Identification of the molecular pathways that drive and steer pseudopods

Many cells are able to crawl very slowly over a solid surface or burrow their way through a tissue. Often they are guided by chemicals that attract them and in this way, they can perform a useful purpose. For instance white blood cells are attracted by chemicals inadvertently released bacteria, which they then engulf and destroy. Similarly, amoebae are attracted to bacteria, which they then consume as food. The first step in movement is to make projection - a pseudopod - in the direction of travel. This pseudopod can form anywhere around the cell and is driven out by tiny motor which uses some of the same components as muscle. The position where the pseudopod does form is somehow governed by the concentration of attractive chemical around the cell. The aim of the work proposed here is to learn more about how a pseudopod is produced in the right place so that the cell moves towards an attractive chemical.

Chemotaxis, the movement of cells towards attractive chemicals, is vital to the function of the immune system and in wound healing, as well as being the route by which many cancer cells spread. When amoeboid cells chemotax, they first extend a pseudopod towards the highest chemoattractant concentration, and then draw the rest of the body after it. The pseudopod itself is driven out by actin polymerization beneath the plasma membrane, which in turn is triggered by the arp2/3 complex, controlled by SCAR, Rac1 and its cognoscente activating GEFs, for which there are many candidates. Using the well-studied case of Dictyostelium cells chemotaxing towards cyclic-AMP, we wish to help elucidate how a pseudopod is triggered and localized. We shall screen the genomic complement of RacGEFs for their localization in pseudopodia and phosphorylation in response to chemoattractant, and then make single and multiple gene deletions in selected candidates to define their genetic function. The set of GEFs thus defined as activating Rac1 in pseudopodia will be subject to detailed study to help define their targeting mechanism to pseudopodia and regulation by phosphorylation. Although the SCAR complex is the principle activator of the arp2/3 complex in pseudopodia, it can be replaced by WASP in SCAR null mutants. WASP is strongly targeted to the pseudopod and by mutagenesis of the protein and assay in SCAR null cells we hope to define a minimum WASP that is targeted. This will then be used to search for the feature that is recognized by WASP in pseudopodia, and to test for a similar targeting mechanism in the much more complicated SCAR complex.

The work in this proposal entails basic research that will significantly advance our understanding of cell movement. The work falls directly within the principal strategic aims to advance fundamental understanding of a complex biological process. As with all basic research, the immediate beneficiaries of this work will be other academic scientists. However it is decisively aimed for impact on society in the longer term.
Strategic priorities
The proposed research meets the terms of several strategic priorities:
a) Data driven biology. This priority aims to stimulate "new ways of working", providing informatic resources to realize maximum value from biological-based technologies. Our systematic imaging of a complex gene family will yield valuable data that can be quantitatively extracted from the published image set by a wide range of groups working on processes that are affected by regulation of the cytoskeleton in general.
b) Systems approaches to biosciences. Systems biology is an approach in which experimental biology is integrated with modelling in a synergistic fashion. Cell motility is a prime example of a process that cannot be understood in terms of biological pathways alone. The fundamental molecular principles of motility that we aim to uncover will be used to test and refine current models that explain cell migration and chemotaxis.
c) Synthetic biology. The emerging discipline of synthetic biology encompasses the design and construction of biologically based parts and systems. Nano-scale motility has the potential to revolutionize several problems, such as targeted drug delivery. Dedicated efforts are currently underway to realize this potential. Our work on pseudopod formation is focussed on a bottom-up approach to find the elemental components and pathways that drive motility. The identified components will form the elementary building blocks that are required for the construction of new synthetic motility systems.
Impact on health and wealth
Metastatic cancer is one of the leading causes of death in the UK. Metastasis remains the least effectively treated characteristic of cancer and only few drugs are currently in clinical trial. The inhibition of cell motility is considered a potential route to block metastasis. Protein kinases are key targets of the pharmaceutical industry. The kinase-targeted drug market has an estimated annual global value of over £30 billion pounds. The proposed work explores the regulation of the main motility regulators, Rac GEFs by phosphorylation through protein kinases. This provides the first clues to open up new avenues for intervention of cell motility in metastasis.
Impact on the knowledge economy
This project will contribute to the training of a new generation of highly skilled researchers. The co-applicant is keen to start his own independent research. The project allows him to move to Cambridge. Exposure to different academic environments is important to get the best start on the road to independence. Cambridge works hard to offer exceptional training capabilities and cutting edge technology. Investment in new leaders in academia is essential to maintain the leading role of the UK in an increasingly competitive global environment.