Abstract:GPR124 is involved in embryonic development and remains expressed by select organs. The importance of GPR124 during development suggests that its aberrant expression might participate in tumor growth. Here we show that both increases and decreases in GPR124 expression in glioblastoma cells reduce cell proliferation by differentially altering the duration mitotic progression. Using mass spectrometry‐based proteomics, we discovered that GPR124 interacts with ch‐TOG, a known regulator of both microtubule (MT)‐plu… Show more
“…Previous microscopy studies suggested that chTOG localizes to kinetochores ( Campbell et al, 2019 ; Gutiérrez-Caballero et al, 2015 ; Ryan et al, 2020 ), similar to the budding yeast ortholog ( Miller et al, 2016 ; Miller et al, 2019 ); however, it was unclear if this population was simply bound to microtubule tips. To address this, we used engineered HCT116 cells where the endogenous chTOG genes were epitope tagged with EGFP ( Cherry et al, 2019 ) to determine whether chTOG specifically localizes to kinetochores throughout mitosis ( Figure 1a ). We found that chTOG is largely excluded from the nucleus until prometaphase, when it appeared on kinetochores as assayed by co-localization with anti-centromere antibody (ACA) ( Figure 1a ).…”
Section: Resultsmentioning
confidence: 99%
“…HCT116 ( Cherry et al, 2019 ), 293T ( Ding et al, 2013 ), and HeLa FlpIn Cells ( Etemad et al, 2015 ) cells were grown in a high-glucose DMEM (Thermo Fisher Scientific 11-965-118/Gibco 11965118) supplemented with antibiotic/antimycotic (Thermo Fisher Scientific 15240062) and 10% Foetal Bovine Serum (Thermo Fisher Scientific 26140095) at 37°C supplemented with 5% CO 2 . For microscopy experiments, cell suspensions were added to 35 mm wells containing acid washed 1.5 × 22 mm square coverslips (Fisher Scientific 152222) and grown for 12–24 hr prior to transfections or immunostaining.…”
Accurate chromosome segregation requires kinetochores on duplicated chromatids to biorient by attaching to dynamic microtubules from opposite spindle poles, which exerts forces to bring kinetochores under tension. However, kinetochores initially bind to microtubules indiscriminately, resulting in errors that must be corrected. While the Aurora B protein kinase destabilizes low-tension attachments by phosphorylating kinetochores, low-tension attachments are intrinsically less stable than those under higher tension in vitro independent of Aurora activity. Intrinsic tension-sensitive behavior requires the microtubule regulator Stu2 (budding yeast Dis1/XMAP215 ortholog), which we demonstrate here is likely a conserved function for the TOG protein family. The human TOG protein, chTOG, localizes to kinetochores independent of microtubules by interacting with Hec1. We identify a chTOG mutant that regulates microtubule dynamics but accumulates erroneous kinetochore-microtubule attachments that are not destabilized by Aurora B. Thus, TOG proteins confer a unique, intrinsic error correction activity to kinetochores that ensures accurate chromosome segregation.
“…Previous microscopy studies suggested that chTOG localizes to kinetochores ( Campbell et al, 2019 ; Gutiérrez-Caballero et al, 2015 ; Ryan et al, 2020 ), similar to the budding yeast ortholog ( Miller et al, 2016 ; Miller et al, 2019 ); however, it was unclear if this population was simply bound to microtubule tips. To address this, we used engineered HCT116 cells where the endogenous chTOG genes were epitope tagged with EGFP ( Cherry et al, 2019 ) to determine whether chTOG specifically localizes to kinetochores throughout mitosis ( Figure 1a ). We found that chTOG is largely excluded from the nucleus until prometaphase, when it appeared on kinetochores as assayed by co-localization with anti-centromere antibody (ACA) ( Figure 1a ).…”
Section: Resultsmentioning
confidence: 99%
“…HCT116 ( Cherry et al, 2019 ), 293T ( Ding et al, 2013 ), and HeLa FlpIn Cells ( Etemad et al, 2015 ) cells were grown in a high-glucose DMEM (Thermo Fisher Scientific 11-965-118/Gibco 11965118) supplemented with antibiotic/antimycotic (Thermo Fisher Scientific 15240062) and 10% Foetal Bovine Serum (Thermo Fisher Scientific 26140095) at 37°C supplemented with 5% CO 2 . For microscopy experiments, cell suspensions were added to 35 mm wells containing acid washed 1.5 × 22 mm square coverslips (Fisher Scientific 152222) and grown for 12–24 hr prior to transfections or immunostaining.…”
Accurate chromosome segregation requires kinetochores on duplicated chromatids to biorient by attaching to dynamic microtubules from opposite spindle poles, which exerts forces to bring kinetochores under tension. However, kinetochores initially bind to microtubules indiscriminately, resulting in errors that must be corrected. While the Aurora B protein kinase destabilizes low-tension attachments by phosphorylating kinetochores, low-tension attachments are intrinsically less stable than those under higher tension in vitro independent of Aurora activity. Intrinsic tension-sensitive behavior requires the microtubule regulator Stu2 (budding yeast Dis1/XMAP215 ortholog), which we demonstrate here is likely a conserved function for the TOG protein family. The human TOG protein, chTOG, localizes to kinetochores independent of microtubules by interacting with Hec1. We identify a chTOG mutant that regulates microtubule dynamics but accumulates erroneous kinetochore-microtubule attachments that are not destabilized by Aurora B. Thus, TOG proteins confer a unique, intrinsic error correction activity to kinetochores that ensures accurate chromosome segregation.
“…To address this, we used engineered HCT116 cells where endogenous chTOG alleles were epitope tagged with EGFP (Cherry et al, 2019) to determine whether chTOG specifically localizes to kinetochores throughout mitosis ( Fig. 1a).…”
Section: Resultsmentioning
confidence: 99%
“…Mammalian cell culture HCT116 (Cherry et al, 2019), 293T (Ding et al, 2013), and HeLa FlpIn Cells (Etemad, Kuijt, & Kops, 2015) To entirely depolymerize the microtubule cytoskeleton, growth media were supplemented with 10 µM nocodazole (Sigma-Aldrich, M1404) for one hour. To synchronize cells, they were treated with 2.5 mM thymidine (Sigma-Aldrich, T9250) for 16 hours, cells were then placed in drug-free media for 8 hours.…”
Accurate chromosome segregation requires kinetochores on duplicated chromatids to biorient by attaching to dynamic microtubules from opposite spindle poles, which exerts forces to bring kinetochores under tension. However, kinetochores initially bind to MTs indiscriminately, resulting in errors that must be corrected. While the Aurora B protein kinase destabilizes low-tension attachments by phosphorylating kinetochores, low-tension attachments are intrinsically less stable than those under higher tension in vitro independent of Aurora activity. Intrinsic tension-sensitive behavior requires the microtubule regulator Stu2 (budding yeast Dis1/XMAP215 ortholog), which we demonstrate here is likely a conserved function for the TOG protein family. The human TOG protein, chTOG, localizes to kinetochores independent of microtubules by interacting with Hec1. We identify a chTOG mutant that regulates microtubule dynamics but accumulates erroneous kinetochore-microtubule attachments that Aurora B fails to destabilize. Thus, TOG proteins confer a unique, intrinsic error correction activity to kinetochores that ensures accurate chromosome segregation.
“…Despite multiple attempts to generate a homozygous knock-in for chTOG–FKBP–GFP, we only recovered heterozygous lines (more than twenty heterozygous clones in three separate attempts). Although there is a report of homozygous knock-in of chTOG–FKBP–GFP in HCT116 cells ( Cherry et al, 2019 ), we assume that homozygous knock-in of chTOG–FKBP–GFP in HeLa cells is lethal. Fig.…”
A multiprotein complex containing TACC3, clathrin and other proteins has been implicated in mitotic spindle stability. To disrupt this complex in an anti-cancer context, we need to understand its composition and how it interacts with microtubules. Induced relocalization of proteins in cells is a powerful way to analyze protein–protein interactions and, additionally, monitor where and when these interactions occur. We used CRISPR/Cas9 gene editing to add tandem FKBP–GFP tags to each complex member. The relocalization of endogenous tagged protein from the mitotic spindle to mitochondria and assessment of the effect on other proteins allowed us to establish that TACC3 and clathrin are core complex members and that chTOG (also known as CKAP5) and GTSE1 are ancillary to the complex, binding respectively to TACC3 and clathrin, but not each other. We also show that PIK3C2A, a clathrin-binding protein that was proposed to stabilize the TACC3–chTOG–clathrin–GTSE1 complex during mitosis, is not a member of the complex. This work establishes that targeting the TACC3–clathrin interface or their microtubule-binding sites are the two strategies most likely to disrupt spindle stability mediated by this multiprotein complex.
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