The Mid1 and Mid2 Ubiquitin Ligases Associate with Astrin and Regulate Astrin Levels During Cytokinesis to Promote Proper Cell Abscission

Abstract

The TRIM/RBCCsuperfamily of ubiquitin ligases has variable roles in cellular homeostasis including signaling, regulation of cytoskeletal structures and cell cycle progression. Misregulation of TRIM family members has been linked to developmental disorders and oncogenesis. The Mid1 (Midline 1 also known as Trim18) and Mid2 (Midline 2 also known as Trim1) ubiquitin ligases share a similar domain architecture including a domain that mediates the binding of these proteins to microtubules. Mid1 and Mid2 also homo and heterodimerize and have been implicated in anchoring proteins to microtubules and in bundling, stabilization and organization of microtubules. We have explored the role of Mid1 and Mid2 during cell division and determined that Mid1 and Mid2 regulate Astrin protein levels in a cell cycle dependent manner, which is critical for proper cytokinesis. Characterization of the Mid1 and Mid2 interactomes identified Astrin as the top interacting protein. Mid1 and Mid2 not only associate with Astrin, they also colocalize with Astrin to intracellular bridge microtubules during telophase. Astrin levels normally increase during mitotic entry and decrease during mitotic exit and depletion of Mid2 stabilized Astrin during mitotic exit, arrested cells during cytokinesis and generated binucleated cells. Additionally, Mid2 was able to ubiquitinate Astrin in vitro. We propose that Mid1 and Mid2 ubiquitinate and target a pool of Astrin, localized to intracellular bridge microtubules, for degradation prior to cell abscission and that this mechanism is important for ensuring that microtubule bundling/crosslinking is relieved as part of the microtubule disassembly process during cytokinesis, which is necessary for proper cell division.