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Molecular mechanism of mRNA localisation in Drosophila: a novel function of the motor protein Kinesin

Reference	BB/D004594/1
Principal Investigator / Supervisor	Dr Isabel Palacios
Co-Investigators / Co-Supervisors
Institution	University of Cambridge
Department	Zoology
Funding type	Research
Value (£)	260,570
Status	Completed
Type	Research Grant
Start date	01/10/2005
End date	31/03/2009
Duration	42 months

Abstract

The aims are to understand how motor proteins recognise their cargoes and how mRNAs are localised. These aims are organised in 2 objectives: 1.Characterising the Kinesin Light Chain (KLC)-independent function of Kinesin Heavy Chain(KHC) in Drosophila; 2.Understanding how KHC associates with oskar(osk) mRNA. My previous work has shown that some of the KHC functions in flies rely in a novel KLC-independent mechanism. In the oocyte, these functions are the localisations of osk mRNA and Dynein Heavy Chain (DHC) to the posterior, and the induction of cytoplasmic flows. We will study this new mechanism of KHC function, as well as answer how KHC associates with osk mRNA, by several complementary approaches. 1.Characterising the KLC-independent function of KHC:a.Screening for new mutants with a khc-like phenotype: We will screen for mutants that produce similar phenotypes to those in the khc, namely a failure to localise osk mRNA, and most importantly, the absence of cytoplasmic flows, a defect only observed so far in khc. For this, we will re-screen several osk mRNA localisation mutants (isolated in D. St Johnston's lab) by analysing the ooplasmic flows, which is done in the confocal microscope by time-lapse movies, and Kalman scans (fig1.Caseforsupport). The mapping of the found mutants to a small interval (50Kb) will be done by meiotic recombination using single-nucleotide polymorphisms. We will then proceed to the sequence of candidate genes, and if necessary to a deletion mapping. In parallel, we will further characterise the mutant phenotype by looking at the microtubule organisation and the localisations of KHC and DHC. Once the mutated gene is identified, we will make a rescue construct by cloning the transcription unit into a germline specific vector. We will look at the distribution of the protein by both GFP tagging and antibodies, in both wild-type and several mutant backgrounds, such as khc and stau. If these experiments suggest a direct association of the factor with KHC and/or osk mRNA, we will further characterise this interaction with techniques described next. b.Identifying new KHC-interactors and analysing their function:We will identify new KHC interactors by both a two-hybrid(TH) screen, and the isolation of KHC complexes. The TH screen will be done using the tail of KHC and an ovarian cDNA library. The KHC complexes will be isolated from ovarian extracts using a KHC antibody, and the co-purified proteins will be identified by mass spec or antibodies. We will then obtain mutants to analyse the KHC-related function of these interactors. If the mutants do not exist, we will generate them by the imprecise excision of a P element in or close to the gene of interest. c.Characterising the function of candidate KHC-interactors in oogenesis: We will analyse the function during oogenesis of proteins that have homology to KHC interactors or associate with KHC in other cell types. These candidate proteins are YETI, Unc-76 and Pat1. In the case of unc-76, there are mutants and antibodies available, and thus the functional analysis can be done promptly. In the case of YETI and Pat1, there are no mutants or antibodies available. We will study the localisation of Pat1-GFP and YETI-GFP in the oocyte, as well as characterise their interaction with KHC by TH, and immunoprecipitations using Pat1-GFP and YETI-GFP extracts. If these experiments show that Pat1 and YETI interact with KHC, we will generate pat1 mutants by the imprecise excision of a P-element inserted in the gene, and yeti mutants by RNAi. 2.Understanding how KHC associates with the osk mRNA localisation complex. To analyse whether KHC interacts directly with osk mRNA, we will analyse whether osk mRNA (by RT-PCR) or any of the components of the osk RNP are part of the isolated KHC complexes described above. Since KHC has a role in vesicle transport, we will analyse by EM if there is any association of the components of the osk RNP, as well as KHC, with vesicles.

Summary

All organisms, including humans, present many aspects of asymmetry in their bodies. Some of these asymmetries define specific body axes, such as the anterior-posterior (head to tail) axis. It is fascinating when one considers that the determination of the body axes occurs early in the development of an organism, and in some cases, even in the egg. Embryologists suggested long ago that the asymmetric distribution of substances, called determinants, in the cytoplasm of an egg can confer a specific characteristic to the cells that receive it following a division. One way to asymmetrically distribute determinants is to localise the transcripts (mRNAs) that encode them to the region where the determinant is required, and to subsequently translate into protein only the localised mRNAs. One of the most striking examples of cell polarisation is found in the egg of the fruitfly Drosophila melanogaster, in which the transcripts that encode determinants for the anterior-posterior axis are localised to opposite poles of the cell. The mRNAs are then anchored at the anterior and posterior poles until translation occurs at a precise stage in the development of the egg. For example, the localisation of oskar mRNA to the posterior pole of the oocyte is essential to determine the development of the abdomen and the pole cells. Thus, the understanding of how mRNAs are localised is of critical importance towards the understanding of how an organism - including humans - develop. Molecular motor proteins generate the movement of a wide variety of materials (called cargoes) in cells. Such movements are crucial for many different cellular and developmental functions. Thus, elucidating the transport pathways mediated by motor proteins, the identity of the cargoes moved, and the nature of the motor-cargo link are areas of increasing importance and intense investigation. Kinesin, a microtubule plus-end directed motor protein, is required for the localisation of oskar mRNA and the induction ofcytoplasmic flows in the Drosophila oocyte. Although some progress has been made in the understanding of how Kinesin functions, the nature of the Kinesin-cargo interactions remain poorly understood. Similarly, although several genes required for the localisation of mRNAs have been identified, the molecular mechanism - such as how localised mRNAs are coupled to the motor proteins that transport them - is not yet understood. My research aim is to characterise a novel mechanism for Kinesin-cargo binding and to understand how RNAs are localised, using the function of Kinesin during Drosophila oogenesis, and the localisation of oskar mRNA as a model system. Why did I choose Drosophila as a model system? I strongly believe, and it has been proven time and again, that the fruitfly is one of the most powerful and elegant systems for combining genetics, biochemistry and cell biology for the study of developmental processes.

Committee	Closed Committee - Genes & Developmental Biology (GDB)
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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