Imperial, Molecular and cellular basis of infection

 The actomyosin cytoskeleton of the malaria parasite, Plasmodium, has been studied extensively in the context of cell motility and host cell invasion, implicating the divergent actin (Act1) and myosin XIV (MyoA) in the process and dissecting their function (Tardieux and Baum, 2016). However, to date, little attention has been focussed on the role of other actomyosin interactions, specifically the role of the five other myosins B-F, in alternative fundamental cellular processes. In recent years, actin has been implicated in facilitating gene regulation across eukaryotic cells. Similarly, myosins have been shown to be key factors within the nucleus (de Lanoerolle et al 2005) and outside the nucleus facilitating nuclear positioning (Thiam et al 2016) as well as working with actin for chromatin reorganisation. Recent proteomic studies have found strong evidence for one of the six myosins, MyoE, in the Plasmodium nucleus (Oehring et al 2012) and strong labelling of actin (Angrisano et al 2012) around the nucleus suggesting some of these processes may be conserved. This PhD proposal aims to answer the question of what is the role of the actomyosin system in nuclear function in the most clinically relevant malaria parasite, Plasmodium falciparum. Using a combination of molecular genetics and protein chemistry, the PhD will explore the localisation, conditional knockout phenotype and detailed cellular characterisation of the Plasmodium myosins (C-F) and their association with actin. This will be complemented by protein expression and characterisation of the key nuclear variants using baculoviral expression towards structural and biochemical/biophysical characterisation in in vitro assays ranging from straightforward sedimentation assays to single molecule imaging.

The work builds on several developments in recent years that provide the ideal background for a work dissecting the function of nuclear myosins. – Recent advances in molecular genetics using the CRISPR/Cas9 system (now adapted for us in the Baum lab at Imperial) (Ghorbal et al 2014) enable rapid tagging of myosins in Plasmodium and has been validated for use with Plasmodium myosin A (one of the six myosins). – Complementing the CRISPR/Cas9 system, a novel method for conditional knockout of genes using a synthetic intron and Cre/Lox gene excision process has been developed (Jones et al 2016) that allows for detailed dissection of function in essential genes. This is now in use in the Baum lab. – Following advances in Toxoplasma research (Bookwalter et al 2014), the chaperonin system of Plasmodium myosins has been developed in the Baum lab for use in a baculoviral protein expression system and validated for myosin A, which enables the soluble and functional expression of these formerly intractable proteins. – Cell lysate based cytoskeletal assays are now used in yeast systems (Michelot and Drubin 2014) and can give profound insights into the dynamics of actomyosin systems ex vivo. Although not adapted for use in Plasmodium the background for development of such as system is in place and expertise available through a collaborator of the Baum lab at CNRS Grenoble.

The tagging of Plasmodium myosins (A and B) and their in vitro expression (A thus far) has already been validated and, as such, there is strong confidence that the same approaches will be applicable to Myosins C–F. Each protein is, of course, different and will present specific challenges. Because there are four myosins that will each be tagged at the commencement of the project, it is anticipated that if one proves intractable, others will remain feasible to study. Similarly, for protein expression, while the focus will be on the nuclear myosin, other myosins will serve as backup strategies to characterise the broader actomyosin system (non-motility related) of the malaria parasite. With conditional strategies, there are multiple potential directions the work could take depending on the phenotypes observed. For example, if a knockout is non-lethal, this may prompt exploration of gene regulation changes with collaborators experienced in transcriptomics. With each aim, the work will build toward full characterisation of a non-motile actomyosin system, with nuclear-related systems as the primary focus, while other non-nuclear systems remain viable contingencies. Reports have been presented at conferences demonstrating Plasmodium berghei (mouse malaria) tagging of non-motility-related myosins, providing confidence that this will not be a stumbling block for the project.

The project represents an excellent balance of parasite tissue culture and protein chemistry toward a holistic characterisation of non-motility-related myosins. It balances low-risk and higher-risk technologies (e.g. cell lysate assays), offering the potential for both reliable publication of data and high-impact discoveries. Overall, the programme of experiments will provide excellent training in a wide range of cell and molecular techniques that are readily transferable across cell and molecular biology disciplines.

Basic Science
Enabling Technologies & Assays

Oct 2015 — Sep 2019

$213,496

United Kingdom