Please use this identifier to cite or link to this item: http://hdl.handle.net/2445/114169
Title: Cytoskeletal modulation of single-cell branching
Author: Ricolo, Delia
Director: Araújo, Sofia J.
Keywords: Drosòfila
Microtúbuls
Citosquelet
Drosophila
Microtubules
Cytoskeleton
Issue Date: 19-May-2017
Publisher: Universitat de Barcelona
Abstract: [eng] The embryonic tracheal system of Drosophila melanogaster consists of a network of interconnected epithelial tubes of different size and architecture characterized by different cellular mechanisms of tube formation. The main branches of the Drosophila tracheal system have an extracellular lumen because their cells fold to form a tube. However, terminal cells (TCs), specialized cells designed to connect the tracheal system to target tissues, form unicellular branches by generating of a subcellular lumen. This topology of unicellular tubes is a good model to clarify the mechanisms that orchestrate single-cell branching, a process parallel to capillary sprouting in blood vessels. During tracheal embryonic development, TCs produce seamless tubes, generating a cytoplasmic extension, by cell elongation, and a concurrent intracellular luminal space surrounded by an apical membrane. Cell elongation and subcellular lumen formation are very much dependent on cytoskeleton reorganization. The main aim of this thesis was to understand new aspects of cytoskeletal modulation that orchestrate subcellular lumen formation. In particular, we have addressed this aim analysing mutants displaying an increase in subcellular lumen branching and mutants characterized by the absence of the subcellular lumen. We found that mutations in Regulator of Cyclin A (Rca1) and Cyclin A (CycA) affect subcellular branching, causing TCs to form more than one subcellular lumen. The effect of Rca1 is post-mitotic in the tracheal system, and depends on an amplification of centrosome number. Other mutant conditions, characterized by the increase of centrosome number, such as Slimb (slmb) and the overexpression of SAK also show excess of subcellular lumen branching. Furthermore, we showed that de novo lumen formation is impaired in mutant embryos with low centrosome numbers such as sas4 and is restored in the double mutant Rca1; sas4. The data presented here define a requirement for the centrosome as a microtubule organizing center (MTOC) for the initiation of subcellular lumen formation. We propose that in wt condition two centrosomes are needed to arrange the specific intracellular TC organization necessary to generate a subcellular lumen, and that an excess of centrosome numbers allows for an increase in single- cell branching. We also analysed the involvement of the spectraplakin Short-stop (Shot) in the cytoskeletal organization of the TCs. Shot is a multifunctional protein involved in many aspects of cytoskeletal organization in different tissues, which can operate as a single cytoskeleton component as well as coordinating cytoskeletal elements between them. This functional versatility of Shot is probably reflected by the abundant generation of isoforms and by the modulation of its numerous domains. We found that the overexpression of shot in the tracheal system induces extra-branching of the subcellular lumen and this effect depends mainly on the C-tail domain at the C- terminus and its involvement in the stabilization/polimerization of MTs. On the other hand, by examining loss of function alleles, analysing its structural function and visualizing Shot accumulation, we suggest that Shot is not just involved in MT organization in the TC but it also acts as a crosslinker between MTs and the actin network. The first calponin domain (CH1) of the acting binding domain (ABD) at the N-terminal is involved in this cross-linking activity. Finally, we provide some data indicating a functional overlap between the spectraplakin and the microtubule associated protein (MAP) Tau during subcellular lumen formation.
[spa] Las células terminales (TCs) de la tráquea del embrión de Drosophila melanogaster son capaces de generar un lumen subcelular y son utilizadas como modelo para la formación de tubos unicelulares de tipo “seamless”. La generación de dicho lumen depende estrictamente de una especifica organización del citoesqueleto que permite la formación de una nueva membrana apical en el interior de la TC. El objetivo del trabajo aquí presentado ha sido lo de aclarar nuevos aspectos de la modulación del citoesqueleto en el contexto de la formación del lumen sub-celular. Los mutantes de Regulator of Cyclin A (Rca1) y CycA (Cyclin A) están caracterizados por TC con mas de un lumen subcelular. El efecto de Rca1 es post-mitótico y esta causado por un aumento del numero de centrosomas. Reportamos, atraves el estudio de Rca1 y otros mutantes afectados en el numero de centrosomas, una estricta asociación entre centrosomas y formación del lumen sub-celular. Nuestros datos revelan, por primer vez, la función de los centrosomas como centros de organización de microtubulos (MTOC) en la TC y que un exceso de centrosomas puede causar un aumento en la capacidad de ramificación del lumen. En este trabajo también hemos analizado la función de la spectraplakina Short-stop (Shot). A través de experimentos de sobre-expresión y falta de función de shot, integrados con estudios estructura-función y de localización de sus productos proteicos hemos concluido que la spectraplakina actúa el la TC acudiendo a diferentes grados de organización citosqueletrica; en nuestro modelo Shot es capaz de promover la estabilización/polymerizacion de microtubulos, y un exceso de esta función puede causar extra ramificación en la TC. Por otro lado, Shot esta implicado en la correcta conexión entre la red de microtubulos y la actina y su falta influye negativamente la formación del lumen sub-celular. También reportamos datos preliminares que indican una superposición funcional entre Shot y la proteína asociada a microtulos (MAP) Tau durante el desarrollo del la TC.
URI: http://hdl.handle.net/2445/114169
Appears in Collections:Tesis Doctorals - Departament - Genètica

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