Dissecting the molecular mechanisms of drosophila border cell migration using time-lapse live cell imaging

Dissection 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

Use of a targeted, computer/web-based guided self-help psychoeducation toolkit for distressing hallucinations (MUSE) in people with an at-risk mental state for psychosis: protocol for a randomised controlled feasibility trial

Individuals who access at-risk mental state (ARMS) services often have unusual sensory experiences and levels of distress that lead them to seek help. The Managing Unusual Sensory Experiences (MUSE) treatment is a brief symptom targeted intervention that draws on psychological explanations to help account for unusual experiences. Practitioners use formulation and behavioural experiments to support individuals to make sense of their experiences and enhance coping strategies. The primary objective of this feasibility trial is to resolve key uncertainties before a definitive trial and inform parameters of a future fully powered trial

Fig.S1 Comparison of global RNA-Seq gene expression profiles. from Transcriptional responses to hyperplastic MRL signalling in <i>Drosophila</i>

Comparison of global RNA-Seq gene expression profiles. A, When control (MS1096-GAL4), MS1096>pico and MS1096>mrtf samples are grouped together, very little difference in the global gene expression profiles can be seen between conditions. B, Nevertheless, dendrograms show close hierarchical clustering between the different replicates for each genotype, suggesting there are consistent changes in gene expression in response to pico and mrtf overexpression

Table S1. Comparison of gene level expression in wings imaginal disks of pico and mrtf to control by RNA-sequencing. from Transcriptional responses to hyperplastic MRL signalling in <i>Drosophila</i>

Comparison of gene level expression in wings imaginal disks of pico and mrtf to control by RNA-sequencing. mRNA from wings imaginal disks of MS1096>pico MS1096>mrft and MS1096-GAL4 alone were sequenced on illumina Hi-seq 2000 (40 M reads each repeat) and statistically compared by t-test where P value were adjusted by Benjamini-Hochberg (FDR<0.05; (Benjamini and Hochberg, 1995)) multiple testing procedure (allDataset worksheet). Genes significantly under- or over-expressed in pico and mrtf are displayed in separated worksheets

Text S1. Details of RNAi lines, matrix of CarG boxes, sequence of SRE element used in SRE-mCherry reporter and primer sequences used in this paper. from Transcriptional responses to hyperplastic MRL signalling in <i>Drosophila</i>

Recent work has implicated the actin cytoskeleton in tissue size control and tumourigenesis, but how changes in actin dynamics contribute to hyperplastic growth is still unclear. Overexpression of Pico, the only <i>Drosophila</i> Mig-10/RIAM/Lamellipodin adapter protein family member, has been linked to tissue overgrowth via its effect on the myocardin-related transcription factor (Mrtf), an F-actin sensor capable of activating serum response factor (SRF). Transcriptional changes induced by acute Mrtf/SRF signalling have been largely linked to actin biosynthesis and cytoskeletal regulation. However, by RNA profiling, we find that the common response to chronic <i>mrtf</i> and <i>pico</i> overexpression in wing discs was upregulation of ribosome protein and mitochondrial genes, which are conserved targets for Mrtf/SRF and are known growth drivers. Consistent with their ability to induce a common transcriptional response and activate SRF signalling <i>in vitro</i>, we found that both <i>pico</i> and <i>mrtf</i> stimulate expression of an SRF-responsive reporter gene in wing discs. In a functional genetic screen, we also identified <i>deterin</i>, which encodes <i>Drosophila</i> Survivin, as a putative Mrtf/SRF target that is necessary for <i>pico</i>-mediated tissue overgrowth by suppressing proliferation-associated cell death. Taken together our findings raise the possibility that distinct targets of Mrtf/SRF may be transcriptionally induced depending on the duration of upstream signalling

Table S2. Biological Process Ontology (BP-GO) term enrichment in genes showing their expression affected by pico and mrtf. from Transcriptional responses to hyperplastic MRL signalling in <i>Drosophila</i>

Biological Process Ontology (BP-GO) term enrichment in genes showing their expression affected by pico and mrtf. Gene Ontology (GO) enrichment was determined using DAVID (Huang et al., 2009). Biological process categories from GO analysis that are significantly overrepresented among the genes for which the expression were either decreased (downregulated worksheet) or increased (upregulated worksheet) in response to pico overexpression or mrtf overexpression. BP-GO were grouped into categories sharing similar function and used to produce the pie chart (Figure 1A). Only the categories with a minimum of 5 genes per category and an EASE score ≤0.05 were considered

Lamellipodin and the Scar/WAVE complex cooperate to promote cell migration in vivo

Cell migration is essential for development, but its deregulation causes metastasis. The Scar/WAVE complex is absolutely required for lamellipodia and is a key effector in cell migration, but its regulation in vivo is enigmatic. Lamellipodin (Lpd) controls lamellipodium formation through an unknown mechanism. Here, we report that Lpd directly binds active Rac, which regulates a direct interaction between Lpd and the Scar/WAVE complex via Abi. Consequently, Lpd controls lamellipodium size, cell migration speed, and persistence via Scar/WAVE in vitro. Moreover, Lpd knockout mice display defective pigmentation because fewer migrating neural crest-derived melanoblasts reach their target during development. Consistently, Lpd regulates mesenchymal neural crest cell migration cell autonomously in Xenopus laevis via the Scar/WAVE complex. Further, Lpd’s Drosophila melanogaster orthologue Pico binds Scar, and both regulate collective epithelial border cell migration. Pico also controls directed cell protrusions of border cell clusters in a Scar-dependent manner. Taken together, Lpd is an essential, evolutionary conserved regulator of the Scar/WAVE complex during cell migration in vivo

Carbonic anhydrase III (Car3) is not required for fatty acid synthesis and does not protect against high-fat diet induced obesity in mice

Carbonic anhydrases are a family of enzymes that catalyze the reversible condensation of water and carbon dioxide to carbonic acid, which spontaneously dissociates to bicarbonate. Carbonic anhydrase III (Car3) is nutritionally regulated at both the mRNA and protein level. It is highly enriched in tissues that synthesize and/or store fat: liver, white adipose tissue, brown adipose tissue, and skeletal muscle. Previous characterization of Car3 knockout mice focused on mice fed standard diets, not high-fat diets that significantly alter the tissues that highly express Car3. We observed lower protein levels of Car3 in high-fat diet fed mice treated with niclosamide, a drug published to improve fatty liver symptoms in mice. However, it is unknown if Car3 is simply a biomarker reflecting lipid accumulation or whether it has a functional role in regulating lipid metabolism. We focused our in vitro studies toward metabolic pathways that require bicarbonate. To further determine the role of Car3 in metabolism, we measured de novo fatty acid synthesis with in vitro radiolabeled experiments and examined metabolic biomarkers in Car3 knockout and wild type mice fed high-fat diet. Specifically, we analyzed body weight, body composition, metabolic rate, insulin resistance, serum and tissue triglycerides. Our results indicate that Car3 is not required for de novo lipogenesis, and Car3 knockout mice fed high-fat diet do not have significant differences in responses to various diets to wild type mice