A protein network-guided screen for cell cycle regulators in Drosophila

Background: Large-scale RNAi-based screens are playing a critical role in defining sets of genes that regulate specific cellular processes. Numerous screens have been completed and in some cases more than one screen has examined the same cellular process, enabling a direct comparison of the genes identified in separate screens. Surprisingly, the overlap observed between the results of similar screens is low, suggesting that RNAi screens have relatively high levels of false positives, false negatives, or both

Neurofly 2008 abstracts : the 12th European Drosophila neurobiology conference 6-10 September 2008 Wuerzburg, Germany

This volume consists of a collection of conference abstracts

Studying fibroblast growth factor (FGF) mediated cell migration in "Drosophila" larval air sacs

Invertebrates and vertebrates use FGF signaling in many developmental processes. Mesoderm formation, limb outgrowth but also the development of the vascular system and the lung rely on FGF ligands. We have chosen to study the Drosophila FGF signaling pathway that has been shown to be required for mesodermal- as well as tracheal cell migration. We aimed at a better understanding of FGF signaling to elucidate how the extracellular information, provided by the FGF/Bnl ligand is interpreted in tracheal cells. Using Downstream of FGFR (Dof), an adaptor protein of the FGF signaling pathway, as an entry point, we have previously identified interacting proteins and focused on one prime candidate as a potential linker of FGFR to the cytoskeleton. This candidate protein Receptor of protein kinase C (Rack1) is conserved throughout evolution. rack1 is expressed in the early embryonic tracheal system and has been proposed to play important roles in cell migration as well as in the regulation of the actin cytoskeleton. We have identified and characterized rack1 mutants; these mutants are zygotic lethal but neither show a detectable embryonic- nor any other larval phenotype, due to a very high maternal contribution. Removing the maternal store by generating germline clones results in eggs that fail to develop. This developmental arrest is due to an incomplete transfer of maternal product into the oocyte (nurse cell dumping). In order to characterize the function of rack1 in the context of FGF signaling, we started to characterize the development of third instar larval air sacs. It has been reported that this structure develops via cell migration as well as cell division in response to FGF/Bnl signaling. First we confirm the occurrence of cell division and found that in early air sacs, division is ubiquitous and becomes restricted later to the central part of the air sac. We also documented cell behavior during cell migration using live imaging. To initiate a genetic analysis of rack1 and other candidate target genes in tracheal cell migration, strains and methods were established, allowing the generation of mosaic air sacs consisting of marked wild-type or mutant cells in an otherwise heterozygous background based on the MARCM system. This system was also applied to characterize cellular shape and dynamics of individual or small groups of air sac tracheoblasts in different parts of the air sac. We found that air sac tip cells extend long and dynamic actin based protrusions and further demonstrated that cells not directly located at the tip do form similar protrusions. Finally, we took advantage of the our knowledge of air sac architecture and development to study the cell-autonomous requirement of candidate genes in genetic mosaics. We showed that marked wild-type clones have a preference to be positioned at the tip. Mutants lacking btl or dof, two genes required for embryonic tracheal cell migration, never populate regions at the migratory front. We inferred that air sac tracheoblast cells lacking btl or dof are deficient in migration and take this as a readout for measuring cell migration. Having established criteria for measuring cell migration in air sacs, we tested rack1 mutants for their involvement in air sac tracheoblast migration and find that this gene is not required for this process. We also analyzed other candidate genes as well as components of the FGF signaling pathway and found evidence that Ras plays a dual role during third instar air sac formation. It appears to integrate signaling input from the EGFR pathway to trigger cell division as well as input from the FGF pathway to activate a cell migratory response. In contrast to border cells, mutants affecting the transcription factor Slow border cells (Slbo), the VEGFR (PVR) or DE-Cadherin (Shg) do not impede air sac tracheoblast migration. Components shown to regulate the actin cytoskeleton in response to PVR signaling such as Myoblast city (Mbc) the Drosophila Dock180 homologue or the small Rho family GTPases Rac1, Rac2 and Mig-2-like (Mtl) as well as the effector Chickadee, the Drosophila homologue of Profilin, are essential for air sac tracheoblast migration. Thus, recruitment of these actin cytoskeleton regulators and effectors is mediated via different ligands/receptors in trachea and border cells. Our studies demonstrate that the development of the air sac during late larval stages is a good system to study guided cell migration and allows the genetic dissection of the FGF signaling pathway. The tools we developed allow to assay any candidate gene for which a mutant is available and also laid the foundation for the isolation and characterization of genes in a genome wide EMS screen

A Drosophila protein-interaction map centered on cell-cycle regulators

BACKGROUND: Maps depicting binary interactions between proteins can be powerful starting points for understanding biological systems. A proven technology for generating such maps is high-throughput yeast two-hybrid screening. In the most extensive screen to date, a Gal4-based two-hybrid system was used recently to detect over 20,000 interactions among Drosophila proteins. Although these data are a valuable resource for insights into protein networks, they cover only a fraction of the expected number of interactions. RESULTS: To complement the Gal4-based interaction data, we used the same set of Drosophila open reading frames to construct arrays for a LexA-based two-hybrid system. We screened the arrays using a novel pooled mating approach, initially focusing on proteins related to cell-cycle regulators. We detected 1,814 reproducible interactions among 488 proteins. The map includes a large number of novel interactions with potential biological significance. Informative regions of the map could be highlighted by searching for paralogous interactions and by clustering proteins on the basis of their interaction profiles. Surprisingly, only 28 interactions were found in common between the LexA- and Gal4-based screens, even though they had similar rates of true positives. CONCLUSIONS: The substantial number of new interactions discovered here supports the conclusion that previous interaction mapping studies were far from complete and that many more interactions remain to be found. Our results indicate that different two-hybrid systems and screening approaches applied to the same proteome can generate more comprehensive datasets with more cross-validated interactions. The cell-cycle map provides a guide for further defining important regulatory networks in Drosophila and other organisms

The influence of cell size on cytokinesis in situ and genomic interrogation of human cell size regulation

La cellule est l’élément fondamental de la vie. Plus d’une vingtaine de trillions de cellules forment les organes et tissus de notre corps. Ces cellules sont de taille spécifique puisqu’elles ont des fonctions précises au sein de leur tissu respectif. Dans la plupart des cas, les cellules doivent proliférer en se divisant pour se renouveler et ainsi assurer le bon fonctionnement d’un organisme. La dernière étape de la division cellulaire, la cytokinèse, est exécutée par la contraction d’un anneau contractile d’actomyosine, nécessaire pour effectuer la séparation physique de la cellule en deux cellules filles. La première partie des travaux décrits dans cet ouvrage portent sur la caractérisation de la cytokinèse en utilisant, comme modèle in vivo, les cellules précurseur de la vulve (VPCs) du nématode C. elegans. Notre étude révèle que plusieurs aspects de l’anneau d’actomyosine s’ajustent en fonction de la taille de la cellule. Entre autres, la largeur de l’anneau contractile, juste avant sa constriction, s’ajuste en fonction de la longueur des VPCs. De plus, la rapidité avec laquelle l’anneau se contracte dépend de la circonférence de la cellule. Ces découvertes nous ont amené à nous demander comment la cellule régule sa taille? Les cellules en prolifération maintiennent leur taille en homéostasie en équilibrant leur taux de croissance et de division cellulaire. Afin d’interroger les gènes impliqués dans le maintien de la taille cellulaire du mammifère, nous avons utilisé la technologie CRISPR/Cas9, afin d’éliminer par délétion tous les gènes humains, à raison d’un par cellule, pour identifier ceux qui causent une augmentation ou une diminution de la taille cellulaire. Cette étude nous a permis d’identifier plusieurs gènes déjà connus régulant la croissance cellulaire. De plus, nous avons identifié un groupe de gènes, incluant TLE4 un corépresseur de la transcription que nous avons caractérisé, n’ayant jamais été associé avec une fonction de contrôle de la taille cellulaire chez les mammifères. En somme, nos travaux ont contribué à l’approfondissement des connaissances sur la division cellulaire, plus précisément la cytokinèse, et des gènes impliqués dans le maintien de la taille cellulaire. Une meilleure connaissance du fonctionnement de ces deux évènements cellulaires est essentielle puisque leur dérégulation peut entrainer plusieurs pathologies, incluant le cancer.Cells are the fundamental building blocks of life. The human body contains over twenty trillion cells that make up the different tissues and organs of our bodies. Cells within organs are of specific sizes to perform their specialized functions. In most cases, these cells must divide to proliferate and replenish the population of cells essential for proper organism function. The final stage of cellular division, termed cytokinesis, entails the assembly and constriction of a contractile ring that drives the dramatic cell shape changes required to physically partition the cell into two daughter cells. The first part of the work presented in this thesis addresses the characterization of cytokinesis in the epithelial vulval precursor cells (VPCs) of the nematode worm C. elegans. This study principally revealed that several aspects of cytokinesis scale with cell size. For instance, I observed that the breadth of the actomyosin ring scaled with VPC length. In addition, the speed of contractile ring constriction scaled with the circumference of VPCs. These scaling events raised the more general question as to how cells regulate their size. Proliferating cells attain cell size homeostasis by balancing cell growth and cell division. In order to define the molecular regulators of size in human cells a genome-wide approach was taken. Recently developed CRISPR/Cas9 technology was used to perform the first pooled knockout screens for human cell size regulators in the NALM-6 pre-B lymphocytic cell line. These screens revealed many genes that affect the size of NALM-6 cells, a number of which were previously known to be involved in growth regulation. In addition, these screens revealed the identity of many genes with no previously established functions associated with cell size regulation. Amongst the previously unknown regulators, I characterized the function of a co-repressor of transcription, TLE4, which I showed functions as a regulator of the B-cell lineage. This work contributes to the knowledge of the mechanics of cytokinesis in C. elegans epithelial cells and of the genes that coordinate cell size in humans. These results provide insights into cell growth and division in normal cells and how these processes may be perturbed in cancer and other diseases

Automated data integration for developmental biological research

In an era exploding with genome-scale data, a major challenge for developmental biologists is how to extract significant clues from these publicly available data to benefit our studies of individual genes, and how to use them to improve our understanding of development at a systems level. Several studies have successfully demonstrated new approaches to classic developmental questions by computationally integrating various genome-wide data sets. Such computational approaches have shown great potential for facilitating research: instead of testing 20,000 genes, researchers might test 200 to the same effect. We discuss the nature and state of this art as it applies to developmental research

Strategies for increasing the applicability of biological network inference

The manipulation of cellular state has many promising applications, including stem cell biology and regenerative medicine, biofuel production, and stress resistant crop development. The construction of interaction maps promises to enhance our ability to engineer cellular behavior. Within the last 15 years, many methods have been developed to infer the structure of the gene regulatory interaction map from gene abundance snapshots provided by high-throughput experimental data. However, relatively little research has focused on using gene regulatory network models for the prediction and manipulation of cellular behavior. This dissertation examines and applies strategies to utilize the predictive power of gene network models to guide experimentation and engineering efforts. First, we developed methods to improve gene network models by integrating interaction evidence sources, in order to utilize the full predictive power of the models. Next, we explored the power of networks models to guide experimental efforts through inference and analysis of a regulatory network in the pathogenic fungus Cryptococcus neoformans. Finally, we develop a novel, network-guided algorithm to select genetic interventions for engineering transcriptional state. We apply this method to select intervention strains for improving biofuel production in a mixed glucose-xylose environment. The contributions in this dissertation provide the first thorough examination, systematic application, and quantitative evaluation of the utilization of network models for guiding cellular engineering