RESUMO
NDC80 complex (NDC80C) is composed of four subunits (SPC24, SPC25, NDC80, and NUF2) and is vital for kinetochore-microtubule (KT-MT) attachment during mitosis. Paradoxically, NDC80C also functions in the activation of the spindle-assembly checkpoint (SAC). This raises an interesting question regarding how mitosis is regulated when NDC80C levels are compromised. Using a degron-mediated depletion system, we found that acute silencing of SPC24 triggered a transient mitotic arrest followed by mitotic slippage. SPC24-deficient cells were unable to sustain SAC activation despite the loss of KT-MT interaction. Intriguingly, our results revealed that other subunits of the NDC80C were co-downregulated with SPC24 at a posttranslational level. Silencing any individual subunit of NDC80C likewise reduced the expression of the entire complex. We found that the SPC24-SPC25 and NDC80-NUF2 subcomplexes could be individually stabilized using ectopically expressed subunits. The synergism of SPC24 downregulation with drugs that promote either mitotic arrest or mitotic slippage further underscored the dual roles of NDC80C in KT-MT interaction and SAC maintenance. The tight coordinated regulation of NDC80C subunits suggests that targeting individual subunits could disrupt mitotic progression and provide new avenues for therapeutic intervention. IMPLICATIONS: These results highlight the tight coordinated regulation of NDC80C subunits and their potential as targets for antimitotic therapies.
Assuntos
Proteínas de Ciclo Celular , Proteínas do Citoesqueleto , Mitose , Proteínas Nucleares , Humanos , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Células HeLa , Proteínas do Citoesqueleto/metabolismo , Proteínas do Citoesqueleto/genética , Cinetocoros/metabolismo , Pontos de Checagem da Fase M do Ciclo Celular/genética , Fuso Acromático/metabolismo , Subunidades Proteicas/metabolismo , Subunidades Proteicas/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/genéticaRESUMO
Listeria monocytogenes is a Gram-positive, intracellular pathogen that is highly adapted to invade and replicate in the cytosol of eukaryotic cells. Intermediate metabolites in the menaquinone biosynthesis pathway are essential for the cytosolic survival and virulence of L. monocytogenes, independent of the production of menaquinone (MK) and aerobic respiration. Determining which specific intermediate metabolite(s) are essential for cytosolic survival and virulence has been hindered by the lack of an identified 1,4-dihydroxy-2-naphthoyl-coenzyme A (DHNA-CoA) thioesterase essential for converting DHNA-CoA to DHNA in the MK synthesis pathway. Using the recently identified Escherichia coli DHNA-CoA thioesterase as a query, homology sequence analysis revealed a single homolog in L. monocytogenes, LMRG_02730 Genetic deletion of LMRG_02730 resulted in an ablated membrane potential, indicative of a nonfunctional electron transport chain (ETC) and an inability to aerobically respire. Biochemical kinetic analysis of LMRG_02730 revealed strong activity toward DHNA-CoA, similar to its E. coli homolog, further demonstrating that LMRG_02730 is a DHNA-CoA thioesterase. Functional analyses in vitro, ex vivo, and in vivo using mutants directly downstream and upstream of LMRG_02730 revealed that DHNA-CoA is sufficient to facilitate in vitro growth in minimal medium, intracellular replication, and plaque formation in fibroblasts. In contrast, protection against bacteriolysis in the cytosol of macrophages and tissue-specific virulence in vivo requires the production of 1,4-dihydroxy-2-naphthoate (DHNA). Taken together, these data implicate LMRG_02730 (renamed MenI) as a DHNA-CoA thioesterase and suggest that while DHNA, or an unknown downstream product of DHNA, protects the bacteria from killing in the macrophage cytosol, DHNA-CoA is necessary for intracellular bacterial replication.
