Award details

Dynamic in vivo imaging and genetic dissection of the motility machinery of Drosophila macrophages

Reference	BB/F020635/2
Principal Investigator / Supervisor	Dr Brian Stramer
Co-Investigators / Co-Supervisors
Institution	King's College London
Department	Randall Div of Cell and Molecular Biophy
Funding type	Research
Value (£)	390,513
Status	Completed
Type	Research Grant
Start date	01/06/2009
End date	30/06/2012
Duration	37 months

Abstract

A better knowledge of the molecular events governing cell motility is crucial to understanding a variety of human pathologies. However, most of our understanding of cell movement stems from tissue culture assays that give us an incomplete picture of this process. To expand our understanding of cell motility this proposal will combine the genetic tractability of Drosophila, along with the ability to live image Drosophila macrophage wound chemotaxis in vivo, to dissect the requirement of key regulators of cell migration. In objective 1 I will develop and characterize fluorescent tools that allow for the live imaging of key components of the migration machinery (actin, microtubules, polarity markers, and integrins). Aside from being useful to the Drosophila research community, these markers when expressed in macrophages will increase the range of this assay as they will allow me to more precisely dissect the role of candidate migratory genes. I will then use these imaging tools to examine the requirement of the microtubule cytoskeleton, which will be removed both chemically and genetically in macrophages, during cell migration. In the next objective I will examine the role of the cancer-associated, actin bundling protein, fascin, during macrophage migration. This gene, which has only been studied using in vitro motility assays, is specifically expressed in Drosophila macrophages. Using preexisting fascin mutant alleles will, for the first time, allow for the examination of fascin during migration in vivo. In the final objective I will undertake a screening approach to isolate novel migration regulatory genes. Drosophila macrophages undergo a number of migratory transitions during their development in which they first gain and then lose the capacity for movement within the animal. I will isolate cells from each of these stages and compare their transcriptional profile, which will elucidate novel candidates that may be regulating their migratory switch.

Summary

A better understanding of how a cell regulates its ability to move is crucial to understanding and controlling a variety of human diseases e.g. cancer and inflammation. Despite this clinical significance, most of our knowledge of how cell motility is regulated stems from artificial assays that involve watching isolated cells migrate on 2 dimensional plastic substrates. While our ability to watch these cells move by creating high-resolution microscopic movies has been informative, it is likely that these 'in vitro' assays do not tell us the entire story. This proposal will increase our understanding of cell movement and begin to extrapolate what we have learned from watching isolated cells migrate on plastic, to physiologically relevant events. To address this issue, I have been using the fruitfly's (Drosophila Melanogaster) inflammatory response as a way to study cell motility. The fly contains a 'blood' cell, analogous to our white blood cells, that responds to wounds and infections in a similar manner to our inflammatory response. Aside from the clinical significance of directly studying the fly's inflammatory response, the movement of these cells can be imaged using high resolution microscopy live within the animal, which allows us to watch cells move in their natural environment. Along with the ability for high resolution imaging within the animal, the fly has further advantages. Drosophila has been a biological model system widely used for nearly 100 years due to its rapid generation time and genetic manipulability. It is therefore easier and much faster than more complex models, such as the mouse, to pinpoint a function to one's gene of interest. In the first objective of the proposal, I will further enhance this motility assay and develop novel tools that will allow me to live image the intracellular machinery important for cell movement. These tools, which will be extremely useful to the Drosophila research community, will allow me to expand the scopeof this assay and examine the exact requirement of various candidate migration regulatory genes during cell movement. In the next objective, I will investigate the requirement of a gene that is thought important during the spread of cancer cells throughout the body. Fascin is a gene that is expressed in a number of cancers and is correlated with a poor clinical outcome. However, all of what we know about this gene during cell movement has come from the analysis of isolated cells. Fortunately, Drosophila blood cells express high levels of this gene and fly mutants exist. Therefore, using the fluorescent tools created in objective 1, I will dissect the requirement of fascin during the migration of macrophages by analyzing pre-existing fly lines that contain mutations in the fascin gene. Next, I will investigate the regulation of fascin activity within the cell during migration. How this gene is activated and deactivated during cell movement has also been widely studied in isolated cells and is thought to involve modification of critical regions within the protein. Mutations in the exact same region of Drosophila fascin will be created and analyzed within migrating Drosophila blood cells. In the final objective, I will undertake an approach that will allow for the identification of novel migration regulatory genes. During embryonic development of the fruitfly, blood cells undergo a number of changes where they transition between a non-migratory and a migratory cell type, which is analogous to the metastatic spreading of cancer cells. Using a technique called microarray analysis will allow me to compare all the genes that are expressed at each stage of blood cell development. The ability to compare the non-migratory to the migratory stage will elucidate the genes that may be regulating their motile potential. The role of candidate genes during cell movement can then be easily examined in this system.

Committee	Closed Committee - Genes & Developmental Biology (GDB)
Research Topics	Immunology
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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