Endogenous tagging of the heterotrimeric kinesin-2 motor and inducible vimentin relocalization to study intracellular organelle organisation

Abstract

Within cells, organelles are organized in a distinct manner where most organelles are clustered around the nucleus. This perinuclear architecture is essential for organelle function and is maintained through interactions with the cytoskeleton. Organelles are transported along the microtubule (MT) network by MT associated motor proteins. A balance in transport mediated by the minus-end-directed dynein motor and plus-end-directed kinesin motors preserves organelle organization. The largest organelle in the cell, the endoplasmic reticulum (ER) has a distinct morphology that is dependent on association with kinesin-1 and dynein. However, new evidence indicates a heterotrimeric kinesin-2 could play a role in shaping the ER as well. In this study, we therefore aimed to tag this kinesin-2 endogenously and we succeeded using the CRISPR/Cas9-based pORANGE technique. While the importance of the MT network in regulating organelle positioning is extensively studied, the less well studied vimentin network might also be involved. Vimentin intermediate filaments are a component of the cytoskeleton and they form a dense network around the nucleus. The vimentin network has recently been suggested to function as an organelle scaffold, supposedly immobilizing organelles in the perinuclear region. Here, we investigated potential interactions between vimentin, the MT network, the ER, mitochondria and lysosomes to examine the role of vimentin in maintaining organelle architecture. We developed an FRB-FKBP Rapalog-inducible system and used it to reposition vimentin. Our results show that vimentin pulls along ER and mitochondria, indicating an interaction. Lysosomes, on the other hand, were not pulled along but they were nonetheless dependent on the vimentin network for their perinuclear localization. We confirmed the Rapalog system is a useful tool to analyze interactions of the vimentin cytoskeletal network with other organelles. Additionally, our preliminary data suggests vimentin anchors organelles in the perinuclear area through direct or indirect interactions.