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Caracterización De Acnmcp1 Una Proteína Implicada En Organización Nuclear De Plantas

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Caracterización De Acnmcp1 Una Proteína Implicada En Organización Nuclear De Plantas UNIVERSIDAD COMPLUTENSE DE MADRID FACULTAD DE CIENCIAS BIOLÓGICAS Departamento de Genética TESIS DOCTORAL Caracterización de AcNMCP1 una proteína implicada en organización nuclear de plantas Characterization of AcNMCP1 a protein implicated in the nuclear organization in plants MEMORIA PARA OPTAR AL GRADO DE DOCTOR PRESENTADA POR Malgorzata Ciska Directoras Susana Moreno Díaz de la Espina Concepción Romero Martínez Madrid, 2014 ©Malgorzata Ciska, 2013 UNIVERSIDAD COMPLUTENSE DE MADRID CONSEJO SUPERIOR DE INVESTIGACIONES CIENTÍFICAS CENTRO DE INVESTIGACIONES BIOLÓGICAS LABORATORIO DE MATRIZ NUCLEAR Y REGULACIÓN DE LA ORGANIZACIÓN Y FUNCIONALIDAD NUCLEAR Caracterización de AcNMCP1 una proteína implicada en organización nuclear de plantas Characterization of AcNMCP1 a protein implicated in the nuclear organization in plants TESIS DOCTORAL Malgorzata Ciska Madrid, 2013 2 UNIVERSIDAD COMPLUTENSE DE MADRID FACULTAD DE CIENCIAS BIOLÓGICAS DEPARTAMENTO DE GENÉTICA Caracterización de AcNMCP1 una proteína implicada en organización nuclear de plantas Characterization of AcNMCP1 a protein implicated in the nuclear organization in plants MEMORIA PARA OPTAR AL GRADO DE DOCTOR PRESENTADA POR MALGORZATA CISKA Vº Bº DIRECTORES DE TESIS Vº Bº TUTOR DE TESIS Fdo. Dra.Susana Moreno Díaz de la Espina Fdo.: Dra. Concepción Romero Martínez Fdo.: Malgorzata Ciska MADRID, 2013 CONSEJO SUPERIOR DE INVESTIGACIONES CIENTÍFICAS CENTRO DE INVESTIGACIONES BIOLÓGICAS LABORATORIO DE MATRIZ NUCLEAR Y REGULACIÓN DE LA ORGANIZACIÓN Y FUNCIONALIDAD NUCLEAR 3 4 Acknowledgements In the first place I would like to thank the people who made it possible to finish this work. Dr. Susana Moreno who has put a trust in me and has allowed me to develop as a scientist while working on such an inspiring project. Dr. Kyoshi Masuda whose collaboration and professional expertise allowed us to perform experiments and obtain results of importance in the plant cell biology. My parents who has always believed in me and supported me through thick and thin even though I know many times it was not easy for them. My biggest failure would be to let them down. I could not forget about Merce, our technician who helped me with the experiments more than once and who always spared a smile and a chat when I needed. I would like to thank CSIC for granting me the JAE fellowship (JAEPre_08_00012/JAEPre027) and providing financial: support [PIE 201020E019] as well as the Spanish Ministry of Science and Innovation [BFU2010-15900]. Dr. Francisco García Tabares and Ms María Fernández López-Lucendo from proteomics fascility, Dr. Ma Teresa Seisdedos Domínguez from confocal microscopy, Dr. Fernando Escolar Antunez from electron microscopy and Dr. Perdo Lastres Varo from the flow cytometry facility for expert technical assistance. Also my tutor at the UCM, Dr. Concepción Romero. The people who helped me in the professional manner, as well as on the personal level. Dr Livia Donaire who helped me performing the northern blot analysis and whose friendship made my first years in a foreign country much easier. Dr Cesar Llave who allowed me to perform the northern blot in his lab. Mrs Elzbieta Smolczynska from the State Plant Health and Seed Inspection Service who kindly supported me with cereal seeds. Dr Alberto Garcia, “un sol” who always had a good advice, professional and concerning private life. 5 Acknowledgements Also I would like to thank dr Javier Gallego from the UCM for making the administrative steps of the thesis so much easier and more pleasant. And also people who might not remember me but who inspired me and awoke the passion for science during The Wilhelm Bernard workshop in Riga: prof Yossif Gruenbaum and prof David Jans. I do have to mention my “CIB team” as if not for them I would probably run back to Poland after the first weeks. People that welcomed me and quickly became my friends: Fati, Meme, Fran and Ana Castro. Jose Ramon, who introduced me to the lab and to his friends and help me to intergrate in spite of the language barrier. Also Hector, Maite and Vanessa who are great companions in scientific and social affairs. My partner, Quike, without whom I wouldn´t have had decided on coming to Spain and whose help made it possible to start the fellowship and also a completely new life. Michiel Janssens, my good friend who always have motivated me even from a 2,000 km distance. Finally I would like to thank all the staff from CIB for creating such a friendly working environment as it was a great pleasure working there for the last five years. 6 Resumen RESUMEN La lámina es una estructura filamentosa adosada a la membrana nuclear interna presente en el núcleo de numerosos eucariotas, incluyendo protozoos, metazoos y plantas. Está constituida por una red proteínica compleja asociada a la membrana nuclear interna y a los poros nucleares complejos. En metazoos la lámina consiste en un polímero de filamentos de laminas con numerosas proteínas asociadas que regulan su asociación con la membrana nuclear interna, poros nucleares y cromatina. Los principales componentes de la lámina de metazoos son las laminas que constituyen la clase V de la superfamilia de filamentos intermedios. Las laminas tienen importantes funciones en el núcleo como son la regulación de la organización y posición de la cromatina, replicación, transcripción y reparación del DNA, mantenimiento de la morfología nuclear, transducción de señales, conexión física del núcleoesqueleto y citoesqueleto, etc. Estas funciones se realizan de forma similar en el núcleo de plantas aunque carecen de genes codificantes de laminas y de la mayoría de las proteínas que se asocian con ellas. Además la lámina ha sido descrita repetidamente en varias especies de plantas mono y dicotiledóneas aunque su composición proteínica no es conocida. Por estos motivos se ha postulado que las plantas tendrían una lámina compuesta por un tipo diferente de proteínas. Hasta el presente se han propuesto varias proteínas específicas como candidatas para realizar las funciones de las laminas en plantas. Entre ellas se encuentran proteínas reconocidas por anticuerpos contra laminas y filamentos intermedios y también proteínas coiled coil específicas de plantas. Entre estas últimas están las proteínas constitutivas de la matriz nuclear (NMCP) que son las candidatas mas sólidas para reemplazar a las laminas en plantas ya que al igual que estas presentan una 7 Resumen estructura coiled coil tripartita, localizan en la periferia nuclear y están implicadas en la regulación de la morfología nuclear. El objetivo de este trabajo era por una parte la caracterización de la familia de proteínas NMCP estableciendo las relaciones filogenéticas entre los distintos ortólogos en varias especies de mono y dicotiledóneas, así como la determinación de sus principales características: estructura secundaria, distribución de dominios conservados y sitios de modificación postranscripcional. Asimismo nos planteamos el análisis de la proteína NMCP1 en Allium cepa (AcNMCP1) incluyendo su secuencia y la caracterización de la proteína endógena. Finalmente efectuamos la comparación de las principales características de NMCPs y laminas que permitieran establecer si las proteínas NMCPs realizan funciones de laminas en plantas. La búsqueda en la base de datos Phytozome v8 demostró que la familia está muy conservada ya que todas las plantas excepto las unicelulares expresan al menos dos proteínas NMCP. El análisis filogenético reveló que la familia está dividida en dos grupos uno conteniendo las proteínas NMCP1 y el otro las NMCP2. El primero está a su vez dividido en dos subgrupos NMCP1 y NMCP3. Las monocotiledóneas expresan una proteína NMCP1 y una NMCP2 mientras que las dicotiledóneas tiene una proteína NMCP2 y dos o más del tipo NMCP1. La forma ancestral de las NMCPs parece pertenecer al grupo NMCP2 ya que los dos ortólogos del musgo Physcomitrella patens están incluidos en ese grupo. El análisis exhaustivo de las secuencias NMCP demostró que comparten muchas características con las laminas. La predición de segmentos coiled coil con el programa MARCOIL reveló que todas las NMCPs tienen una estructura tripartita conservada con un segmento central coiled coil análogo al 8 Resumen de las laminas aunque el doble de largo. Este está dividido en dos segmentos separados por un linker corto que no tiene estructura coiled coil y en algunos casos interrumpidos por linkers internos. La predicción de sitios de modificación postranslacional demostró que las NMCPs tienen sitios de fosforilación para cdk1 flanqueando el dominio coiled coil central al igual que las laminas. La búsqueda de motivos conservados con MEME demostró la presencia de regiones muy conservadas en ambos extremos del dominio coiled coil, en las posiciones de los linkers y en la cola de la proteína, entre estas una región de localización nuclear, un dominio de aminoácidos ácidos (en las NMCP1) y el extremo carboxy terminal de la proteína (excepto en las NMCP2 de dicotiledóneas). Todas estas características son compartidas con las laminas. El análisis mediante “Western blot” usando un anticuerpo dirigido contra una región muy conservada de la proteína incluyendo la cabeza y el principio aminoterminal del segmento coiled coil reveló que el peso molecular de las proteínas endógenas es muy variable entre especies, aunque los deducidos de los cDNAs son similares, sugiriendo que las proteínas experimentan modificaciones post-tranlacionales. También analizamos la secuencia y propiedades bioquímicas de un ortólogo de Allium cepa: AcNMCP1. El cDNA codifica una proteína de 1.217 aminoácidos que presenta la estructura compartida con el resto de las NMCPs. Su peso molecular es mucho mas alto (200 kDa) que el deducido de la secuencia (139 kDa). La posibilidad de dimerización de la proteína fue descartada en base a las extracciones con altas concentraciones de urea, tiocianato de guanidina y alta temperatura y pH, sugiriendo que la proteína experimenta modificaciones post-translacionales. AcNMCP1 es altamente insoluble y un componente del núcleoesqueleto como demuestra la extracción secuencial con detergentes no iónicos, nucleasa y tampones de baja y alta fuerza iónica.
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