Identification of novel septate junction components through genome-wide glial screens

Epithelial barriers are central to the development of metazoans by compartmentalizing the body in distinct chemical milieus essential for the function of many organs. One such barrier is the blood-brain barrier, which isolates the nervous system from the body fluid to maintain its ionic homeostasis and ensure nerve pulse transmission. In Drosophila, the blood-brain barrier is formed late in embryogenesis by a thin epithelium of subperineurial glia that ensheath the nervous system. Similar to other epithelia, subperineurial glia seal the paracellular space by forming large multiprotein complexes at the lateral membrane, the septate junctions (SJs), which impede free diffusion and mediate barrier function. To identify novel genes required for blood-brain barrier formation, we followed a genome-wide in vivo RNAi approach. We initially screened almost the whole genome for genes required in glia for adult viability and impressively identified 3679 potential candidates. Subsequently, we tested these candidates for requirement in subperineurial glia for adult survival and identified 383 genes. At a last step, we directly asked if blood-brain barrier formation is compromised in the knock-down of the genes by performing the embryonic dye penetration assay in a selection of candidates and identified five genes that play a role during barrier development. Three of these genes, macroglobulin complement-related (mcr) and the previously uncharacterized pasiflora1 and pasiflora2 are further characterized in the context of this thesis. Here we show that all three proteins are novel components of the Drosophila SJ. Pasiflora1 and Pasiflora2 belong to a previously uncharacterized family of tetra-spanning membrane proteins, while Mcr was reported to be a secreted protein in S2 cells required for phagocytosis and clearance of specific pathogens. Through detailed phenotypic analysis we demonstrate that the mutants show leaky blood-brain and tracheal barriers, overelongated tracheal tubes and mislocalization of SJ proteins, phenotypes that are characteristic of SJ mutants. Consistent with the observed phenotypes, the genes are co-expressed in SJ-forming embryonic epithelia and glia and are required cell-autonomously to exert their function. In columnar epithelia, the proteins localize at the apicolateral membrane compartment, where they colocalize with other SJ proteins, and similar to known SJ components, their restricted localization depends on other complex members. Using fluorescence recovery after photobleaching experiments, we demonstrate for Pasiflora proteins that they are core SJ components, as they are required for complex formation and themselves show restricted mobility within the membrane of wild-type epithelial cells, but fast diffusion in cells with disrupted SJs. Taken together, our results show that Pasiflora1 and Pasiflora2 are novel integral SJ components and implicate a new family of tetraspan proteins in the development of cell junctions. In addition, we find a new unexpected role for Mcr as a transmembrane SJ protein, which raises questions about a potential intriguing link between epithelial barrier function, phagocytosis and innate immunity and has potential implications for the function of occluding junctions

Centrosome Amplification Increases Single-Cell Branching in Post-mitotic Cells

Centrosome amplification is a hallmark of cancer, although we are still far from understanding how this process affects tumorigenesis [1, 2]. Besides the contribution of supernumerary centrosomes to mitotic defects, their biological effects in the post-mitotic cell are not well known. Here, we exploit the effects of centrosome amplification in post-mitotic cells during single-cell branching. We show that Drosophila tracheal cells with extra centrosomes branch more than wild-type cells. We found that mutations in Rca1 and CycA affect subcellular branching, causing tracheal tip cells to form more than one subcellular lumen. We show that Rca1 and CycA post-mitotic cells have supernumerary centrosomes and that other mutant conditions that increase centrosome number also show excess of subcellular lumen branching. Furthermore, we show that de novo lumen formation is impaired in mutant embryos with fewer centrioles. 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 centrosomes are necessary to drive subcellular lumen formation. In addition, centrosome amplification increases single-cell branching, a process parallel to capillary sprouting in blood vessels [3]. These results shed new light on how centrosomes can contribute to pathology independently of mitotic defects

Identification of novel septate junction components through genome-wide glial screens

Epithelial barriers are central to the development of metazoans by compartmentalizing the body in distinct chemical milieus essential for the function of many organs. One such barrier is the blood-brain barrier, which isolates the nervous system from the body fluid to maintain its ionic homeostasis and ensure nerve pulse transmission. In Drosophila, the blood-brain barrier is formed late in embryogenesis by a thin epithelium of subperineurial glia that ensheath the nervous system. Similar to other epithelia, subperineurial glia seal the paracellular space by forming large multiprotein complexes at the lateral membrane, the septate junctions (SJs), which impede free diffusion and mediate barrier function. To identify novel genes required for blood-brain barrier formation, we followed a genome-wide in vivo RNAi approach. We initially screened almost the whole genome for genes required in glia for adult viability and impressively identified 3679 potential candidates. Subsequently, we tested these candidates for requirement in subperineurial glia for adult survival and identified 383 genes. At a last step, we directly asked if blood-brain barrier formation is compromised in the knock-down of the genes by performing the embryonic dye penetration assay in a selection of candidates and identified five genes that play a role during barrier development. Three of these genes, macroglobulin complement-related (mcr) and the previously uncharacterized pasiflora1 and pasiflora2 are further characterized in the context of this thesis. Here we show that all three proteins are novel components of the Drosophila SJ. Pasiflora1 and Pasiflora2 belong to a previously uncharacterized family of tetra-spanning membrane proteins, while Mcr was reported to be a secreted protein in S2 cells required for phagocytosis and clearance of specific pathogens. Through detailed phenotypic analysis we demonstrate that the mutants show leaky blood-brain and tracheal barriers, overelongated tracheal tubes and mislocalization of SJ proteins, phenotypes that are characteristic of SJ mutants. Consistent with the observed phenotypes, the genes are co-expressed in SJ-forming embryonic epithelia and glia and are required cell-autonomously to exert their function. In columnar epithelia, the proteins localize at the apicolateral membrane compartment, where they colocalize with other SJ proteins, and similar to known SJ components, their restricted localization depends on other complex members. Using fluorescence recovery after photobleaching experiments, we demonstrate for Pasiflora proteins that they are core SJ components, as they are required for complex formation and themselves show restricted mobility within the membrane of wild-type epithelial cells, but fast diffusion in cells with disrupted SJs. Taken together, our results show that Pasiflora1 and Pasiflora2 are novel integral SJ components and implicate a new family of tetraspan proteins in the development of cell junctions. In addition, we find a new unexpected role for Mcr as a transmembrane SJ protein, which raises questions about a potential intriguing link between epithelial barrier function, phagocytosis and innate immunity and has potential implications for the function of occluding junctions

Centrosome Amplification Increases Single-Cell Branching in Post-mitotic Cells

Centrosome amplification is a hallmark of cancer, although we are still far from understanding how this process affects tumorigenesis [1, 2]. Besides the contribution of supernumerary centrosomes to mitotic defects, their biological effects in the post-mitotic cell are not well known. Here, we exploit the effects of centrosome amplification in post-mitotic cells during single-cell branching. We show that Drosophila tracheal cells with extra centrosomes branch more than wild-type cells. We found that mutations in Rca1 and CycA affect subcellular branching, causing tracheal tip cells to form more than one subcellular lumen. We show that Rca1 and CycA post-mitotic cells have supernumerary centrosomes and that other mutant conditions that increase centrosome number also show excess of subcellular lumen branching. Furthermore, we show that de novo lumen formation is impaired in mutant embryos with fewer centrioles. 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 centrosomes are necessary to drive subcellular lumen formation. In addition, centrosome amplification increases single-cell branching, a process parallel to capillary sprouting in blood vessels [3]. These results shed new light on how centrosomes can contribute to pathology independently of mitotic defects.S.J.A. is a Ramon y Cajal Researcher (RYC-2007-00417); D.R. is the recipient of an FPU PhD fellowship from the Spanish Ministerio de Educación (FPU12/O5765); M.D. was supported by the Erasmus Programme. This work was supported by the Generalitat de Catalunya and grants from the Spanish Ministerio de Ciencia e Innovación/Ministerio de Economia y Competitividad (BFU2009-07629 and BFU2012-32115). IRB Barcelona is the recipient of a Severo Ochoa Award of Excellence from MINECO (Spanish Government).Peer Reviewe

Centrosome Amplification Increases Single-Cell Branching in Post-mitotic Cells

Centrosome amplification is a hallmark of cancer, although we are still far from understanding how this process affects tumorigenesis [1, 2]. Besides the contribution of supernumerary centrosomes to mitotic defects, their biological effects in the post-mitotic cell are not well known. Here, we exploit the effects of centrosome amplification in post-mitotic cells during single-cell branching. We show that Drosophila tracheal cells with extra centrosomes branch more than wild-type cells. We found that mutations in Rca1 and CycA affect subcellular branching, causing tracheal tip cells to form more than one subcellular lumen. We show that Rca1 and CycA post-mitotic cells have supernumerary centrosomes and that other mutant conditions that increase centrosome number also show excess of subcellular lumen branching. Furthermore, we show that de novo lumen formation is impaired in mutant embryos with fewer centrioles. 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 centrosomes are necessary to drive subcellular lumen formation. In addition, centrosome amplification increases single-cell branching, a process parallel to capillary sprouting in blood vessels [3]. These results shed new light on how centrosomes can contribute to pathology independently of mitotic defects