The X-Linked-Intellectual-Disability-Associated Ubiquitin Ligase Mid2 Interacts with Astrin and Regulates Astrin Levels to Promote Cell Division

Abstract

SUMMARY Mid1 and Mid2 are ubiquitin ligases that regulate microtubule dynamics and whose mutation is associated with X-linked developmental disorders. We show that Astrin, a microtubule-organizing protein, co-purifies with Mid1 and Mid2, has an overlapping localization with Mid1 and Mid2 at intercellular bridge microtubules, is ubiquitinated by Mid2 on lysine 409 and is degraded during cytokinesis. Mid2 depletion led to Astrin stabilization during cytokinesis, cytokinetic defects, multinucleated cells, and cell death. Similarly, expression of a K409A mutant Astrin in Astrin-depleted cells led to the accumulation of K409A on intercellular bridge microtubules and an increase in cytokinetic defects, multinucleated cells, and cell death. These results indicate that Mid2 regulates cell division through the ubiquitination of Astrin on K409, which is critical for its degradation and proper cytokinesis. These results may help explain how mutation of MID2 leads to misregulation of microtubule organization and the downstream disease pathology associated with X-linked intellectual disabilities.

Fatostatin Inhibits Cancer Cell Proliferation by Affecting Mitotic Microtubule Spindle Assembly and Cell Division

“…Fixed and Live Cell Imaging-Fixed and live cell imaging was carried out as described previously (21,26). For quantifying spindle and mitotic defects, 100 cells from three independent experiments were counted, and the data are presented as the average Ϯ S.D.…”

Inducible LAP-tagged Stable Cell Lines for Investigating Protein Function, Spatiotemporal Localization and Protein Interaction Networks

“…With respect to other LAP/TAP-tagging approaches, this protocol has been streamlined to be compatible with high-throughput approaches to map protein localization and protein-protein interactions within any cellular pathway. This approach has been widely applied to the functional characterization of proteins critical for cell cycle progression, mitotic spindle assembly, spindle pole homeostasis, and ciliogenesis to name a few and has aided the understanding of how misregulation of these proteins can lead to human diseases 15,16,19,20 . For example, our group recently utilized this system to define the function and regulation of the STARD9 mitotic kinesin (a candidate cancer target) in spindle assembly 15,21 , to define a new molecular link between the Tctex1d2 dynein light chain and short rib polydactyly syndromes (SRPS) 19 , and to define a new molecular link to understanding how mutation of the Mid2 ubiquitin ligase can lead to X-linked intellectual disabilities 16 .…”

“…This approach has been widely applied to the functional characterization of proteins critical for cell cycle progression, mitotic spindle assembly, spindle pole homeostasis, and ciliogenesis to name a few and has aided the understanding of how misregulation of these proteins can lead to human diseases 15,16,19,20 . For example, our group recently utilized this system to define the function and regulation of the STARD9 mitotic kinesin (a candidate cancer target) in spindle assembly 15,21 , to define a new molecular link between the Tctex1d2 dynein light chain and short rib polydactyly syndromes (SRPS) 19 , and to define a new molecular link to understanding how mutation of the Mid2 ubiquitin ligase can lead to X-linked intellectual disabilities 16 . Other laboratories have also successfully applied this method, including one that determined that Tctn1, a regulator of mouse Hedgehog signaling, was a part of a ciliopathy-associated protein complex that regulated ciliary membrane composition and ciliogenesis in a tissue-dependent manner 22,23 .…”

“…Test the quality of the purification by analyzing the collected samples by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), silver staining the gel (see Materials Table) and by immunoblotting the eluates and probing with anti-GFP antibodies to ensure that the LAP-tagged purification worked 16 , see Figure 4. 2.…”

“…Stain the gel with a mass spectrometry compatible protein stain. Excise the most prominent bands and the space in between them from the gel and process them for analysis by mass spectrometry separately 16 . NOTE: There are numerous approaches for separating the final purification eluates and preparing them for mass spectrometry 5 .…”