Browsing by Subject "Golgi Apparatus"
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Item Dissecting the Mitotic Golgi Membranes Mediated Microtubule Polymerization(2019-11-25) Guo, Haijing; Henne, W. Mike; Goodman, Joel M.; Yu, Hongtao; Seemann, JoachimA properly assembled astral microtubule network is required for correct mitotic spindle orientation, which is important in multiple development processes as it determines cell fate and function. The initiation and growth of astral microtubules was previously attributed to centrosomes and microtubule stabilizing proteins. Here in my dissertation research, I demonstrate that microtubules initiated by mitotic Golgi membranes contribute to the growth of astral microtubules and the proper orientation of the spindle. In turn, the microtubule initiation activity of mitotic Golgi membranes facilitates the proper inheritance of the single copy Golgi apparatus, which is essential in polarized cellular functions, including directional cell migration and secretion. Microtubule assembly is initiated by the Golgi resident protein GM130, which locally activates the spindle assembly factor TPX2 at the mitotic Golgi membranes. GM130 relieves TPX2 from inhibition by competing for importin α binding. The mitotic phosphorylation of importin α on Serine 62 by Cdk1 switches its substrate preference towards GM130 and enables the competition-based activation. The importin α S62A mutant impedes the local TPX2 activation and compromises the astral microtubules, which ultimately leads to misoriented spindles. Blocking of the GM130-importin α-TPX2 activation pathway reduces the astral microtubule growth rate. I also identified that the human GM130 homolog GLP harbors a domain that is highly similar to the TPX2 activating domain of GM130, which could potentially initiate microtubule assembly. My research reveals the novel role of mitotic Golgi membranes in astral spindle organization and the underlying mechanism that regulates this process in a spatio-temporal manner.Item Investigating the Mechanism and Mode of Action of Golgi Toxins(December 2023) Cervantes, Christopher Luis; Liszczak, Glen; Posner, Bruce A.; Wang, Fei; Nijhawan, DeepakAuxin-inducible forward genetics uncovered point mutations within Golgi Brefeldin A Resistant Guanine Nucleotide Exchange Factor 1 or GBF1 following lethal dose selection with a synthetic disubstituted pyrimidine toxin called Golgitox (GTOX). Resistant clones were also cross-resistant to the fungal toxin, Brefeldin A (BFA), and synthetic GBF1 inhibitor, Golgicide A (GCA). Like BFA and GCA, GTOX triggered Golgi disassembly via GBF1. Given that BFA is a reported molecular glue, we profiled Gbf1-Arf interactions in 293T cell lysates pre-treated with Golgi toxin. Both GTOX and GCA promoted GBF1-dependent interactions with Arfs 4 and 5, whereas BFA also interacted with Arf1. GBF1 domain mapping revealed that the HUS-SEC7-HDS1 domains were sufficient for promoting GTOX-dependent engagement with Arfs 4 and 5. Meanwhile, structural activity relationship studies showed that modifying the methyl group on the benzimidazole ring preserved GTOX activity and interactions between Gbf1 and Arfs 4 and 5. To assess which Arfs regulate BFA and GTOX cytotoxicities, genome-wide CRISPR/Cas9 compound enrichment screens were carried out, which identified ARF4 as being the most enriched hit. Next, we validated that ARF4 loss-of-function partially confers resistance to BFA and GTOX. Next, we asked whether GTOX preferentially interacts with ARF4-GDP versus ARF4-GTP. We found that exogenous ARF4 T31N (GDP-locked mutant) sensitized HCT116 scramble control cells 4-fold to GTOX, interacted with Gbf1 just as well as WT Arf4, but failed to rescue Arf4-mediated BiP retrieval. Collectively, these results suggest that the Gbf1-GTOX-Arf4-GDP complex is functionally inactive but deleterious to cell viability. Taken together, GTOX may act as a molecular glue to suppress GBF1 functions through downstream effector substrates like Arfs 4 and 5.Item The Mitotic Spindle Mediates Inheritance of the Golgi Ribbon Structures(2010-05-14) Wei, Jen-Hsuan; Seemann, JoachimThe mammalian Golgi ribbon disassembles during mitosis and reforms in both daughter cells after division. Mitotic Golgi membranes concentrate around the spindle poles, suggesting that the spindle may control Golgi partitioning. To test this, cells were induced to divide asymmetrically with the entire spindle segregated into only one daughter cell. A ribbon reforms in the nucleated karyoplasts, whereas the Golgi stacks in the cytoplasts are scattered. However, the scattered Golgi stacks are polarized and transport cargo. Microinjection of Golgi extract together with tubulin or incorporation of spindle materials rescues Golgi ribbon formation. Therefore, the factors required for postmitotic Golgi ribbon assembly are transferred by the spindle, but the constituents of functional stacks are partitioned independently, suggesting that Golgi inheritance is regulated by two distinct mechanisms.