Kinetochore- and chromosome-driven transition of microtubules into bundles promotes spindle assembly

Mitotic spindle assembly is crucial for chromosome segregation and relies on bundles of microtubules that extend from the poles and overlap in the middle. However, how these structures form remains poorly understood. Here we show that overlap bundles arise through a network-to-bundles transition driven by kinetochores and chromosomes. STED super-resolution microscopy reveals that PRC1-crosslinked microtubules initially form loose arrays, which become rearranged into bundles. Kinetochores promote microtubule bundling by lateral binding via CENP-E/kinesin-7 in an Aurora B-regulated manner. Steric interactions between the bundle-associated chromosomes at the spindle midplane drive bundle separation and spindle widening. In agreement with experiments, theoretical modeling suggests that bundles arise through competing attractive and repulsive mechanisms. Finally, perturbation of overlap bundles leads to inefficient correction of erroneous kinetochore-microtubule attachments. Thus, kinetochores and chromosomes drive coarsening of a uniform microtubule array into overlap bundles, which promote not only spindle formation but also chromosome segregation fidelity

Functional study of the NIMA protein kinases Nek9, Nek6 and Nek7 at the onset of mitosis. Control of the kinesin Eg5 and prophase centrosome separation

[eng] Mitosis is a tightly regulated process that aims to ensure the correct distribution of the chromosomes between the two newly generated cells. Many protein kinases have been defined as essential for this process: cyclin- dependent kinases, Aurora family and Polo family kinases are some of the most relevant players. The objective of this thesis is to characterize one of the less studied kinase pathways involved in this process, which is constituted by the NIMA-related kinases Nek9, Nek6 and Nek7. Nek9 is activated at the onset of mitosis by a double step mechanism mediated by CDK1 and Plk1. Once Nek9 is activated it can bind to Nek6 and Nek7 and phosphorylate them, promoting their activation. Finally, Nek6 and Nek7 are responsible for the phosphorylation of the kinesin Eg5, promoting Eg5 accumulation at centrosome, and consequently, centrosome separation. The kinesin eg5 motor protein is considered as one of the major players for centrosome separation and formation of the bipolar spindle. The tetramer configuration allows Eg5 to bind antiparallel microtubules and slide them apart, exerting a force that promotes centrosome separation and the maintenance of the bipolar spindle. Centrosome separation, however, is a highly intricate process that involves several pathways, including Eg5 activity. Dynein presents a directed activity towards the minus ends of microtubules, which has a redundant role to Eg5 in centrosome separation. Dynein accumulation at the cell cortex and the nuclear membrane, through its adaptor BicD2, is also involved in centrosome tethering at the nuclear envelope, a necessary step prior to separation. Furthermore, dynein can control the position of Eg5 at the spindle via TPX2, an event that could also happen before nuclear envelope breakdown (NEB). Here we describe the conditions required for Eg5 accumulation at the centrosmes after Ser1033 phosphorylation. During the development of this project we have explored the essential circumstances for correct Eg5 localization in cells. By using protein-protein interaction techniques and shRNA depletion of protein candidates we have determined that another motor protein, dynein, together with the adaptor BicD2 and the protein TPX2 are responsible for Eg5 accumulation around centrosomes. Additionally, we proposed TPX2 as a novel Nek9 substrate and we have investigated the role of this phosphorylation, which affects TPX2 localization during prophase, before NEB. We present with this thesis a model for Eg5 accumulation at microtubule minus ends and centrosome separation during prophase summarized in the following points: 1) Dynein complex transports Eg5 towards the centrosome. Dynein interacts with Eg5 independently of the Ser1033 phosphorylation. The adaptor BicD2, which interacts directly with Eg5 tail domain, mediates the interaction. Dynein motility towards microtubule minus ends and the presence of BicD2 on the complex are required for Eg5 localization at centrosomes. Thus, the dynein complex is required for Eg5 transport to the centrosomes during G2-M transition. 2) TPX2 inhibits Eg5 motility in response to Ser1033 phosphorylation. TPX2 is necessary for the correct localization of Eg5 at centrosomes during prophase. TPX2 mislocalization at centrosomes without altering its overall levels leads to failed Eg5 localization, therefore the presence of TPX2 at centrosomes during prophase is required for Eg5 localization. TPX2 interacts with Eg5 during mitosis and the interaction is abolished when the Ser1033 can’t be phosphorylated. Thus, TPX2 is able to respond to Eg5 Ser1033 phosphorylation, which we propose is promoting the interaction between these two proteins, and consequently inhibiting Eg5 motility at centrosomal levels. 3) TPX2 phosphorylation by Nek9 promotes its centrosomal localization. Nek9 phosphorylation of TPX2 is responsible for TPX2 localization at the spindle poles during prophase. Nek9 phosphorylates TPX2 at residues that are proximal to a NLS, making TPX2 localization more cytoplasmic and promoting its accumulation to the area where Nek9 is more active, the centrosome.[spa] La mitosis es un proceso altamente regulado cuyo objetivo es asegurar la correcta distribución de los cromosomas entre las dos células nuevamente generadas. Diferentes proteínas quinasas han sido definidas como esenciales en este proceso pero el objetivo de esta tesis es caracterizar una de las rutas de señalización menos estudiada, la cual la componen las NIMA quinasas Nek9, Nek6 y Nek7. Nek9 es activada al inicio de mitosis por un doble mecanismo mediado por CDK1 y Plk1. Una vez activada, se puede unir a Nek6 y Nek7 y fosforilarlas, promoviendo su activación. Finalmente, Nek6 y Nek7 son responsables de la fosforilación de la quinesina Eg5, promoviendo la acumulación de Eg5 en los centrosomas, y en consecuencia, la separación de los mismos en profase. Aquí describimos las condiciones necesarias para la acumulación de Eg5 en los centrosomas después de la fosforilación en la Ser1033. Durante el desarrollo de este trabajo hemos explorado las circunstancias esenciales para una correcta localización de Eg5 en las células. Usando técnicas de interacción proteína-proteína y técnicas de silenciamiento proteico de candidatos con shRNA hemos determinado que otra proteína motora, dineína, junto con el adaptador BicD2 y la proteína TPX2, son responsables de la acumulación de Eg5 alrededor de los centrosomas. Además, hemos propuesto a TPX2 como un nuevo substrato regulado por Nek9 y hemos investigado el papel de esta fosforilación, la cual afecta la localización de TPX2 durante profase, antes de la rotura de la membrana nuclear. Con esta tesis presentamos un modelo para la acumulación de Eg5 y la separación de los centrosomas en profase que puede ser resumido en los siguientes puntos: - El complejo de dineína transporta Eg5 hacia el centrosoma independientemente de la fosforilación en la Ser1033. El adaptador BicD2 media esta interacción uniéndose directamente al dominio C terminal de Eg5. -TPX2 inhibe movilidad de Eg5 en respuesta a la fosforilación en la Ser1033. - La presencia de TPX2 en los centrosomas es necesaria para la localización de Eg5. La fosforilación de TPX2 por Nek9 promueve la localización de TPX2 en los centrosomas durante la profase

Regulation of the dynein adaptor BICD2 through phosphorylation

Trabajo presentado en el Current Trends in Biomedicine Workshop: “From cancer to developmental defects: the control of DNA segregation and human disease”, celebrado en Baeza (España), del 14 al 16 de octubre de 201

TH588 and Low-Dose Nocodazole Impair Chromosome Congression by Suppressing Microtubule Turnover within the Mitotic Spindle

Microtubule-targeting agents (MTAs) have been used for decades to treat different hematologic and solid cancers. The mode of action of these drugs mainly relies on their ability to bind tubulin subunits and/or microtubules and interfere with microtubule dynamics. In addition to its MTH1-inhibiting activity, TH588 has been recently identified as an MTA, whose anticancer properties were shown to largely depend on its microtubule-targeting ability. Although TH588 inhibited tubulin polymerization in vitro and reduced microtubule plus-end mobility in interphase cells, its effect on microtubule dynamics within the mitotic spindle of dividing cells remained unknown. Here, we performed an in-depth analysis of the impact of TH588 on spindle-associated microtubules and compared it to the effect of low-dose nocodazole. We show that both treatments reduce microtubule turnover within the mitotic spindle. This microtubule-stabilizing effect leads to premature formation of kinetochore-microtubule end-on attachments on uncongressed chromosomes, which consequently cannot be transported to the cell equator, thereby delaying cell division and leading to cell death or division with uncongressed chromosomes

MZT1 regulates microtubule nucleation by linking γTuRC assembly to adapter-mediated targeting and activation

Regulation of the γ-tubulin ring complex (γTuRC) through targeting and activation restricts nucleation of microtubules to microtubule-organizing centers (MTOCs), aiding in the assembly of ordered microtubule arrays. However, the mechanistic basis of this important regulation remains poorly understood. Here, we show that, in human cells, γTuRC integrity, determined by the presence of γ-tubulin complex proteins (GCPs; also known as TUBGCPs) 2–6, is a prerequisite for interaction with the targeting factor NEDD1, impacting on essentially all γ-tubulin-dependent functions. Recognition of γTuRC integrity is mediated by MZT1, which binds not only to the GCP3 subunit as previously shown, but cooperatively also to other GCPs through a conserved hydrophobic motif present in the N-termini of GCP2, GCP3, GCP5 and GCP6. MZT1 knockdown causes severe cellular defects under conditions that leave γTuRC intact, suggesting that the essential function of MZT1 is not in γTuRC assembly. Instead, MZT1 specifically binds fully assembled γTuRC to enable interaction with NEDD1 for targeting, and with the CM1 domain of CDK5RAP2 for stimulating nucleation activity. Thus, MZT1 is a ‘priming factor’ for γTuRC that allows spatial regulation of nucleation.This study was supported by grants from the Ministerio de Economıa y ́ Competitividad (MINECO) (BFU2009-08522, BFU2012-33960 and BFU2015- 69275-P to J.L. and BFU2014-58422-P to J.R.), and IRB Barcelona intramural funds. J.L. acknowledges additional support from the Ramón y Cajal Programme (RYC-2010-07108 MINECO, Spain). R.R.C. was supported by a PhD fellowship from the Consejo Nacional de Ciencia y Tecnologıa (Mexican National Council for ́ Science and Technology; CONACYT) (CVU 269229).Peer reviewe

Nek9 phosphorylation defines a new role for TPX2 in Eg5-dependent centrosome separation before nuclear envelope breakdown

Centrosomes [1, 2] play a central role during spindle assembly in most animal cells [3]. In early mitosis, they organize two symmetrical microtubule arrays that upon separation define the two poles of the forming spindle. Centrosome separation is tightly regulated [4, 5], occurring through partially redundant mechanisms that rely on the action of microtubule-based dynein and kinesin motors and the actomyosin system [6]. While centrosomes can separate in prophase or in prometaphase after nuclear envelope breakdown (NEBD), prophase centrosome separation optimizes spindle assembly and minimizes the occurrence of abnormal chromosome attachments that could end in aneuploidy [7, 8]. Prophase centrosome separation relies on the activity of Eg5/KIF11, a mitotic kinesin [9] that accumulates around centrosomes in early mitosis under the control of CDK1 and the Nek9/Nek6/7 kinase module [10-17]. Here, we show that Eg5 localization and centrosome separation in prophase depend on the nuclear microtubule-associated protein TPX2 [18], a pool of which localizes to the centrosomes before NEBD. This localization involves RHAMM/HMMR [19] and the kinase Nek9 [20], which phosphorylates TPX2 nuclear localization signal (NLS) preventing its interaction with importin and nuclear import. The pool of centrosomal TPX2 in prophase has a critical role for both microtubule aster organization and Eg5 localization, and thereby for centrosome separation. Our results uncover an unsuspected role for TPX2 before NEBD and define a novel regulatory mechanism for centrosome separation in prophase. They furthermore suggest NLS phosphorylation as a novel regulatory mechanism for spindle assembly factors controlled by the importin/Ran system.This work was funded by the Ministerio de Economía, Industria y Competitividad (MINECO) from Spain through Plan Nacional de I+D grants BFU2014-58422 (to J.R.) and BFU2012-37163 (to I.V.). N.G.-S. and M.R.-S. are recipients of FPI Fellowships BES-2015-072446 and BES-2013-064601 from MINECO. IRB Barcelona and CRG are Severo Ochoa Award of Excellence recipients from MINECO. J.R. would like to thank the IRB Barcelona for its enduring institutional support

Nek9 phosphorylation defines a new role for TPX2 in Eg5-dependent centrosome separation before nuclear envelope breakdown

Centrosomes [1, 2] play a central role during spindle assembly in most animal cells [3]. In early mitosis, they organize two symmetrical microtubule arrays that upon separation define the two poles of the forming spindle. Centrosome separation is tightly regulated [4, 5], occurring through partially redundant mechanisms that rely on the action of microtubule-based dynein and kinesin motors and the actomyosin system [6]. While centrosomes can separate in prophase or in prometaphase after nuclear envelope breakdown (NEBD), prophase centrosome separation optimizes spindle assembly and minimizes the occurrence of abnormal chromosome attachments that could end in aneuploidy [7, 8]. Prophase centrosome separation relies on the activity of Eg5/KIF11, a mitotic kinesin [9] that accumulates around centrosomes in early mitosis under the control of CDK1 and the Nek9/Nek6/7 kinase module [10-17]. Here, we show that Eg5 localization and centrosome separation in prophase depend on the nuclear microtubule-associated protein TPX2 [18], a pool of which localizes to the centrosomes before NEBD. This localization involves RHAMM/HMMR [19] and the kinase Nek9 [20], which phosphorylates TPX2 nuclear localization signal (NLS) preventing its interaction with importin and nuclear import. The pool of centrosomal TPX2 in prophase has a critical role for both microtubule aster organization and Eg5 localization, and thereby for centrosome separation. Our results uncover an unsuspected role for TPX2 before NEBD and define a novel regulatory mechanism for centrosome separation in prophase. They furthermore suggest NLS phosphorylation as a novel regulatory mechanism for spindle assembly factors controlled by the importin/Ran system.This work was funded by the Ministerio de Economía, Industria y Competitividad (MINECO) from Spain through Plan Nacional de I+D grants BFU2014-58422 (to J.R.) and BFU2012-37163 (to I.V.). N.G.-S. and M.R.-S. are recipients of FPI Fellowships BES-2015-072446 and BES-2013-064601 from MINECO. IRB Barcelona and CRG are Severo Ochoa Award of Excellence recipients from MINECO. J.R. would like to thank the IRB Barcelona for its enduring institutional support