Mecanismo de formación de cilio primario en células epiteliales polarizadas

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 23-06-2017El cilio primario es una extensión de membrana altamente conservada que protruye de la superficie apical de la mayoría de las células eucariotas. Su estructura consiste en una membrana ciliar que rodea un armazón de microtúbulos, el axonema, que deriva del centriolo más maduro. Aunque su función ha permanecido desconocida durante mucho tiempo, actualmente se sabe que el cilio primario actúa como un biosensor regulando múltiples rutas de señalización y la homeostasis de los tejidos. Durante los últimos años la relevancia clínica y fisiológica del cilio primario se ha hecho evidente debido a que los defectos en su estructura o su disfunción tienen como consecuencia el desarrollo de un grupo de enfermedades genéticas que se agrupan colectivamente con el nombre de ciliopatías. Entre estas enfermedades destacan la poliquistosis renal, la ceguera, la sordera, la obesidad, y los defectos en el desarrollo embrionario. A pesar de que el riñón es el órgano más frecuentemente afectado por las ciliopatías, la biogénesis del cilio primario se ha estudiado mayoritariamente en tipos celulares no polarizados. Hace casi 50 años se propuso que en los epitelios polarizados, como los de los túbulos renales, el proceso de ciliogénesis tiene lugar exclusivamente en la membrana plasmática, mientras que por el contrario, los fibroblastos ensamblan el cilio de manera intracelular. Utilizado el modelo de células epiteliales polarizadas MDCK, he investigado el desconocido proceso de biogénesis del cilio primario en células epiteliales polarizadas. He observado que el cuerpo medio, una estructura basada en microtúbulos localizada en la parte central del puente intercelular formado entre las dos células hijas durante las etapas finales de la división celular, es heredado por una de las dos células en forma de remanente, el cual se posiciona en la periferia de la membrana apical acumulando maquinaria relevante para la ciliogénesis. Posteriormente, este remanente se mueve sobre la membrana apical reuniéndose con el centrosoma. Una vez que se han juntado estos dos orgánulos, el remanente posibilita la formación del cilio primario. Estos hallazgos revelan un mecanismo biológico que conecta funcionalmente el cuerpo medio con el centrosoma y el cilio primario, los otros dos orgánulos celulares basados en microtúbulos. Además, he investigado el papel de la proteína MAL, un componente de la maquinaria general de transporte apical, en el proceso de ciliogénesis. Los resultados obtenidos indican que MAL es necesaria para la correcta condensación de las membranas en la base del cilio, lo que a su vez, es crucial para la elongación eficiente del citado orgánulo. En resumen, este trabajo establece una nueva ruta de ciliogénesis primaria en células epiteliales renales y confiere importancia a las membranas situadas en la base del cilio primario.The primary cilium is a highly conserved membrane extension protruding from the cell surface of most mammalian cells. It consists of a ciliary membrane that surrounds a microtubule-based structure termed the axoneme, which is nucleated from the older of the two centrioles. Although its function has been an enigma for a long time, nowadays it is known to act as a biosensor regulating multiple signaling pathways during development and tissue homeostasis. The physiological and clinical relevance of cilia is evident, since defects in primary cilium function cause a wide spectrum of genetic diseases collectively grouped under the term of ciliopathies. Among the disorders produced by primary cilium dysfunction are cystic kidney disease, blindness, deafness, obesity, and developmental and skeletal abnormalities. The kidney is the organ most frequently affected in ciliopathies. However, despite its importance in the kidney, primary cilium biogenesis has mainly been studied in non-polarized cells. Almost 50 years ago, it was proposed that the process of primary ciliogenesis in polarized epithelia, such as that in kidney tubules, takes place entirely at the plasma membrane, in contrast to fibroblasts that assemble the cilium intracellularly. Using the renal epithelial MDCK cell line, I have investigated the unexplored process of primary cilium biogenesis in polarized epithelial cells. I observed that the midbody, which is a microtubule-based structure that occupies the central part of the intercellular bridge connecting the two sister cells during the final stages of cell division, is inherited by one of the cells as a remnant that localizes at the periphery of the apical membrane, and that accumulates important machinery for cilium biogenesis. The remnant then moves along the apical plasma membrane to a central position to encounter the centrosome. Once the two organelles have met, the remnant enables the centrosome for primary cilium formation. These findings reveal a biological mechanism that functionally links the midbody with the centrosome and the primary cilium, which are the other two main microtubule-based organelles. I have also investigated the role of MAL, a component of the machinery of apical transport, in primary cilium assembly. The results indicate that MAL is required for correct membrane condensation at the ciliary base, which, in turn, is essential for efficient primary cilium elongation. In summary, the work presented establishes a novel pathway of primary ciliogenesis in renal polarized epithelial cells and establishes the importance of the condensation of the membranes at the ciliary base

MALL, a membrane-tetra-spanning proteolipid overexpressed in cancer, is present in membraneless nuclear biomolecular condensates

Proteolipids are proteins with unusual lipid-like properties. It has long been established that PLP and plasmolipin, which are two unrelated membrane-tetra-spanning myelin proteolipids, can be converted in vitro into a water-soluble form with a distinct conformation, raising the question of whether these, or other similar proteolipids, can adopt two different conformations in the cell to adapt their structure to distinct environments. Here, we show that MALL, another proteolipid with a membrane-tetra-spanning structure, distributes in membranes outside the nucleus and, within the nucleus, in membrane-less, liquid-like PML body biomolecular condensates. Detection of MALL in one or other environment was strictly dependent on the method of cell fixation used, suggesting that MALL adopts different conformations depending on its physical environment —lipidic or aqueous— in the cell. The acquisition of the condensate-compatible conformation requires PML expression. Excess MALL perturbed the distribution of the inner nuclear membrane proteins emerin and LAP2β, and that of the DNA-binding protein BAF, leading to the formation of aberrant nuclei. This effect, which is consistent with studies identifying overexpressed MALL as an unfavorable prognostic factor in cancer, could contribute to cell malignancy. Our study establishes a link between proteolipids, membranes and biomolecular condensates, with potential biomedical implication

The MAL protein is crucial for proper membrane condensation at the ciliary base, which is required for primary cilium elongation

The base of the primary cilium contains a zone of condensedmembranes whose importance is not known. Here, we have studiedthe involvement of MAL, a tetraspanning protein that exclusivelypartitions into condensed membrane fractions, in the condensation ofmembranes at the ciliary base and investigated the importance ofthese membranes in primary cilium formation. We show that MALaccumulates at the ciliary base of epithelial MDCK cells. Knockdownof MAL expression resulted in a drastic reduction in the condensationof membranes at the ciliary base, the percentage of ciliated cells andthe length of the cilia, but did not affect the docking of the centrosometo the plasma membrane or produce missorting of proteins to thepericentriolar zone or to the membrane of the remaining cilia. Rab8(for which there are two isoforms, Rab8A and Rab8b), IFT88 andIFT20, which are important components of the machinery of ciliarygrowth, were recruited normally to the ciliary base of MAL-knockdowncells but were unable to elongate the primary cilium correctly. MAL,therefore, is crucial for the proper condensation of membranes at theciliary base, which is required for efficient primary cilium extension

Effectiveness of an intervention for improving drug prescription in primary care patients with multimorbidity and polypharmacy:Study protocol of a cluster randomized clinical trial (Multi-PAP project)

This study was funded by the Fondo de Investigaciones Sanitarias ISCIII (Grant Numbers PI15/00276, PI15/00572, PI15/00996), REDISSEC (Project Numbers RD12/0001/0012, RD16/0001/0005), and the European Regional Development Fund ("A way to build Europe").Background: Multimorbidity is associated with negative effects both on people's health and on healthcare systems. A key problem linked to multimorbidity is polypharmacy, which in turn is associated with increased risk of partly preventable adverse effects, including mortality. The Ariadne principles describe a model of care based on a thorough assessment of diseases, treatments (and potential interactions), clinical status, context and preferences of patients with multimorbidity, with the aim of prioritizing and sharing realistic treatment goals that guide an individualized management. The aim of this study is to evaluate the effectiveness of a complex intervention that implements the Ariadne principles in a population of young-old patients with multimorbidity and polypharmacy. The intervention seeks to improve the appropriateness of prescribing in primary care (PC), as measured by the medication appropriateness index (MAI) score at 6 and 12months, as compared with usual care. Methods/Design: Design:pragmatic cluster randomized clinical trial. Unit of randomization: family physician (FP). Unit of analysis: patient. Scope: PC health centres in three autonomous communities: Aragon, Madrid, and Andalusia (Spain). Population: patients aged 65-74years with multimorbidity (≥3 chronic diseases) and polypharmacy (≥5 drugs prescribed in ≥3months). Sample size: n=400 (200 per study arm). Intervention: complex intervention based on the implementation of the Ariadne principles with two components: (1) FP training and (2) FP-patient interview. Outcomes: MAI score, health services use, quality of life (Euroqol 5D-5L), pharmacotherapy and adherence to treatment (Morisky-Green, Haynes-Sackett), and clinical and socio-demographic variables. Statistical analysis: primary outcome is the difference in MAI score between T0 and T1 and corresponding 95% confidence interval. Adjustment for confounding factors will be performed by multilevel analysis. All analyses will be carried out in accordance with the intention-to-treat principle. Discussion: It is essential to provide evidence concerning interventions on PC patients with polypharmacy and multimorbidity, conducted in the context of routine clinical practice, and involving young-old patients with significant potential for preventing negative health outcomes. Trial registration: Clinicaltrials.gov, NCT02866799Publisher PDFPeer reviewe

Pasados y presente. Estudios para el profesor Ricardo García Cárcel

Ricardo García Cárcel (Requena, 1948) estudió Historia en Valencia bajo el magisterio de Joan Reglà, con quien formó parte del primer profesorado de historia moderna en la Universidad Autónoma de Barcelona. En esta universidad, desde hace prácticamente cincuenta años, ha desarrollado una extraordinaria labor docente y de investigación marcada por un sagaz instinto histórico, que le ha convertido en pionero de casi todo lo que ha estudiado: las Germanías, la historia de la Cataluña moderna, la Inquisición, las culturas del Siglo de Oro, la Leyenda Negra, Felipe II, Felipe V, Austrias y Borbones, la guerra de la Independencia, la historia cultural, los mitos de la historia de España... Muy pocos tienen su capacidad para reflexionar, ordenar, analizar, conceptualizar y proponer una visión amplia y llena de matices sobre el pasado y las interpretaciones historiográficas. A su laboriosidad inimitable se añade una dedicación sin límites en el asesoramiento de alumnos e investigadores e impulsando revistas, dosieres, seminarios o publicaciones colectivas. Una mínima correspondencia a su generosidad lo constituye este volumen a manera de ineludible agradecimiento

Multi-dimensional and spatiotemporal correlative imaging at the plasma membrane of live cells to determine the continuum nano-to-micro scale lipid adaptation and collective motion

The primary cilium is a specialized plasma membrane protrusion with important receptors for signalling pathways. In polarized epithelial cells, the primary cilium assembles after the midbody remnant (MBR) encounters the centrosome at the apical surface. The membrane surrounding the MBR, namely remnant-associated membrane patch (RAMP), once situated next to the centrosome, releases some of its lipid components to form a centrosome-associated membrane patch (CAMP) from which the ciliary membrane stems. The RAMP undergoes a spatiotemporal membrane refinement during the formation of the CAMP, which becomes highly enriched in condensed membranes with low lateral mobility. To better understand this process, we have developed a correlative imaging approach that yields quantitative information about the lipid lateral packing, its mobility and collective assembly at the plasma membrane at different spatial scales over time. Our work paves the way towards a quantitative understanding of the spatiotemporal lipid collective assembly at the plasma membrane as a functional determinant in cell biology and its direct correlation with the membrane physicochemical state. These findings allowed us to gain a deeper insight into the mechanisms behind the biogenesis of the ciliary membrane of polarized epithelial cells.Marie Curie Career Integration Grant (NanodynacTCELLvation; PCIG13-GA-2013-618914) to JBS and a grant (PGC2018-095643-B-I00) to MAA from the Spanish Ministerio de Ciencia e Innovación, Agencia Estatal de Investigación, y Fondo Europeo de Desarrollo Regional, European Union (MICINN/AEI/ FEDER, EU) and by the financial support of the Central Laser Facility (Science and Technology Facilities Council, Harwell, UK

Novel role for the midbody in primary ciliogenesis by polarized epithelial cells

The primary cilium is a membrane protrusion that is crucial for vertebrate tissue homeostasis and development. Here, we investigated the uncharacterized process of primary ciliogenesis in polarized epithelial cells. We show that after cytokinesis, the midbody is inherited by one of the daughter cells as a remnant that initially locates peripherally at the apical surface of one of the daughter cells. The remnant then moves along the apical surface and, once proximal to the centrosome at the center of the apical surface, enables cilium formation. The physical removal of the remnant greatly impairs ciliogenesis. We developed a probabilistic cell population-based model that reproduces the experimental data. In addition, our model explains, solely in terms of cell area constraints, the various observed transitions of the midbody, the beginning of ciliogenesis, and the accumulation of ciliated cells. Our findings reveal a biological mechanism that links the three microtubule-based organelles-the midbody, the centrosome, and the cilium-in the same cellular process.Spanish Ministerio de Economía y Competitividad/Fondo Europeo de Desarrollo Regional: BFU2012-32532 and BFU2015-67266-R; Comunidad de MadridPeer Reviewe

The MAL protein is crucial for proper membrane condensation at the ciliary base, which is required for primary cilium elongation

© 2015. Published by The Company of Biologists Ltd. The base of the primary cilium contains a zone of condensed membranes whose importance is not known. Here, we have studied the involvement of MAL, a tetraspanning protein that exclusively partitions into condensed membrane fractions, in the condensation of membranes at the ciliary base and investigated the importance of these membranes in primary cilium formation. We show that MAL accumulates at the ciliary base of epithelial MDCK cells. Knockdown of MAL expression resulted in a drastic reduction in the condensation of membranes at the ciliary base, the percentage of ciliated cells and the length of the cilia, but did not affect the docking of the centrosome to the plasma membrane or produce missorting of proteins to the pericentriolar zone or to the membrane of the remaining cilia. Rab8 (for which there are two isoforms, Rab8A and Rab8b), IFT88 and IFT20, which are important components of the machinery of ciliary growth, were recruited normally to the ciliary base of MAL-knockdown cells but were unable to elongate the primary cilium correctly. MAL, therefore, is crucial for the proper condensation of membranes at the ciliary base, which is required for efficient primary cilium extension.Ministerio de Economı́a y Competitividad, Spain [grant numbers BFU2012-32532 and CONSOLIDER COAT CSD2009-00016 to M. A.A.]. G.A. was supported by the Amarouto Program for senior researchers from the Comunidad Autónoma de Madrid.Peer Reviewe

Adaptive Lipid Immiscibility and Membrane Remodeling Are Active Functional Determinants of Primary Ciliogenesis

Lipid liquid–liquid immiscibility and its consequent lateral heterogeneity have been observed under thermodynamic equilibrium in model and native membranes. However, cholesterol‐rich membrane domains, sometimes referred to as lipid rafts, are difficult to observe spatiotemporally in live cells. Despite their importance in many biological processes, robust evidence for their existence remains elusive. This is mainly due to the difficulty in simultaneously determining their chemical composition and physicochemical nature, whilst spatiotemporally resolving their nanodomain lifetime and molecular dynamics. In this study, a bespoke method based on super‐resolution stimulated emission depletion (STED) microscopy and raster imaging correlation spectroscopy (RICS) is used to overcome this issue. This methodology, laser interleaved confocal RICS and STED‐RICS (LICSR), enables simultaneous tracking of lipid lateral packing and dynamics at the nanoscale. Previous work indicated that, in polarized epithelial cells, the midbody remnant licenses primary cilium formation through an unidentified mechanism. LICSR shows that lipid immiscibility and its adaptive collective nanoscale self‐assembly are crucial for the midbody remnant to supply condensed membranes to the centrosome for the biogenesis of the ciliary membrane. Hence, this work poses a breakthrough in the field of lipid biology by providing compelling evidence of a functional role for liquid ordered‐like membranes in primary ciliogenesis.This work was supported by a grant (PGC2018‐095643‐B‐I00) to MAA Spanish Ministerio de Ciencia e Innovación, Agencia Estatal de Investigación, and the Fondo Europeo de Desarrollo Regional, European Union and by the financial support through a grant (App. Number16230026) for access to the Central Laser Facility (Science and Technology Facilities Council, UKRI) to use in‐house confocal and STED equipment. E.G. and J.B.S. acknowledge funding from the (ERC) by means of a Marie Curie Career Integration Grant (NanodynacTCELLvation; PCIG13‐GA‐2013‐618914).Peer reviewe