Towards Auxin-Inducible Degradation in Mouse Small Intestinal Organoids

Abstract

The Golgi complex is essential for the processing, sorting, and packaging of proteins and lipids to their intra- and extracellular target destinations. To execute these functions efficiently, the Golgi complex is highly depended on its integrity and spatial organization. Recently, the Scharaw laboratory discovered that unlike the classical single textbook Golgi complex, mouse small intestinal stem cells (ISCs) display multiple Golgi stacks oriented to the lateral membranes. Furthermore, in ISCs comprising multiple Golgi complexes, the scaffolding protein A-kinase anchoring protein 9 (AKAP9/AKAP450) is enhanced expressed, compared to ISCs with a single Golgi complex. This indicates the importance of AKAP9 in multiple Golgi complex arrangement of ISCs. However, the specific role of AKAP9 in this process remains unknown. To address this, we take steps towards the implementation of the auxin-inducible degron (AID) protein degradation system in mouse small intestinal organoids. Once established, this system will enable the conditional depletion of AKAP9. To investigate the feasibility of this system for use in mouse small intestinal organoids, we first aimed to establish the AID system in 4T1 epithelial mouse breast cancer cells. To introduce the AID machinery in the 4T1 cells, we performed electroporations and opmized an antibiotic selection procedure, leading to complete selection of positive clones. In parallel, we opmized the electroporation of mouse small intestinal organoids, required to introduce the AID machinery. Lastly, we developed a strategy to endogenously tag Akap9 for AID dependent degradation, which shows no major alterations in AKAP9s predicted protein structure. Establishing AID in mouse small intestinal organoids may form a valuable tool that allows us to study the molecular origins and regulators of the multiple Golgi organization observed in ISCs.