substance use disorders.
- Understanding Alk's molecular interactions is crucial for deciphering its diverse roles.
Purpose of the Study:
- To identify molecules interacting with Alk in the Drosophila central nervous system (CNS) under physiological conditions.
- To leverage BioID proximity labeling for unbiased identification of Alk-associated proteins.
- To characterize the neuronal Alk proximitome without overexpression artifacts.
Main Methods:
- Utilized CRISPR/Cas9-mediated homology-directed repair (HDR) to generate endogenous Alk::BioID chimeras (Alk::BirA*, Alk::TurboID, Alk::miniTurbo).
- Employed in vivo proximity labeling (BioID) to capture interacting proteins in Drosophila larval brains.
- Analyzed labeled proteins using liquid chromatography-mass spectrometry (LC-MS3) for proteomic screening.
Main Results:
- Next-generation BioID proteins (TurboID, miniTurbo) demonstrated superior labeling speed and efficiency compared to BirA*.
- Identified an extensive neuronal Alk proximitome, revealing numerous novel components of Alk signaling complexes.
- Validated co-expression of Stardust (Sdt), Discs large 1 (Dlg1), Syntaxin (Syx), and Rugose (Rg) with Alk in the CNS.
- Discovered Corkscrew (Csw), a protein-tyrosine-phosphatase, as a modulator of Alk signaling.
Conclusions:
- The study successfully mapped the Alk proximitome in the Drosophila CNS using advanced BioID techniques.
- The findings provide a comprehensive resource for understanding Alk signaling networks in neurons.
- Identified key interacting proteins and a novel modulator (Csw) offer new avenues for research into Alk function.
