Membrane compartmentalization of Ect2/Cyk4/Mklp1 and NuMA/dynein regulates cleavage furrow formation.

In animal cells, spindle elongation during anaphase is temporally coupled with cleavage furrow formation. Spindle elongation during anaphase is regulated by NuMA/dynein/dynactin complexes that occupy the polar region of the cell membrane and are excluded from the equatorial membrane. How NuMA/dynein/dynactin are excluded from the equatorial membrane and the biological significance of this exclusion remains unknown. Here, we show that the centralspindlin (Cyk4/Mklp1) and its interacting partner RhoGEF Ect2 are required for NuMA/dynein/dynactin exclusion from the equatorial cell membrane. The Ect2-based (Ect2/Cyk4/Mklp1) and NuMA-based (NuMA/dynein/dynactin) complexes occupy mutually exclusive membrane surfaces during anaphase. The equatorial membrane enrichment of Ect2-based complexes is essential for NuMA/dynein/dynactin exclusion and proper spindle elongation. Conversely, NuMA-based complexes at the polar region of the cell membrane ensure spatially confined localization of Ect2-based complexes and thus RhoA. Overall, our work establishes that membrane compartmentalization of NuMA-based and Ect2-based complexes at the two distinct cell surfaces restricts dynein/dynactin and RhoA for coordinating spindle elongation with cleavage furrow formation.

Innate immune sensing of influenza A viral RNA through IFI16 promotes pyroptotic cell death.

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 interaction with chromatin is vital for proper chromosome decondensation at the mitotic exit.

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.

PP2A--B55γ counteracts Cdk1 and regulates proper spindle orientation through the cortical dynein adaptor NuMA.

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.

Plk1 regulates spindle orientation by phosphorylating NuMA in human cells.

Proper orientation of the mitotic spindle defines the correct division plane and is essential for accurate cell division and development. In metazoans, an evolutionarily conserved complex comprising of NuMA/LGN/Gαi regulates proper orientation of the mitotic spindle by orchestrating cortical dynein levels during metaphase. However, the molecular mechanisms that modulate the spatiotemporal dynamics of this complex during mitosis remain elusive. Here, we report that acute inactivation of Polo-like kinase 1 (Plk1) during metaphase enriches cortical levels of dynein/NuMA/LGN and thus influences spindle orientation. We establish that this impact of Plk1 on cortical levels of dynein/NuMA/LGN is through NuMA, but not via dynein/LGN. Moreover, we reveal that Plk1 inhibition alters the dynamic behavior of NuMA at the cell cortex. We further show that Plk1 directly interacts and phosphorylates NuMA. Notably, NuMA-phosphorylation by Plk1 impacts its cortical localization, and this is needed for precise spindle orientation during metaphase. Overall, our finding connects spindle-pole pool of Plk1 with cortical NuMA and answers a long-standing puzzle about how spindle-pole Plk1 gradient dictates proper spindle orientation for error-free mitosis.