Cookies disclaimer

I agree Our site saves small pieces of text information (cookies) on your device in order to deliver better content and for statistical purposes. You can disable the usage of cookies by changing the settings of your browser. By browsing our website without changing the browser settings you grant us permission to store that information on your device.

Current Filters

Recipients:
University of Cambridge

Results

Mechanisms of cell polarity and mRNA localisation in the Drosophila oocyte. 01 Dec 2006

The anterior-posterior axis of Drosophila is determined by the localisation of bicoid and oskar mRNAs to opposite poles of the oocyte, and provides an excellent system to investigate the conserved mechanisms of cell polarity and mRNA localisation. 1) We plan to screen the whole genome for mutants that affect the localisation of fluorescently-labelled bicoid and oskar mRNAs in living oocytes, in order to identify genes required for oocyte polarity and the localisation of each mRNA 2) The recruitment of the PAR-1 kinase to the oocyte posterior is the first sign of anterior-posterior polarity. We will characterise a number of genes required for this localisation, and use a proteomics approach to identify proteins that interact with PAR-1 in vivo. 3) Since PAR-1 regulates the organisation of the microtubule cytoskeleton, we will perform biochemical screens for PAR-1 substrates that associate with microtubules, and analyse several candidate proteins identified by genetic screens and bioinformatics. 4) We will characterise the components of the oskar mRNA and bicoid mRNA localisation complexes using genetic and proteomics approaches, and will visualise their localisation in vivo. 6) We will develop an oskar mRNA in vitro motility assay to determine how the motors that move it are regulated.

Amount: £4,120,402
Funder: The Wellcome Trust
Recipient: University of Cambridge

Rate of degradation of Aurora kinases 27 Apr 2017

Aurora kinases regulate the segregation of chromatids and are key enzymes in mitosis. AurA assembles the spindle poles; AurB faciliates cytokinesis of the daughter cells. Their ubiquitin-mediated degradation regulates the transition from mitosis back to interphase and show different kinetic profiles: AurA degrades 5-fold faster than AurB. Previous unpublished data from the Lindon lab showed that a AurA1-133-AurB78-345 chimera tagged with GFP degraded with similar kinetics to full-length AurA. Therefore all of the information required for rapid degradation of AurA resides in the 1-133 region. We plan to construct various AurA-AurB chimeras and express them in dividing cells. We will carry out a quantitative analysis of degradation of these chimeras using single-cell fluorescence timelapse assays. We aim to identify the the minimal sequence within AurA1-133 required to specify accelerated degradation kinetics. We plan to compare this with other known regulatory sequences for ubiquitin-mediated degradation ('degrons') and to gain a better understanding of how AurA engages the destruction machinery to affect its degradation kinetics. This information can assist the design of new therapeutic tools, such as PROTACs, that harness ubiquitin-mediated degradation to destroy targets not druggable by conventional means.

Amount: £0
Funder: The Wellcome Trust
Recipient: University of Cambridge

Early cell fate decisions and cell positioning in the mouse embryo. 01 Jun 2006

I propose three complementary approaches to study how early events can help to specify polarity of the mouse embryo. I first propose to study developmental cues in the egg whose animal-vegetal polarity dictates one axis of the future blastocyst, and the position of sperm entry determines the second axis. The sperm entry position sets the first cleavage plane and conveys a division advantage upon the cell inheriting it. I will investigate the cytoplasmic events underlying these two consequences of sperm entry and test their relative importance in establishing the embryonic abembryonic polarity of the blastocyst. I also propose to study why, although the animal pole of the egg can be removed without affecting development, its duplication is inhibitory. Secondly I will combine lineage tracing and transplantation studies to ask how polarity of the blastocyst, set up by the above processes, is transformed to give organised signalling centres in the postimplantation embryo. My focus will be to discover the origins of visceral endoderm with potential to signal to the epiblast of the egg cylinder. To determine when such signalling centres become active, I will first concentrate on transplantation experiments that test the ability of anterior visceral endoderm precursors to repress posterior gene activity. The extent of such experiments could be broadened by better knowledge of the patterns of gene expression from the blastocyst onwards. The third part of my proposal aims to identify genes that are expressed asymmetrically along axes of the blastocyst and/or at the earliest times within postimplantation signalling centres or their progenitors. My attention will centre upon finding genes that are differentially expressed in the animal and vegetal halves of the blastocyst or become uniquely expressed in progenitors of anterior visceral endoderm. These will be identified through the construction of subtractive cDNA libraries and by screening microarrays. I will select genes with expression patterns likely to be meaningful in the development of signalling centres and examine the consequences of both their ectopic expression and loss of expression using dsRNAi. In the longer term I propose to analyse how the patterns of expression of these or other genes are rebuilt following perturbation of development. Thus not only do I hope my work wil contribute to a molecular understanding of how asymmetries are established and transmitted to later stages of the embryo in normal development, but also will provide insight into the remarkable regulative properties of the mammalian embryo.

Amount: £2,417,906
Funder: The Wellcome Trust
Recipient: University of Cambridge

A high resolution platform to capture the dynamic spatial proteome. 11 Jun 2015

This proposal aims to create a platform for mapping the subcellular location of a substantial proportion of the proteome in a single experiment with high resolution. It is based on preliminary work carried out by the Lilley group in collaboration with Thermo, giving tantalising insight into what this technology could deliver if developed further into a fit-for-purpose spatial proteomics platform. The key objectives are: 1. Expand the sampling of subcellular proteome to locate proteins to multi ple compartments. 2. Capture information of the effect of post-transcriptional and post-translational modification on spatial location. 3. Develop approaches to enable the mapping of the dynamic subcellular redistribution of proteins upon biological perturbation. 4. Incorporate work-flows that will deliver spatial information about targeted sub-sets of proteins and integration with whole cell maps. 5. Develop cross-linking strategies to preserve interactions of peripheral membrane proteins and between components of multi-protein complexes. 6. Develop a set of bespoke informatics tools facilitating the application of pattern recognition for robust analysis. 7. Create a GUI to facilitate community-wide interrogation of cellular maps. 8. Develop on-line protocols. 9. Apply the technology to the co-applicants and collaborators research, adding value to projects already funded by the Wellcome Trust.

Amount: £200,788
Funder: The Wellcome Trust
Recipient: University of Cambridge

The control of polarity in eggs and epithelia. 02 May 2012

The anterior-posterior axis of Drosophila is determined by the localisation of bicoid and oskar mRNAs to opposite poles of the oocyte, and provides an excellent system to investigate the conserved mechanisms of cell polarity and mRNA localisation. 1) We plan to screen the whole genome for mutants that affect the localisation of fluorescently-labelled bicoid and oskar mRNAs in living oocytes, in order to identify genes required for oocyte polarity and the localisation of each mRNA 2) The recruitment of the PAR-1 kinase to the oocyte posterior is the first sign of anterior-posterior polarity. We will characterise a number of genes required for this localisation, and use a proteomics approach to identify proteins that interact with PAR-1 in vivo. 3) Since PAR-1 regulates the organisation of the microtubule cytoskeleton, we will perform biochemical screens for PAR-1 substrates that associate with microtubules, and analyse several candidate proteins identified by genetic screens and bioinformatics. 4) We will characterise the components of the oskar mRNA and bicoid mRNA localisation complexes using genetic and proteomics approaches, and will visualise their localisation in vivo. 6) We will develop an oskar mRNA in vitro motility assay to determine how the motors that move it are regulated.

Amount: £2,881,633
Funder: The Wellcome Trust
Recipient: University of Cambridge

A molecular and genetic analysis of cell polarity and mRNA localisation in the Drosophila oocyte. 16 Sep 2008

mRNA localisation is a general mechanism that targets proteins to the specific regions of a cell where they are required. The significance of this process has been most clearly demonstrated in Drosophila, where the localisation of bicoid, oskar, and gurken mRNAs to three distinct positions within the oocyte defines the two axes of the embryo. To investigate the molecular mechanisms that direct mRNA localisation, we propose to take several approaches to determine how oskar mRNA is transported to the posterior of the oocyte. 1) We will investigate how the RNA-binding protein Staufen recognises oskar mRNA to form the substrate for localisation, and will also characterise a novel gene, wkl, that is specifically required for the movement of Staufen/oskar mRNA complexes from the anterior to the posterior. 2) We shall observe the process of localisation in living oocytes to determine whether oskar mRNA is actively transported to the posterior pole. 3) Since it is clear that we have so far identified only a few of the factors that are involved oskar mRNA localisation, we will use two strategies to find the missing components in this pathway. We shall carry out biochemical screens for proteins that bind to Staufen/oskar mRNA complexes, as these may correspond to the cytoskeletal components that mediate localisation. We shall also perform a large scale genetic screen for new genes involved in this process. Our second objective is to understand how the A-P axis of the oocyte becomes polarised to define the destination of these transcripts. We will use a variety of approaches to identify the follicle cells that induce this polarisation, to investigate the effects of this polarising signal on the organisation of the oocyte cytoskeleton, and to characterise the components of this signalling pathway. In addition, we shall analyse the spindle genes, which are required for the three steps that generate the first polarities during oogenesis.

Amount: £9,393
Funder: The Wellcome Trust
Recipient: University of Cambridge

The role of Myosin VI in Membrane traffic and Cell Division. 29 Aug 2008

Myosin motor proteins move membrane and vesicles along actin tracks. Myosin VI unlike almost all other myosins moves towards the minus end of actin filaments. Our intracellular localisation of myosin VI at the Golgi complex, in clathrin coated pits/vesicles and at kinetochores in mitotic cells indicates that it functions in a wide variety of intracellular processes. The diverse roles of myosin VI are mediated by interaction with a range of different binding partners. The aim of my research is to characterise the roles of myosin VI and its interacting partners, which we have recently identified: 1. In the maintenance of Golgi morphology, sorting of cargo in the trans-Golgi network and in post Golgi transport of vesicles to the plasma membrane; 2. In endocytosis at the apical domain of polarised cells. Using cell lines from the small intestine (Caco-2) and the kidney (MDCK) as well as primary kidney cells isolated from the Snell's waltzer myosin VI knockout mouse, I will determine the sequential steps of the endocytic process and the class of receptor for which myosin VI is important; 3. In mitosis to confirm their localisation at kinetochores and spindle poles and determine their function. I will study how the interaction and activity of these multi-protein complexes containing myosin VI are regulated at various cellular localisations by overexpressing myosin VI of deletion mutants followed by co-immunoprecipitation for subsequent analysis. I will visualise how myosin VI moves in cells using live cell imaging and investigate how its surprisingly wide variety of intracellular functions is so precisely regulated. To investigate which type of motor is required to fulfil this variety of intracellular functions I will study the kinetic and mechanical properties of myosin VI at the single molecule level in collaboration with the group of John Kendrick-Jones at the MRC-Laboratory of Molecular Biology in Cambridge. The information I will gain from these studies will help us to understand how the loss of active myosin VI results in a form of deafness in humans and how alternations in its binding partners cause other disorders such as glaucoma. An understanding of the role of myosin VI in these diseases may allow us to devise therapeutic strategies.

Amount: £216,533
Funder: The Wellcome Trust
Recipient: University of Cambridge