Estudio del ciclo celular de las células troncales germinales del ovario de Drosophila melanogaster: papel del regulador de matriz extracelular Timp

Programa de Doctorado en Biotecnología, Ingeniería y Tecnología QuímicaLínea de Investigación: Biología del DesarrolloClave Programa: DBICódigo Línea: 107En el desarrollo embrionario y en el mantenimiento de tejidos adultos es esencial que se generen nuevas células que contribuyan al crecimiento y reparación de los tejidos cuando sea necesario. Para ello, los eucariotas superiores poseen poblaciones de células indiferenciadas, capaces tanto de autorrenovarse como de dar lugar a uno o varios tipos celulares diferentes; éstas son las llamadas células troncales. Las células troncales se suelen encontrar en microambientes especializados o nichos que regulan su capacidad de proliferación y evitan su diferenciación. Así, para el desarrollo y mantenimiento de los tejidos son claves tanto la correcta actividad de las células troncales como la de los nichos. Los nichos están formados por células de soporte y/o una matriz extracelular especializada. Esta tesis doctoral se centra en estudiar la influencia que la matriz extracelular puede ejercer sobre el comportamiento de las células troncales, utilizando para ello un modelo de células troncales adultas en su nicho natural, el nicho de células troncales germinales (GSCs, del inglés germ line stem cells) del ovario de Drosophila melanogaster. En primer lugar, este trabajo profundiza en la caracterización de algunos parámetros del ciclo celular de las GSCs en condiciones silvestres. Se detallan especialmente parámetros para los que no existe un consenso claro en la literatura, gracias a observaciones directas ex vivo. Parámetros como el comportamiento del espectrosoma, orgánulo específico de GSCs cuya morfología varía a lo largo del ciclo celular; comportamiento de marcadores de ciclo para detallar la progresión de las fases mediante el uso de la herramienta Fly-FUCCI (del inglés fluorescence ubiquitination cell cycle indicator); dinámica de espectrosomas; y eventos asociados a la división de estas células GSCs. En segundo lugar, se estudia el papel que la proteína reguladora de matriz extracelular Timp (del inglés tissue inhibitor of metalloproteinases) podría tener en el comportamiento de las GSCs, analizando la actividad de estas células en un contexto de ausencia de dicha proteína. En esta tesis se ha observado que la ausencia de Timp no afecta a la división de las GSCs, pero sí produce una alteración en la progresión del ciclo celular de estas células troncales, en las que también se observa una acumulación de daño genómico que podría estar desencadenando dichas alteraciones del ciclo. En ausencia de Timp, los ovarios de Drosophila presentan una menor rigidez del tejido, especialmente en el nicho de GSCs, por lo que esta tesis presenta un análisis de la posible relación existente entre el estado de la matriz extracelular y el comportamiento de células troncales en su nicho.Universidad Pablo de Olavide de Sevilla. Escuela de Doctorad

Análisis del papel de la proteína Cinderella en la migración de las células del borde en el ovario de Drosophila melanogaster

Motivación: La migración celular constituye un fenómeno crucial tanto en procesos normales como patológicos, desde el desarrollo embrionario a la metástasis tumoral. Por ello, el avance en el conocimiento de los mecanismos que regulan el fenómeno de migración es de gran relevancia. El ovario de Drosophila melanogaster constituye un buen sistema para el estudio de la migración celular, ya que, durante la maduración de las cámaras ováricas, tiene lugar la delaminación y desplazamiento polarizado de unas células epiteliales llamadas células del borde. Un posible candidato involucrado en esta migración es el gen cinderella. Se ha descrito que Cinderella proporciona una conexión funcional entre adhesiones dinámicas de superficie y cambios del citoesqueleto, durante la migración celular que ocurre en el desarrollo normal del ojo de Drosophila, aunque se desconocen los mecanismos moleculare y celulares por los cuales Cinderella realiza esta función. El objetivo principal de este proyecto es testar si Cinderella se requiere durante la migración de las células del borde y, si este fuera el caso, identificar los mecanismos por los cuales realiza esta función. Métodos: Se han afectado los niveles y la función de Cinderella de manera específica en células del borde mediante la expresión, usando el sistema GAL4/UAS, de RNAis y formas dominantes negativas de la proteína. Se han analizado los fenotipos de migración que las células del borde presentan en estas condiciones experimentales, realizando para ello tinciones inmunohistoquímicas de cámaras ováricas de estadío 10 fijadas.Resultados: Los resultados obtenidos hasta ahora muestran que la disminución de la función de Cinderella en las células del borde produce un retraso en su migración. Así mismo, se ha observado que la expresión de una forma dominante negativa que carece de la región N-terminal, la cual contiene dominios SH3 de unión a cadherinas, es la que produce los fenotipos de migración más fuertes. Conclusiones: Podemos concluir que Cinderella se requiere de manera específica en las células del borde para una migración correcta. El papel que Cinderella pueda tener en dicha migración de las células de borde en el ovario de Drosphila parece estar relacionado con su habilidad para interaccionar con cadherinas, mecanismo que deberá ser estudiado en análisis posteriores

Live analysis of Drosophila female germline stem cells reveals a role for ECM-regulator Timp in stem cell division

Resumen del póster presentado al 11th Meeting of the Spanish Society for Developmental Biology, celebrado en Girona (España) del 19 al 21 de octubre de 2016.-- Tambien presentado a la 25th European Drosophila Research Conference, celebrada en londres (UK) del 22 al 25 de septiembre de 2017.Stem cell activity must be strictly regulated to ensure a proper balance between proliferation and differentiation. This regulation is possible because stem cells reside in specific and restricted microenvironments called niches. The extra cellular matrix (ECM) is an essential component of these niches, where it provides structural support and facilitates proper signalling. For this reason, ECM remodelling in niches ought to be tightly regulated. Matrix metalloproteinases (MMPs), well-known ECM proteases, have been implicated in ECM metabolism in several tissues. MMP activity is generally controlled by Timp (tissue inhibitor of metalloproteinases) proteins. Studying the function of the only Drosophila timp gene in the female germline stem cell (GSC) niche, our laboratory has shown that timp mutant ovaries contain lower amounts of the structural ECM component Collagen IV and possess softer ECM than controls. Moreover, mutant ovaries display an aberrant organisation of the niche and show inefficient gamete production, phenotypes that could be linked to the increased MMP activity typical of timp ovaries. Interestingly, the reduction in gamete production is not due to GSC loss or to increased cell death. Instead, our in vivo analyses support the hypothesis that the reduced generation of germline cysts (and thus of female gametes) is a consequence of mutant GSCs taking longer to divide. In order to demonstrate a link between a prolonged cell cycle in timp mutant GSCs and tissue stiffness, we are studying in detail the cell cycle of control and timp mutant GSCs, with a focus on centrosome/centriole behaviour, spectrosome dynamics and mitosis.Peer Reviewe

Live characterization of Drosophila melanogaster female germline stem cells

Trabajo presentado en el 62nd Drosophila Research Conference, celebrada en Estados Unidos del 23 de marzo al 01 de abril de 2021.The germline stem cells (GSCs) present in the Drosophila melanogaster adult ovary have long been used as a suitable model to study stem cell (SC) behaviour inside its natural microenvironment or niche. GSCs generally divide asymmetrically, as they give rise to one daughter SC that remains in the niche and to a sister cell that enters oogenesis and differentiates into the oocyte and the nurse cell cluster. A large body of research using the fly ovary has allowed the deciphering of different processes that take place in the niche such as cell fate determination, division orientation, intercellular communication, etc. However, because of the long duration of a GSC’s cell cycle (around 20 hours), some descriptions were limited by the difficulties of performing long-term ex vivo analysis of these processes. We have used a combination of genetic tools (such as FlyFUCCI and a myriad of fluorescent proteins) and an ex vivo culture protocol that permits filming live GSCs for long periods of time (at least 10 hours) to describe in detail several processes that occur during GSC proliferation. We will present our recent results that define the behaviour of the spectrosome - the membranous organelle that is essential for proper orientation and division of the GSCs – and its growth throughout the cell cycle, the correlation of cell cycle progression with changes in spectrosome morphology and the behaviour of centrosomes during GSC proliferation

Minimisation of surface energy drives apical epithelial organisation and gives rise to Lewis’ law

The packing of cells in epithelia exhibits striking regularities, regardless of the organism and organ. One of these regularities is expressed in Lewis’ law, which states that the average apical cell area is linearly related to the number of neighbours, such that cells with larger apical area have on average more neighbours. The driving forces behind the almost 100-year old Lewis’ law have remained elusive. We now provide evidence that the observed apical epithelial packing minimizes surface energy at the intercellular apical adhesion belt. Lewis’ law emerges because the apical cell surfaces then assume the most regular polygonal shapes within a contiguous lattice, thus minimising the average perimeter per cell, and thereby surface energy. We predict that the linear Lewis’ law generalizes to a quadratic law if the variability in apical areas is increased beyond what is normally found in epithelia. We confirm this prediction experimentally by generating heterogeneity in cell growth in Drosophila epithelia. Our discovery provides a link between epithelial organisation, cell division and growth and has implications for the general understanding of epithelial dynamics

Does tension keep you in shape? Relevance of Drosophila melanogaster peripodial epithelium on final organ size

Resumen del póster presentado al 11th Meeting of the Spanish Society for Developmental Biology, celebrado en Girona (España) del 19 al 21 de octubre de 2016.How animal organs sense and control that they have reached their species specific target size is a longstanding biological question that hasn¿t been fully addressed yet. Possible mechanisms have been proposed in which molecular concentrations vary throughout organ development and regulate the termination of the process. But during growth, organs not only experience chemical variations but also undergo mechanical stress. We are currently exploring the role tension may play regulating organ growth. Most of what is currently known about growth control mechanisms has been deciphered using the primordia of the adult organs in Drosophila, called imaginal discs. Drosophila imaginal discs are flat sacs of monolayered epithelium. Of the two epithelial sheets, the Peripodial epithelium (PE) plays an ancillary role, not giving rise to adult structures, but participating in the final eversion of the discs during metamorphosis. Therefore, the PE has a mechanical role. We have shown that PE cells accumulate thick acto-myosin bundles with an anterior-posterior orientation, indicative of a strong polarized tension. Therefore, the PE could exert some mechanical influence during disc growth. We will present data on the quantitative characterization of cell and tissue parameters (size, polarity, acto-myosin accumulation, neighbors distribution, cell connectivity, etc) of the PE during disc development, with the aim to infer global properties of the tissue. To relate cell/tissue properties with tension sensing, we will analyze how the subcellular localization of Yki, the co-transcriptional activator of the Hippo pathway and tension sensor, correlates with those properties.Peer Reviewe

Live imaging of the Drosophila ovarian niche shows spectrosome and centrosome dynamics during asymmetric germline stem cell division

Drosophila female germline stem cells (GSCs) are found inside the cellular niche at the tip of the ovary. They undergo asymmetric divisions to renew the stem cell lineage and to produce sibling cystoblasts that will in turn enter differentiation. GSCs and cystoblasts contain spectrosomes, membranous structures essential for orientation of the mitotic spindle and that, particularly in GSCs, change shape depending on the cell cycle phase. Using live imaging and a fusion protein of GFP and the spectrosome component Par-1, we follow the complete spectrosome cycle throughout GSC division and quantify the relative duration of the different spectrosome shapes. We also determine that the Par-1 kinase shuttles between the spectrosome and the cytoplasm during mitosis and observe the continuous addition of new material to the GSC and cystoblast spectrosomes. Next, we use the Fly-FUCCI tool to define, in live and fixed tissues, that GSCs have a shorter G1 compared with the G2 phase. The observation of centrosomes in dividing GSCs allowed us to determine that centrosomes separate very early in G1, before centriole duplication. Furthermore, we show that the anterior centrosome associates with the spectrosome only during mitosis and that, upon mitotic spindle assembly, it translocates to the cell cortex, where it remains anchored until centrosome separation. Finally, we demonstrate that the asymmetric division of GSCs is not an intrinsic property of these cells, as the spectrosome of GSC-like cells located outside of the niche can divide symmetrically. Thus, GSCs display unique properties during division, a behaviour influenced by the surrounding niche.We acknowledge core funding to the Centro Andaluz de Biología del Desarrollo from the Junta de Andalucía

The programmed destabilisation of centrioles in the Drosophila egg chamber through the APC/C ubiquitination pathway

Resumen del póster presentado a la 25th European Drosophila Research Conference, celebrada en Londres (UK) del 22 al 25 de septiembre de 2017.Centrioles are organelles that are present in most of animal cells and are often part of a larger complex called the centrosome, which is composed of the centrioles and pericentriolar material (PCM). The centrosomes regulate various cellular processes, including cell division and establishing cell polarity through their microtubule organising capabilities. In particular, centrosomes are necessary for ciliogenesis and asymmetric cell divisions. In the Drosophila melanogaster egg chamber centriole elimination is thought to prevent parthenogenesis, due to a reticulate mechanism of egg activation prior to fertilisation. lf the centrioles are not eliminated during Drosophila female germline development then the eggs are sterile. However, the centrioles of the germline not only have to be eliminated, but must undergo a peculiar orchestrated relocalisation to the oocyte prior to this elimination. Therefore, centrioles must be strictly controlled for Drosophila fertility. Despite the importance of centriole maintenance in the female germline, the mechanism of this germline-specific regulation is still nebulous. With a modified version of the anaphase promoting complex/cyclosome (APC/C), we show that precise control of the APC/C is responsible for centriole/centrosome regulation during Drosophila oogenesis. The APC/C regulates the programmed destruction of various cell cycle proteins, and we show that in the female germline this includes Polo, a key regulator necessary for PCM and thus centriole stability. lt is through the specifically timed destruction of Polo that the APC/C enables germline centriole elimination. In addition to this, we show the developmental consequence of ectopic centriole regulation.Peer Reviewe

Aboave-Weaire’s law in epithelia results from an angle constraint in contiguous polygonal lattices

It has long been noted that the cell arrangements in epithelia, regardless of their origin, exhibit some striking regularities: first, the average number of cell neighbours at the apical side is (close to) six. Second, the average apical cell area is linearly related to the number of neighbours, such that cells with larger apical area have on average more neighbours, a relation termed Lewis’ law. Third, Aboav-Weaire’s (AW) law relates the number of neighbours that a cell has to that of its direct neighbours. While the first rule can be explained with topological constraints in contiguous polygonal lattices, and the second rule (Lewis’ law) with the minimisation of the lateral contact surface energy, the driving forces behind the AW law have remained elusive. We now show that also the AW law emerges to minimise the lateral contact surface energy in polygonal lattices by driving cells to the most regular polygonal shape, but while Lewis’ law regulates the side lengths, the AW law controls the angles. We conclude that global apical epithelial organization is the result of energy minimisation under topological constraints

The careful control of Polo kinase by APC/C-Ube2C ensures the intercellular transport of germline centrosomes during Drosophila oogenesis

© 2021 The Authors..A feature of metazoan reproduction is the elimination of maternal centrosomes from the oocyte. In animals that form syncytial cysts during oogenesis, including Drosophila and human, all centrosomes within the cyst migrate to the oocyte where they are subsequently degenerated. The importance and the underlying mechanism of this event remain unclear. Here, we show that, during early Drosophila oogenesis, control of the Anaphase Promoting Complex/Cyclosome (APC/C), the ubiquitin ligase complex essential for cell cycle control, ensures proper transport of centrosomes into the oocyte through the regulation of Polo/Plk1 kinase, a critical regulator of the integrity and activity of the centrosome. We show that novel mutations in the APC/C-specific E2, Vihar/Ube2c, that affect its inhibitory regulation on APC/C cause precocious Polo degradation and impedes centrosome transport, through destabilization of centrosomes. The failure of centrosome migration correlates with weakened microtubule polarization in the cyst and allows ectopic microtubule nucleation in nurse cells, leading to the loss of oocyte identity. These results suggest a role for centrosome migration in oocyte fate maintenance through the concentration and confinement of microtubule nucleation activity into the oocyte. Considering the conserved roles of APC/C and Polo throughout the animal kingdom, our findings may be translated into other animals.This work was supported by the Cancer Research UK Career Development Fellowship (CRUK-A12874) and the ShanghaiTech University start-up grant (2018F0202-000-06) to Y.K. and the Wellcome Trust Institutional Strategic Support Fund (ISSF) to A.L.B. and Cancer Research UK (CRUK-RG78567)