PP2A-phosphatase and Polo-like kinase 1 regulates spindle positioning by orchestrating the cortical levels of the dynein adaptor NuMA

Abstract

The mitotic spindle is a microtubule-based diamond shape structure that captures chromosomes and facilitates their segregation during anaphase. Proper positioning of the mitotic spindle is critical for defining the positioning of the cleavage furrow at the onset of cytokinesis. This process further ensures that cell fate determinants are correctly segregated in the daughter cells during development and in stem cells. In animal cells, spindle positioning is controlled by the cortically anchored ternary complex comprising NuMA/LGN/Gαi1-3 in humans [LIN-5/GPR-1/2/-Gαi in C. elegans]. Here, NuMA acts as a cortical dynein-dynactin adaptor. It is the dynein-dynactin motor activity on the dynamic astral microtubules that generates pulling force and thus maintains proper spindle positioning. Importantly, it has been seen that the localization of the ternary complex components and dynein-dynactin is spatiotemporally regulated during mitosis. For instance, NuMA is weakly localized at the polar region of the cell cortex in metaphase, and significantly enriched during anaphase. The weak localization of NuMA ensures proper spindle positioning in metaphase, and its cortical enrichment in anaphase controls spindle elongation. This spatiotemporal localization of the ternary complex components and dynein-dynactin is regulation by the action of several mitotic kinases such as Cdk1 and Aurora A. However, our knowledge in the field of mitotic phosphatases, and their role in spindle positioning is very much limited. Therefore, in the first part of my thesis, I have tried to characterize the function of phosphatase in the viii proper spindle positioning using state-of-the-art molecular and cell biology-based methods. Here, I have identified a PP2A-based phosphatase complex that counteracts Cdk1 and regulates precise localization of NuMA, and dynein-dynactin in mitosis. In the second part, I have investigated the molecular mechanisms by which Polo-like kinase 1 (Plk1) regulates correct spindle positioning in human cells. 1) PP2A-B55γ counteracts Cdk1 to orchestrate spindle positioning through NuMA Previous reports showed that Cdk1 phosphorylates NuMA at T2055, and this phosphorylation negatively regulates cortical NuMA/dynein levels in mitosis. Also, phosphomimetic mutation (D or E) at T2055 in NuMA prevents its accumulation at the cell cortex in mitosis, indicating that NuMA must be in the dephosphorylated state at T2055. Phosphatases such as PPP2CA or PP1/Repo-Man have been linked with cortical NuMA regulation in mitosis, but whether they impact T2055 dephosphorylation remained unknown. In this study, by using a chemical-genetic approach, we identified a PP2A-based heterotrimeric complex consisting of PPP2CA (catalytical subunit), B55γ (regulatory subunit) and PPP2R1B (scaffold subunit) that dephosphorylates NuMA at T2055 residue. RNAi-mediated depletion of any of these subunits caused diminished levels of cortical NuMA/dynein and an increase in pT2055 signal. Further, we found that the loss of the regulatory subunit B55γ, caused spindle mispositioning during metaphase. Also, our data revealed that B55γ directly associates with NuMA. Next, by performing in vitro assays, we showed that the B55γ-based PP2A complex directly dephosphorylates NuMA at T2055. It is widely known that Greatwall kinase (MASTL) downregulates PP2A (B5a and B55d) activity through its substrates ENSA/ARPP19 in mitosis. On the contrary, we report that PP2A-B55γ activity is independent of Greatwall kinase (MASTL) and ENSA. Furthermore, we establish that the Cdk1 phosphorylation at T2055 and dephosphorylation by PP2A-B55γ are dynamically reversible. Notably, we identified polybasic residues in the vicinity of T2055 that are essential for interaction with B55γ. When these polybasic residues are mutated to alanine NuMA's cortical localization is significantly impaired, which has an impact on proper spindle elongation. Since PP2A-B55 has been shown to prefer Threonine over Serine residue, we tested the impact of replacing Threonine with Serine. We found that such modification delayed cortical NuMA localization in anaphase. Overall, our study shed some light on a biochemical tug of war between Cdk1 and PP2A-B55γ that is crucial for proper spindle positioning, which is critical for error-free mitosis. 2) Polo-like kinase 1(Plk1) regulates spindle positioning Plk1 was shown to control spindle positioning through regulation either at the level of dynein-dynactin or LGN. Prior reports have also shown that Plk1 regulates cortical machinery NuMA/LGN/dynein indirectly through its various substrates HMMMR1, MISP, and NDR1. In this work, we uncovered that Plk1 regulates spindle positioning directly through regulating cortical NuMA level. We established that the acute inhibition of Plk1 enriches cortical NuMA/LGN and dynein-dynactin levels, and this causes spindle mispositioning. We show that Plk1 can directly phosphorylate NuMA in vitro. Moreover, we report that LGN is dispensable for NuMA and Plk1 interaction in mitosis. Next, we uncovered that Plk1 phosphorylates NuMA at 1833-34 residues and negatively regulates cortical NuMA localization both in metaphase as well as anaphase. Overall, this study has revealed that Plk1 regulates spindle positioning by directly phosphorylating NuMA.