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The Role of Deubiquitinases in Invasive Migration and Epithelial Integrity in Drosophila
Get PDFCell migration and epithelial maturation are central to normal development, whilst aberrant migration or loss of epithelial integrity are involved in a number of human diseases, including cancer and birth defects. We are studying these processes in the harmless fruit fly, Drosophila melanogaster, which has been intensively studied for century because of its many attractive features for genetic research. Importantly, Drosophila has proven to be a powerful system in which to identify novel molecules that are involved in both development and diseases. We have identified genes required for cell invasion and the maintenance of epithelial integrity that are conserved from Drosophila to humans. We have uncovered novel roles of these genes which may help guide studies in more complex organisms
Dissecting the molecular mechanisms of drosophila border cell migration using time-lapse live cell imaging
No full textDissection of the cellular dynamics and molecular pathways that drive collective cell migration is necessary to better understand cellular rearrangements that underpin normal development, as well as disease states such as cancer metastasis. Border cell migration in the Drosophila ovary has proven to be a good model of invasive cell migration, because of its genetic tractability, and also because recent advances in culturing egg chambers ex vivo have facilitated live cell imaging in this system. The aim of this thesis was to further develop and implement live cell imaging approaches, and to apply these to characterise the role of Pico, the Drosophila Mig10/RIAM/Lpd (MRL) protein in border cell migration. MRL proteins are known to regulate actin dynamics, but their role in epithelial cell migration had not been established. Through careful optimisation, suitable approaches were developed for: medium preparation; dissection and mounting of egg chambers; acquisition of images by confocal microscopy. A fluorescently-labelled reporter strain with improved optical properties was generated to monitor actin dynamics, and a number of other reporters were characterised, either alone or in combination, to determine their behaviour and effect on migration. After trialling several analytical tools and quantitative methods, a streamlined approach to analysing the image data was developed allowing: tracking of border cell migration in four dimensions (XYZ and time) to obtain information about behaviour of the migratory cells; measurement of cellular protrusion dynamics to obtain mechanistic insight into why cellular dynamics might change in different genetic backgrounds. Finally, these approaches were applied to the characterisation of Pico and its interacting partner SCAR, demonstrating that pico affects border cell migration through the modulation of actin protrusion dynamics in a SCAR-dependent manner
Multidisciplinary approaches for the in vivo interrogation of protein phosphatase holoenzymes
Get PDFThe development and application of real-time protein interaction technology
Get PDFProtein interactions are a fundamental part of cellular processes, and represent a key target in the understanding of cell behaviour, communication and function. These interactions are dynamic in nature, and change over time. Furthermore, interactions can be dependent on co-localisation in cellular compartments. Both of these characteristics can be obscured through observation by bulk molecular cell assays such as co-immunoprecipitation (co-IP) and can obscure the intricacies of single cell dynamics.
The development of tools and new methodologies to observe single cell protein interaction dynamics are key to understanding the underlying mechanisms that direct cell fate. Systems biology aims to incorporate into predictive models of the whole system. In this way, the development of quantitative experimental tools is a key component of systems biology. Förster Resonance Energy Transfer is a widely used technique in the field of protein-protein interaction studies. Dependent on the non-radiative transfer of energy from a fluorescent molecule of higher excitation energy to a fluorescent molecule of lower excitation energy with sufficiently overlapping spectra, the process occurs across a 1-10nm (100Å) range. As a result, FRET interactions between fluorophores attached to biologically functional proteins are a strong indication of protein interaction.
The photoswitchable protein Dronpa offers a unique opportunity to develop a real-time live cell variant of this technique. Through the modulation of Dronpa fluorescence, repeated donor quenching can be observed, allowing quantification of FRET interactions between fluorophores without spillover. This is achieved sequentially through the optimisation of Dronpa imaging parameters for live cell imaging, followed by the identification and testing of candidate FRET partners using an optimised imaging protocol. Direct fusions were used to qualify potential FRET responses between tested pairs of fluorophores. FRET responses using positive control constructs were rigorously tested and quantified to ensure repeatable and consistent reporting of FRET. The spectral properties of Dronpa and chosen FRET partners were also rigorously tested to ensure FRET could be accurately measured through sensitised emission.
Following the confirmation of a reliable FRET response using positive control constructs, the assay was applied to the NF-κB protein p105, and other interacting family members to measure any changes in protein dynamics. The system was applied both on a single switch basis to make comparisons between different combinations of co-expressed fusion proteins and, in time series experiments, to measure potential changes in dynamics over time. p105 showed interesting behaviour not reported in the literature, specifically the detection of full length p105 in the nucleus before stimulation and the strong intramolecular interaction of N and C terminal regions which become perturbed by co-expression of p65. p65 displayed a stronger interaction with the c-terminal region of p105, indicating preferential binding of p65 with ankyrin repeat region rather than the Rel homology domain of p105. Fluorescence Cross Correlation Spectroscopy (FCCS) was used as a complimentary technique to confirm findings in the FRET assay. Considered together, these data suggest Dronpa is a viable component for the accurate detection of FRET in real time, free from complication by spillover. The use of this technique could help to elucidate dynamic protein interactions key to the development and improvement of existing models in systems biology approaches
Dissecting the role of the tumour microenvironment in Drosophila cancer models
Get PDFCancer research has been a primary focus in medical research for more than a century. The complex and ever-changing nature of cancer requires the use of diverse model systems and interdisciplinary approaches to gain a comprehensive understanding of the mechanisms that drive disease initiation and progression, which is necessary for the development of effective therapies. Identifying new mechanisms that do not involve the tumour itself to combat cancer progression is a promising research area. However, the complexity of physiological processes and limited genetic accessibility of mammalian model systems makes it challenging to study non-tumour autonomous processes in vivo using conventional whole animal models. In recent decades, Drosophila melanogaster, with its potent genetic tools, has emerged as an attractive model system for investigating both tumour-intrinsic and non-tumour-derived processes that contribute to tumour development in vivo. This thesis is focused on exploring the relationships between genetically defined tumours and their microenvironment, including macrophage-like and adipocyte-like cells, called haemocytes and fat body cells, respectively. Our objective has been to examine the impact of signalling from these cells on tumour growth and invasion. In this study, we have used two Drosophila genetically-defined cancer models, (RasV12/ S100A4 and RasV12/ dlgKD) to identify ways in which tumour-host interactions are shared or unique in these two genetic cancer models. Specifically, the RasV12/ S100A4 model represents metastatic tumours, while the RasV12/ dlgKD model represents the characteristics of invasive tumours marked by the loss of cell polarity. This approach has involved RNAi- mediated knockdown of molecules participating in key signalling pathways, such as Drosophila-JAK/STAT (d-JAK/STAT), Drosophila-Toll (d-Toll), and Drosophila-TNF (d- TNF), accompanied by multiparameter optical imaging of in vivo tumours to measure the phenotypic effects. Notably, amongst other findings, we showed that targeting the JAK/STAT
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receptor within the tumour itself (locally) in a RasV12/dlgKD tumour model restored adult survival. Surprisingly, we found that the tumours were in direct contact with the fat body, and this association was enhanced by knockdown of Drosophila-Toll receptor (d-toll ) in fat body or Drosophila-Toll ligand (d-spz) in haemocytes. the fat body could be equivalent to tumour- associated adipocytes, observed in other models that provide nutrients or other factors to promote tumour growth. Further, we found that knock down d-TNF receptor (d-grnd) in haemocytes leads to anti-tumour activity in RasV12/dlgKD. However, in the RasV12/S100A4 tumour model, we found that knocking down of molecules linked to common immune signalling such as d-TNF, d-Toll, and d-JAK/STAT in haemocytes, led to an increase the number of tumour-associated haemocytes. Notably, when the d-TNF ligand (d-egr) was knocked down in haemocytes, it resulted in a significant increase in primary tumour size but a decrease in invasion. Conversely, knocking down the d-grnd had no effect on tumour volume or invasion. Knocking down d-spz or d-toll had no significant impact on tumour size but led to a significant increase in invasion. In terms of d-JAK/STAT signalling, knockdown of the d- JAK/STAT ligand (d-upd1) or the d-JAK/STAT receptor (d-dome) resulted in a significant decrease in tumour size, while knockdown of the d-JAK/STAT ligand (d-upd2) led to a significant increase in tumour size. No change in tumour size was observed following knockdown of the d-JAK/STAT ligand (d-upd3). The results emphasize the importance of considering the genetic context in such studies, as our findings revealed diverse immune reactions between the two tumour models. However, to comprehensively comprehend the underlying mechanisms behind this differential immune response, further investigation is needed. This has the potential to reveal novel insights and therapeutic opportunities in the field of tumour immunology
Dissecting the role and regulation of MRL function in Drosophila
Get PDFThe Mig10/RIAM/Lpd (MRL) family of adapter proteins have been recognised in transducing signals derived from growth factor receptors to alterations in cell motility and adhesion via changes in actin dynamics. Reduction in the levels of MRL proteins results in diminished cell division rates, growth retardation, an increase in monomeric (G): filamentous (F) actin ratios, loss of cell migration, and lethality. Conversely, overexpression of MRL proteins reduces the ratio of G:F actin, thereby promoting Serum Response Factor (SRF) signalling, lamellipodia formation, cellular invasion and coordinated cell growth and proliferation. Members of the MRL family all share common structural characteristics, most notably the presence of highly conserved central Ras-association (RA) and Pleckstrin homology (PH) domains in addition to an N-terminal Talin binding site and multiple C-terminal SH3 and proline rich motifs capable of interacting with Ena/VASP and Profilin.
This report extends previous work carried out on Pico, the Drosophila MRL homologue, by demonstrating physical interactions with Chickadee (Profilin), Rhea (Talin), Ras, and Protein Phosphatase 1 (PP1). Many of these binding partners were found to co-localise with Pico within highly dynamic membrane ruffles during Drosophila cell spreading, while only Enabled (Ena/VASP) co-localised at the periphery of cells once they had reached a maximal size and spreading had ceased, pointing to the existence of distinct Pico-associated complexes.
This work also details the presence of a highly conserved MAPK-binding site adjacent to the RA domain which had not been previously recorded in the literature. Site-directed mutagenesis revealed this MAPK-binding motif to be required for Pico's interactions with the Erk1/2 homologue Rolled, while conservation of MAPK binding ability was demonstrated in both human Lpd and RIAM orthologues. Further analysis showed that Rolled might phosphorylate Pico at a serine residue (Ser 819) previously identified by high-throughput phosphoproteomics, which may in turn promote Pico's interactions with PP1. Wing growth assays performed using site-directed Pico mutants indicated that PP1 plays a role in negative regulation of Pico-mediated growth, although the relevant targets of the phosphatase remain to be identified. Interestingly, reporter gene experiments confirmed that Pico induced SRF-dependent gene expression in
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Drosophila cells while ectopic SRF signalling has been found to increase expression of rolled, suggesting the potential existence of a Rolled/PP1 mediated negative feedback loop regulating Pico functionality
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
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