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Eibes González Susana - One of the best experts on this subject based on the ideXlab platform.
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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
'Edicions de la Universitat de Barcelona', 2017Co-Authors: Eibes González SusanaAbstract:[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
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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
'Edicions de la Universitat de Barcelona', 2016Co-Authors: Eibes González SusanaAbstract: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.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
Kassandra M Orimckenney - One of the best experts on this subject based on the ideXlab platform.
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competition between microtubule associated proteins directs motor transport
Nature Communications, 2018Co-Authors: Brigette Y Monroy, Danielle L Sawyer, Bryce E Ackermann, Melissa M Borden, Tracy Tan, Kassandra M OrimckenneyAbstract:Within cells, motor and non-motor microtubule-associated proteins (MAPs) simultaneously converge on the microtubule. How the binding activities of non-motor MAPs are coordinated and how they contribute to the balance and Distribution of motor transport is unknown. Here, we examine the relationship between MAP7 and tau owing to their antagonistic roles in vivo. We find that MAP7 and tau compete for binding to microtubules, and determine a mechanism by which MAP7 displaces tau from the lattice. MAP7 promotes kinesin-based transport in vivo and strongly recruits kinesin-1 to the microtubule in vitro, providing evidence for direct enhancement of motor motility by a MAP. Both MAP7 and tau strongly inhibit kinesin-3 and have no effect on cytoplasmic dynein, demonstrating that MAPs differentially control distinct classes of motors. Overall, these results reveal a general principle for how MAP competition dictates access to the microtubule to determine the Correct Distribution and balance of motor activity.
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competition between microtubule associated proteins directs motor transport
bioRxiv, 2017Co-Authors: Brigette Y Monroy, Danielle L Sawyer, Bryce E Ackermann, Melissa M Borden, Tracy Tan, Kassandra M OrimckenneyAbstract:Within cells, numerous motor and non-motor microtubule-associated proteins (MAPs) simultaneously converge on the microtubule lattice. How the binding activities of non- motor MAPs are coordinated and how they contribute to the balance and Distribution of microtubule motor transport is unknown. Here, we examine the relationship between MAP7 and tau due to their antagonistic effects on neuronal branch formation and kinesin motility in vivo (1-8). We find that MAP7 and tau compete for binding to microtubules, and determine a mechanism by which MAP7 displaces tau from the lattice. In striking contrast to the inhibitory effect of tau, MAP7 promotes kinesin-based transport in vivo and strongly enhances kinesin-1 binding to the microtubule in vitro, providing evidence for direct enhancement of motor motility by a MAP. In contrast, both MAP7 and tau strongly inhibit the dendrite-specific motor, kinesin-3, and have no effect on cytoplasmic dynein, demonstrating that MAPs exhibit differential control over distinct classes of motors. Overall, these results reveal a general principle for how MAP competition dictates access to the microtubule to determine the Correct Distribution and balance of molecular motor activity.
Martinez Delgado Paula - One of the best experts on this subject based on the ideXlab platform.
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Identification of novel NEK9 substrates and functions through the use of genetically engineered mice. Novel roles in the control of the centrosome cycle
Universitat de Barcelona, 2018Co-Authors: Martinez Delgado PaulaAbstract:Mitosis is a process that ensure the Correct Distribution of the chromosomes between the two newly generated cells, is tightly regulated by two main processes, protein degradation controlled by the APC and protein phosphorylation by different mitotic kinases. CDK1 is the master regulator of mitosis but in the last decades proteins from the Aurora or Polo or the NIMA family have been shown to play key roles in mitosis. The objective of this thesis is to identify new roles during the cell cycle and more specifically the late phases of mitosis of Nek9, a NIMA-related kinase. We aim to characterize new substrates and functions of the kinase by using different cell lines and genetically modified mice and interfering with Nek9 expression. The centrosome acts as the major microtubule-organizing center (MTOC) of the cell to maintain cytoskeleton in interphase and to organize the bipolar spindle in mitosis, and its duplication cycle is coupled with the cell cycle. When the cell enters mitosis, the duplicated centrosomes separate to the spindle poles and assemble the bipolar mitotic spindle for accurate chromosome separation and to maintain genomic stability. However, centrosome aberrations occur frequently and often lead to abnormal mitotic spindle formation, which can result in abnormal chromosome segregation and as a consequence tumorigenesis, microcephaly or ciliopathies. Nek9 is inactive during interphase and activated at centrosomes and spindle poles during mitosis by a two-step mechanism mediated by Plk1 and CDK1. Once active, Nek9 is able to bind Nek6 and Nek7 and directly phosphorylate these kinases inducing in turn their activation. Our group has shown that Nek6/7 phosphorylates the kinesin Eg5 at Ser1033 in the C-terminal domain, modulating the accumulation of Eg5 at or around centrosomes and their separation during prophase. Nek9 also phosphorylates the adapter NEDD1/GCP-WD, independently of Nek6/7, contributing to its recruitment to the centrosome and in consequence, to the recruitment of the microtubule nucleating complex formed by y-tubulin to the same organelle. Thus, Nek9, Nek7 and Nek6 regulate different aspects of the centrosome machinery during the entry in mitosis and have a role in spindle organization and Correct mitotic progression. Here we show that animals with a single Nek9 KO allele are healthy and fertile but intercrosses between them have not resulted in any homozygous null animals among born offspring indicating that the deletion of Nek9 is embryonic lethal. Also embryos obtained from these intercrosses had a higher frequency of mitotic abnormalities that result in death during the first days of development. As Nek9 is important for the proper development of mitosis we checked whether the expression in heterozygosity of Nek9 results in tumors affecting the viability of the animals. Some differences in tumor-free lifespan between heterozygous and wild type animals have been observed, with the appearance of tumors and aneuploidy. In addition, elimination of Nek9 expression lead to the apparition of abnormal mitosis, aneuploidy and multiple centrosomes both in genetically engineered MEFs and human cells, resulting in accumulation of centrobin, a protein mostly associated with the daughter centrioles, in the amplified centrioles. In the present thesis we describe possible new functions and substrates of Nek9 in the centrosome cycle, closely linked to the cell division cycle, after interfering with its expression using different strategies.La mitosis es un proceso que asegura la distribución Correcta de los cromosomas entre dos células recién generadas, está regulada por dos procesos principales, la degradación y la fosforilación de proteínas por diferentes quinasas mitóticas. CDK1 es el principal regulador de la mitosis, pero en las últimas décadas se ha demostrado que las proteínas de la familia Aurora o Polo o NIMA desempeñan un papel clave en la mitosis. El objetivo de esta tesis es identificar nuevas funciones de Nek9, una quinasa de la familia NIMA, durante el ciclo celular y más específicamente durante las fases tardías de la mitosis. Nuestro objetivo es caracterizar nuevos sustratos y funciones de la quinasa mediante el uso de diferentes líneas celulares y ratones genéticamente modificados que nos permiten interferir con la expresión de Nek9. El centrosoma actúa como el principal centro organizador de microtúbulos de la célula para mantener el citoesqueleto en interfase y para organizar el huso bipolar en la mitosis, su ciclo de duplicación va en sintonía con el ciclo celular. Cuando la célula entra en mitosis, los centrosomas duplicados se separan ensamblando el huso mitótico para segregar los cromosomas y para mantener la estabilidad genómica. Sin embargo, diferentes aberraciones ocurren con frecuencia en el centrosoma y a menudo conducen a la formación anormal del huso mitótico, que puede dar como resultado una segregación cromosómica anormal y, como consecuencia, tumorogénesis, microcefalia o ciliopatias. Nek9 está inactiva en interfase y se activa en los centrosomas durante la mitosis mediante un mecanismo de dos pasos mediado por Plk1 y CDK1. Una vez activo, Nek9 se puede unir a Nek6 y Nek7 y fosforilarlas induciendo a su vez su activación. Nuestro grupo ha demostrado que Nek6/7 fosforilan la quinesina Eg5, modulando la acumulación de Eg5 en los centrosomas y su separación durante la profase. Nek9 también fosforila el adaptador NEDD1 / GCP-WD, independientemente de Nek6/7, lo que contribuye a su reclutamiento en el centrosoma y, en consecuencia, al reclutamiento del complejo de nucleación de microtúbulos formado por y-tubulina. Aquí mostramos que los animales con un único alelo Nek9 KO están sanos y son fértiles. Sin embargo, los cruces entre ellos no dan lugar a ningún animal KO homocigoto, lo que indica que la eliminación de Nek9 es letal durante el desarrollo embrionario. Además, los embriones procedentes de estos cruces tienen una mayor frecuencia de defectos mitóticos que provocan la muerte durante los primeros días de desarrollo. Como Nek9 es importante para el Correcto desarrollo de la mitosis, queríamos ver si la expresión en heterocigosis daba como resultado tumores que afectan la viabilidad de los animales. Se han observado algunas diferencias en la esperanza de vida libre de tumores entre los heterocigotos con cierta incidencia de cáncer y aneuploidía. Por otro lado, la eliminación de la expresión de Nek9 en células conduce a la aparición de mitosis anormales, aneuploidía y múltiples centrosomas, tanto en fibroblastos embrionarios de ratón genéticamente modificados como en células humanas teniendo como consecuencia la acumulación de centrobina, una proteína presente en los procentriolos. En la presente tesis describimos posibles nuevas funciones y sustratos de Nek9 en el ciclo del centrosoma, íntimamente ligado al ciclo de división celular, tras interferir con su expresión de diferentes formas
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Identification of novel Nek9 substrates and functions through the use of genetically engineered mice
CSIC - Instituto de Biología Molecular de Barcelona (IBMB), 2018Co-Authors: Martinez Delgado PaulaAbstract:[EN] Mitosis is a process that ensure the Correct Distribution of the chromosomes between the two newly generated cells, is tightly regulated by two main processes, protein degradation controlled by the APC and protein phosphorylation by different mitotic kinases. CDK1 is the master regulator of mitosis but in the last decades proteins from the Aurora or Polo or the NIMA family have been shown to play key roles in mitosis. The objective of this thesis is to identify new roles during the cell cycle and more specifically the late phases of mitosis of Nek9, a NIMA-related kinase. We aim to characterize new substrates and functions of the kinase by using different cell lines and genetically modified mice and interfering with Nek9 expression. The centrosome acts as the major microtubule-organizing center (MTOC) of the cell to maintain cytoskeleton in interphase and to organize the bipolar spindle in mitosis, and its duplication cycle is coupled with the cell cycle. When the cell enters mitosis, the duplicated centrosomes separate to the spindle poles and assemble the bipolar mitotic spindle for accurate chromosome separation and to maintain genomic stability. However, centrosome aberrations occur frequently and often lead to abnormal mitotic spindle formation, which can result in abnormal chromosome segregation and as a consequence tumorigenesis, microcephaly or ciliopathies. Nek9 is inactive during interphase and activated at centrosomes and spindle poles during mitosis by a two-step mechanism mediated by Plk1 and CDK1. Once active, Nek9 is able to bind Nek6 and Nek7 and directly phosphorylate these kinases inducing in turn their activation. Our group has shown that Nek6/7 phosphorylates the kinesin Eg5 at Ser1033 in the C-terminal domain, modulating the accumulation of Eg5 at or around centrosomes and their separation during prophase. Nek9 also phosphorylates the adapter NEDD1/GCP-WD, independently of Nek6/7, contributing to its recruitment to the centrosome and in consequence, to the recruitment of the microtubule nucleating complex formed by y-tubulin to the same organelle. Thus, Nek9, Nek7 and Nek6 regulate different aspects of the centrosome machinery during the entry in mitosis and have a role in spindle organization and Correct mitotic progression. Here we show that animals with a single Nek9 KO allele are healthy and fertile but intercrosses between them have not resulted in any homozygous null animals among born offspring indicating that the deletion of Nek9 is embryonic lethal. Also embryos obtained from these intercrosses had a higher frequency of mitotic abnormalities that result in death during the first days of development. As Nek9 is important for the proper development of mitosis we checked whether the expression in heterozygosity of Nek9 results in tumors affecting the viability of the animals. Some differences in tumor-free lifespan between heterozygous and wild type animals have been observed, with the appearance of tumors and aneuploidy. In addition, elimination of Nek9 expression lead to the apparition of abnormal mitosis, aneuploidy and multiple centrosomes both in genetically engineered MEFs and human cells, resulting in accumulation of centrobin, a protein mostly associated with the daughter centrioles, in the amplified centrioles. In the present thesis we describe possible new functions and substrates of Nek9 in the centrosome cycle, closely linked to the cell division cycle, after interfering with its expression using different strategies.[ES] La mitosis es un proceso que asegura la distribución Correcta de los cromosomas entre dos células recién generadas, está regulada por dos procesos principales, la degradación y la fosforilación de proteínas por diferentes quinasas mitóticas. CDK1 es el principal regulador de la mitosis, pero en las últimas décadas se ha demostrado que las proteínas de la familia Aurora o Polo o NIMA desempeñan un papel clave en la mitosis. El objetivo de esta tesis es identificar nuevas funciones de Nek9, una quinasa de la familia NIMA, durante el ciclo celular y más específicamente durante las fases tardías de la mitosis. Nuestro objetivo es caracterizar nuevos sustratos y funciones de la quinasa mediante el uso de diferentes líneas celulares y ratones genéticamente modificados que nos permiten interferir con la expresión de Nek9. El centrosoma actúa como el principal centro organizador de microtúbulos de la célula para mantener el citoesqueleto en interfase y para organizar el huso bipolar en la mitosis, su ciclo de duplicación va en sintonía con el ciclo celular. Cuando la célula entra en mitosis, los centrosomas duplicados se separan ensamblando el huso mitótico para segregar los cromosomas y para mantener la estabilidad genómica. Sin embargo, diferentes aberraciones ocurren con frecuencia en el centrosoma y a menudo conducen a la formación anormal del huso mitótico, que puede dar como resultado una segregación cromosómica anormal y, como consecuencia, tumorogénesis, microcefalia o ciliopatias. Nek9 está inactiva en interfase y se activa en los centrosomas durante la mitosis mediante un mecanismo de dos pasos mediado por Plk1 y CDK1. Una vez activo, Nek9 se puede unir a Nek6 y Nek7 y fosforilarlas induciendo a su vez su activación. Nuestro grupo ha demostrado que Nek6/7 fosforilan la quinesina Eg5, modulando la acumulación de Eg5 en los centrosomas y su separación durante la profase. Nek9 también fosforila el adaptador NEDD1 / GCP-WD, independientemente de Nek6/7, lo que contribuye a su reclutamiento en el centrosoma y, en consecuencia, al reclutamiento del complejo de nucleación de microtúbulos formado por y-tubulina. Aquí mostramos que los animales con un único alelo Nek9 KO están sanos y son fértiles. Sin embargo, los cruces entre ellos no dan lugar a ningún animal KO homocigoto, lo que indica que la eliminación de Nek9 es letal durante el desarrollo embrionario. Además, los embriones procedentes de estos cruces tienen una mayor frecuencia de defectos mitóticos que provocan la muerte durante los primeros días de desarrollo. Como Nek9 es importante para el Correcto desarrollo de la mitosis, queríamos ver si la expresión en heterocigosis daba como resultado tumores que afectan la viabilidad de los animales. Se han observado algunas diferencias en la esperanza de vida libre de tumores entre los heterocigotos con cierta incidencia de cáncer y aneuploidía. Por otro lado, la eliminación de la expresión de Nek9 en células conduce a la aparición de mitosis anormales, aneuploidía y múltiples centrosomas, tanto en fibroblastos embrionarios de ratón genéticamente modificados como en células humanas teniendo como consecuencia la acumulación de centrobina, una proteína presente en los procentriolos. En la presente tesis describimos posibles nuevas funciones y sustratos de Nek9 en el ciclo del centrosoma, íntimamente ligado al ciclo de división celular, tras interferir con su expresión de diferentes formas.Peer reviewe
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Identification of novel NEK9 substrates and functions through the use of genetically engineered mice. Novel roles in the control of the centrosome cycle
'Edicions de la Universitat de Barcelona', 2018Co-Authors: Martinez Delgado PaulaAbstract:[eng] Mitosis is a process that ensure the Correct Distribution of the chromosomes between the two newly generated cells, is tightly regulated by two main processes, protein degradation controlled by the APC and protein phosphorylation by different mitotic kinases. CDK1 is the master regulator of mitosis but in the last decades proteins from the Aurora or Polo or the NIMA family have been shown to play key roles in mitosis. The objective of this thesis is to identify new roles during the cell cycle and more specifically the late phases of mitosis of Nek9, a NIMA-related kinase. We aim to characterize new substrates and functions of the kinase by using different cell lines and genetically modified mice and interfering with Nek9 expression. The centrosome acts as the major microtubule-organizing center (MTOC) of the cell to maintain cytoskeleton in interphase and to organize the bipolar spindle in mitosis, and its duplication cycle is coupled with the cell cycle. When the cell enters mitosis, the duplicated centrosomes separate to the spindle poles and assemble the bipolar mitotic spindle for accurate chromosome separation and to maintain genomic stability. However, centrosome aberrations occur frequently and often lead to abnormal mitotic spindle formation, which can result in abnormal chromosome segregation and as a consequence tumorigenesis, microcephaly or ciliopathies. Nek9 is inactive during interphase and activated at centrosomes and spindle poles during mitosis by a two-step mechanism mediated by Plk1 and CDK1. Once active, Nek9 is able to bind Nek6 and Nek7 and directly phosphorylate these kinases inducing in turn their activation. Our group has shown that Nek6/7 phosphorylates the kinesin Eg5 at Ser1033 in the C-terminal domain, modulating the accumulation of Eg5 at or around centrosomes and their separation during prophase. Nek9 also phosphorylates the adapter NEDD1/GCP-WD, independently of Nek6/7, contributing to its recruitment to the centrosome and in consequence, to the recruitment of the microtubule nucleating complex formed by y-tubulin to the same organelle. Thus, Nek9, Nek7 and Nek6 regulate different aspects of the centrosome machinery during the entry in mitosis and have a role in spindle organization and Correct mitotic progression. Here we show that animals with a single Nek9 KO allele are healthy and fertile but intercrosses between them have not resulted in any homozygous null animals among born offspring indicating that the deletion of Nek9 is embryonic lethal. Also embryos obtained from these intercrosses had a higher frequency of mitotic abnormalities that result in death during the first days of development. As Nek9 is important for the proper development of mitosis we checked whether the expression in heterozygosity of Nek9 results in tumors affecting the viability of the animals. Some differences in tumor-free lifespan between heterozygous and wild type animals have been observed, with the appearance of tumors and aneuploidy. In addition, elimination of Nek9 expression lead to the apparition of abnormal mitosis, aneuploidy and multiple centrosomes both in genetically engineered MEFs and human cells, resulting in accumulation of centrobin, a protein mostly associated with the daughter centrioles, in the amplified centrioles. In the present thesis we describe possible new functions and substrates of Nek9 in the centrosome cycle, closely linked to the cell division cycle, after interfering with its expression using different strategies.[spa] La mitosis es un proceso que asegura la distribución Correcta de los cromosomas entre dos células recién generadas, está regulada por dos procesos principales, la degradación y la fosforilación de proteínas por diferentes quinasas mitóticas. CDK1 es el principal regulador de la mitosis, pero en las últimas décadas se ha demostrado que las proteínas de la familia Aurora o Polo o NIMA desempeñan un papel clave en la mitosis. El objetivo de esta tesis es identificar nuevas funciones de Nek9, una quinasa de la familia NIMA, durante el ciclo celular y más específicamente durante las fases tardías de la mitosis. Nuestro objetivo es caracterizar nuevos sustratos y funciones de la quinasa mediante el uso de diferentes líneas celulares y ratones genéticamente modificados que nos permiten interferir con la expresión de Nek9. El centrosoma actúa como el principal centro organizador de microtúbulos de la célula para mantener el citoesqueleto en interfase y para organizar el huso bipolar en la mitosis, su ciclo de duplicación va en sintonía con el ciclo celular. Cuando la célula entra en mitosis, los centrosomas duplicados se separan ensamblando el huso mitótico para segregar los cromosomas y para mantener la estabilidad genómica. Sin embargo, diferentes aberraciones ocurren con frecuencia en el centrosoma y a menudo conducen a la formación anormal del huso mitótico, que puede dar como resultado una segregación cromosómica anormal y, como consecuencia, tumorogénesis, microcefalia o ciliopatias. Nek9 está inactiva en interfase y se activa en los centrosomas durante la mitosis mediante un mecanismo de dos pasos mediado por Plk1 y CDK1. Una vez activo, Nek9 se puede unir a Nek6 y Nek7 y fosforilarlas induciendo a su vez su activación. Nuestro grupo ha demostrado que Nek6/7 fosforilan la quinesina Eg5, modulando la acumulación de Eg5 en los centrosomas y su separación durante la profase. Nek9 también fosforila el adaptador NEDD1 / GCP-WD, independientemente de Nek6/7, lo que contribuye a su reclutamiento en el centrosoma y, en consecuencia, al reclutamiento del complejo de nucleación de microtúbulos formado por y-tubulina. Aquí mostramos que los animales con un único alelo Nek9 KO están sanos y son fértiles. Sin embargo, los cruces entre ellos no dan lugar a ningún animal KO homocigoto, lo que indica que la eliminación de Nek9 es letal durante el desarrollo embrionario. Además, los embriones procedentes de estos cruces tienen una mayor frecuencia de defectos mitóticos que provocan la muerte durante los primeros días de desarrollo. Como Nek9 es importante para el Correcto desarrollo de la mitosis, queríamos ver si la expresión en heterocigosis daba como resultado tumores que afectan la viabilidad de los animales. Se han observado algunas diferencias en la esperanza de vida libre de tumores entre los heterocigotos con cierta incidencia de cáncer y aneuploidía. Por otro lado, la eliminación de la expresión de Nek9 en células conduce a la aparición de mitosis anormales, aneuploidía y múltiples centrosomas, tanto en fibroblastos embrionarios de ratón genéticamente modificados como en células humanas teniendo como consecuencia la acumulación de centrobina, una proteína presente en los procentriolos. En la presente tesis describimos posibles nuevas funciones y sustratos de Nek9 en el ciclo del centrosoma, íntimamente ligado al ciclo de división celular, tras interferir con su expresión de diferentes formas
Pascal Dolle - One of the best experts on this subject based on the ideXlab platform.
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retinaldehyde dehydrogenase 2 and hoxc8 are required in the murine brachial spinal cord for the specification of lim1 motoneurons and the Correct Distribution of islet1 motoneurons
Development, 2005Co-Authors: Julien Vermot, Brigitte Schuhbaur, Herve Le Mouellic, Peter Mccaffery, Jeanmarie Garnier, Didier Hentsch, Philippe Brulet, Karen Niederreither, Pierre Chambon, Pascal DolleAbstract:Retinoic acid (RA) activity plays sequential roles during the development of the ventral spinal cord. Here, we have investigated the functions of local RA synthesis in the process of motoneuron specification and early differentiation using a conditional knockout strategy that ablates the function of the retinaldehyde dehydrogenase 2 (Raldh2) synthesizing enzyme essentially in brachial motoneurons, and later in mesenchymal cells at the base of the forelimb. Mutant ( Raldh2 L –/– ) embryos display an early embryonic loss of a subset of Lim1+ brachial motoneurons, a mispositioning of Islet1+ neurons and inappropriate axonal projections of one of the nerves innervating extensor limb muscles, which lead to an adult forepaw neuromuscular defect. The molecular basis of the Raldh2 L –/– phenotype relies in part on the deregulation of Hoxc8, which in turn regulates the RA receptor RARβ. We further show that Hoxc8 mutant mice, which exhibit a similar congenital forepaw defect, display at embryonic stages molecular defects that phenocopy the Raldh2 L –/– motoneuron abnormalities. Thus, interdependent RA signaling and Hox gene functions are required for the specification of brachial motoneurons in the mouse.
Brigette Y Monroy - One of the best experts on this subject based on the ideXlab platform.
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competition between microtubule associated proteins directs motor transport
Nature Communications, 2018Co-Authors: Brigette Y Monroy, Danielle L Sawyer, Bryce E Ackermann, Melissa M Borden, Tracy Tan, Kassandra M OrimckenneyAbstract:Within cells, motor and non-motor microtubule-associated proteins (MAPs) simultaneously converge on the microtubule. How the binding activities of non-motor MAPs are coordinated and how they contribute to the balance and Distribution of motor transport is unknown. Here, we examine the relationship between MAP7 and tau owing to their antagonistic roles in vivo. We find that MAP7 and tau compete for binding to microtubules, and determine a mechanism by which MAP7 displaces tau from the lattice. MAP7 promotes kinesin-based transport in vivo and strongly recruits kinesin-1 to the microtubule in vitro, providing evidence for direct enhancement of motor motility by a MAP. Both MAP7 and tau strongly inhibit kinesin-3 and have no effect on cytoplasmic dynein, demonstrating that MAPs differentially control distinct classes of motors. Overall, these results reveal a general principle for how MAP competition dictates access to the microtubule to determine the Correct Distribution and balance of motor activity.
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competition between microtubule associated proteins directs motor transport
bioRxiv, 2017Co-Authors: Brigette Y Monroy, Danielle L Sawyer, Bryce E Ackermann, Melissa M Borden, Tracy Tan, Kassandra M OrimckenneyAbstract:Within cells, numerous motor and non-motor microtubule-associated proteins (MAPs) simultaneously converge on the microtubule lattice. How the binding activities of non- motor MAPs are coordinated and how they contribute to the balance and Distribution of microtubule motor transport is unknown. Here, we examine the relationship between MAP7 and tau due to their antagonistic effects on neuronal branch formation and kinesin motility in vivo (1-8). We find that MAP7 and tau compete for binding to microtubules, and determine a mechanism by which MAP7 displaces tau from the lattice. In striking contrast to the inhibitory effect of tau, MAP7 promotes kinesin-based transport in vivo and strongly enhances kinesin-1 binding to the microtubule in vitro, providing evidence for direct enhancement of motor motility by a MAP. In contrast, both MAP7 and tau strongly inhibit the dendrite-specific motor, kinesin-3, and have no effect on cytoplasmic dynein, demonstrating that MAPs exhibit differential control over distinct classes of motors. Overall, these results reveal a general principle for how MAP competition dictates access to the microtubule to determine the Correct Distribution and balance of molecular motor activity.
