This work reveals that Plk1 phosphorylates an evolutionarily conserved protein NuMA in human cells and this phosphorylation negatively regulates NuMA and thus dynein cortical localization in metaphase that is crucial for proper spindle orientation.

Proper orientation of the mitotic spindle is critical for accurate development and morphogenesis. In human cells, spindle orientation is regulated by the evolutionarily conserved protein NuMA, which interacts with dynein and enriches it at the cell cortex. Pulling forces generated by cortical dynein orient the mitotic spindle. Cdk1-mediated phosphorylation of NuMA at threonine 2055 (T2055) negatively regulates its cortical localization. Thus, only NuMA not phosphorylated at T2055 localizes at the cell cortex. However, the identity and the mechanism of action of the phosphatase complex involved in T2055 dephosphorylation remains elusive. Here, we characterized the PPP2CA-B55γ (PPP2R2C)–PPP2R1B complex that counteracts Cdk1 to orchestrate cortical NuMA for proper spindle orientation. In vitro reconstitution experiments revealed that this complex is sufficient for T2055 dephosphorylation. Importantly, we identified polybasic residues in NuMA that are critical for T2055 dephosphorylation, and for maintaining appropriate cortical NuMA levels for accurate spindle elongation. Furthermore, we found that Cdk1-mediated phosphorylation and PP2A-B55γ-mediated dephosphorylation at T2055 are reversible events. Altogether, this study uncovers a novel mechanism by which Cdk1 and its counteracting PP2A-B55γ complex orchestrate spatiotemporal levels of cortical force generators for flawless mitosis.

Summary Programmed cell death pathways are triggered by various stresses or stimuli, including viral infections. The mechanism underlying the regulation of these pathways upon Influenza A virus (IAV) infection is not well characterized. We report that a cytosolic DNA sensor IFI16 is essential for the activation of programmed cell death pathways in IAV infected cells. We have identified that IFI16 functions as an RNA sensor for the influenza A virus by interacting with genomic RNA. The activation of IFI16 triggers the production of type I, III interferons, and also pro-inflammatory cytokines via the STING-TBK1 and Pro-caspase-1 signaling axis, thereby promoting cell death (apoptosis and pyroptosis in IAV infected cells). On the contrary, IFI16 knockdown cells showed reduced inflammatory responses and also prevented cell mortality during IAV infection. Collectively, these results demonstrate the pivotal role of IFI16-mediated IAV sensing and its essential role in activating programmed cell death pathways.

NuMA is an abundant long coiled-coil protein that plays a prominent role in spindle organization during mitosis. In interphase, NuMA is localized to the nucleus and hypothesized to control gene expression and chromatin organization. However, because of the prominent mitotic phenotype upon NuMA loss, its precise function in the interphase nucleus remains elusive. Here, we report that NuMA is associated with chromatin in interphase and prophase but released upon nuclear envelope breakdown (NEBD) by the action of Cdk1. We uncover that NuMA directly interacts with DNA via evolutionarily conserved sequences in its C-terminus. Notably, the expression of the DNA-binding deficient mutant of NuMA affects chromatin decondensation at the mitotic exit, and nuclear shape in interphase. We show that the nuclear shape defects observed upon mutant NuMA expression are due to its potential to polymerize into higher-order fibrillar structures. Overall, this work establishes the spindle independent function of NuMA in choreographing proper chromatin decompaction and nuclear shape by directly associating with the DNA. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text]

Programmed cell death pathways are triggered by various stresses or stimuli, including viral infections. The mechanism underlying the regulation of these pathways upon Influenza A virus IAV infection is not well characterized. We report that a cytosolic DNA sensor IFI16 is essential for the activation of programmed cell death pathways in IAV infected cells. We have identified that IFI16 functions as an RNA sensor for influenza A virus by binding to genomic RNA. The activation of IFI16 triggers the production of type I, III interferons, and also other pro-inflammatory cytokines via the STING-TBK1 and Pro-caspase-1 signaling axis, thereby promoting cell death (apoptosis and pyroptosis in IAV infected cells). Whereas, IFI16 knockdown cells showed reduced inflammatory responses and also prevented cell mortality during IAV infection. These results demonstrate the pivotal role of IFI16-mediated IAV sensing and its essential role in activating programmed cell death pathways.