Importin beta is the major vector for protein import in interphase nuclei and acts as an effector of the GTPase RAN. After nuclear envelope breakdown, when nuclear transport ceases, Importin beta acts in control of mitosis. Importin beta is overexpressed in many cancer types that display high genetic instability. The precise mechanisms through which Importin beta acts in cells in which it is overexpressed are incompletely understood. Importin beta has a modular structure that enables it to interact with many partners, making it difficult to pinpoint its targets in distinct pathways. The goal of this work was to obtain a global view of Importin beta functional mechanisms in mitosis. I first undertook a proteome-wide analysis of importin beta mitotic interactors and identified a large series of known and new interactors, all with mitotic roles. To gain information on the molecular pathways regulated by Importin beta during mitosis, I generated stable cell lines with inducible expression of Importin beta, either wild-type or in mutant forms defective for specific domains. Mitosis was analysed in these cell lines by interactomics, Proximity Ligation Assay (PLA), time-lapse imaging and functional assays. Results can be summarised as follows. First, Importin beta overexpression hinders microtubule (MT) growth and stability. I identified HURP (hepatoma-upregulated protein), a MT-stabilizing factor, as a target of importin beta control in MT stabilization. Importin beta overexpression delocalizes HURP, displacing it from its sites of action. Second, Importin beta influences kinetochore (KT) functions via RANBP2, a nucleoporin endowed with SUMO-E3-ligase activity, with which importin beta interacts. In physiological mitosis, RANBP2 localizes at KTs in metaphase. I found that Importin beta hinders RANBP2 localization and interactions at KTs; this ultimately impairs SUMO modification of KT proteins, including Topoisomerase II-alpha (TOP2A), with severe consequences on chromosome segregation. Third, defects in MT and KT functions induced by Importin beta overexpression ultimately hinder mitotic progression and chromosome segregation, generating genetic instability in daughter cells. Together, these results depict complex roles of Importin beta in mitotic MT function and interaction with KTs and contribute to clarify distinct pathways through which deregulated expression of high Importin beta influences the transmission of the genetic integrity in mitosis.

Importin beta regulates mitosis via distinct molecular mechanisms / Verrico, Annalisa. - (2018 Feb 15).

Importin beta regulates mitosis via distinct molecular mechanisms

VERRICO, ANNALISA
2018

Abstract

Importin beta is the major vector for protein import in interphase nuclei and acts as an effector of the GTPase RAN. After nuclear envelope breakdown, when nuclear transport ceases, Importin beta acts in control of mitosis. Importin beta is overexpressed in many cancer types that display high genetic instability. The precise mechanisms through which Importin beta acts in cells in which it is overexpressed are incompletely understood. Importin beta has a modular structure that enables it to interact with many partners, making it difficult to pinpoint its targets in distinct pathways. The goal of this work was to obtain a global view of Importin beta functional mechanisms in mitosis. I first undertook a proteome-wide analysis of importin beta mitotic interactors and identified a large series of known and new interactors, all with mitotic roles. To gain information on the molecular pathways regulated by Importin beta during mitosis, I generated stable cell lines with inducible expression of Importin beta, either wild-type or in mutant forms defective for specific domains. Mitosis was analysed in these cell lines by interactomics, Proximity Ligation Assay (PLA), time-lapse imaging and functional assays. Results can be summarised as follows. First, Importin beta overexpression hinders microtubule (MT) growth and stability. I identified HURP (hepatoma-upregulated protein), a MT-stabilizing factor, as a target of importin beta control in MT stabilization. Importin beta overexpression delocalizes HURP, displacing it from its sites of action. Second, Importin beta influences kinetochore (KT) functions via RANBP2, a nucleoporin endowed with SUMO-E3-ligase activity, with which importin beta interacts. In physiological mitosis, RANBP2 localizes at KTs in metaphase. I found that Importin beta hinders RANBP2 localization and interactions at KTs; this ultimately impairs SUMO modification of KT proteins, including Topoisomerase II-alpha (TOP2A), with severe consequences on chromosome segregation. Third, defects in MT and KT functions induced by Importin beta overexpression ultimately hinder mitotic progression and chromosome segregation, generating genetic instability in daughter cells. Together, these results depict complex roles of Importin beta in mitotic MT function and interaction with KTs and contribute to clarify distinct pathways through which deregulated expression of high Importin beta influences the transmission of the genetic integrity in mitosis.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11573/1070608
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