Molecular and cellular dissection of kinesin motors in Apicomplexa to reveal roles in parasite proliferation
Lead Research Organisation:
University of Nottingham
Department Name: School of Life Sciences
Abstract
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Technical Summary
The goal of this project is elucidation of the mechanisms of the microtubule-based replicative machinery in apicomplexan parasites. Apicomplexa are evolutionarily diverse protozoan parasites, many of which are important pathogens affecting humans and livestock. This includes Babesia, Eimeria and Neospora, which affect domesticated livestock causing huge economic losses. The parasite life cycles are complex and alternate between sexual and asexual replicative stages in distinct hosts. Their replicative mechanisms are just beginning to be understood.
Kinesins are a large superfamily of ATP-driven, microtubule-dependent motors. They perform many essential functions in eukaryotic replication but their roles in Apicomplexa are poorly understood. The proposed work will study the molecular and cellular basis of kinesin function in apicomplexan replication in vitro and in vivo. We will reconstitute putative mitotic kinesin-microtubule complexes from purified components and biophysically characterize their interactions. The structures of microtubule-bound parasite kinesins will be studied using near-atomic (<5Å) resolution cryo-electron microscopy to 1) visualise parasite-specific features of the kinesin-microtubule interface, and 2) reveal nucleotide-dependent conformational changes that drive force generation. P. berghei is a genetically tractable model organism and an excellent system in which to study sexual and sporogonic stages in the mosquito as well as the asexual stages in vivo. This allows the link between cell division roles and post-division morphogenesis to be studied comprehensively. We will perform a systematic functional screen of all kinesins to study how their activities contribute to replication throughout the parasite life cycle. In vivo studies will also enable us to look for regulatory mechanisms and interaction partners for these motors during different replicative phases and thereby provide insight into a poorly understood aspect of parasite biology.
Kinesins are a large superfamily of ATP-driven, microtubule-dependent motors. They perform many essential functions in eukaryotic replication but their roles in Apicomplexa are poorly understood. The proposed work will study the molecular and cellular basis of kinesin function in apicomplexan replication in vitro and in vivo. We will reconstitute putative mitotic kinesin-microtubule complexes from purified components and biophysically characterize their interactions. The structures of microtubule-bound parasite kinesins will be studied using near-atomic (<5Å) resolution cryo-electron microscopy to 1) visualise parasite-specific features of the kinesin-microtubule interface, and 2) reveal nucleotide-dependent conformational changes that drive force generation. P. berghei is a genetically tractable model organism and an excellent system in which to study sexual and sporogonic stages in the mosquito as well as the asexual stages in vivo. This allows the link between cell division roles and post-division morphogenesis to be studied comprehensively. We will perform a systematic functional screen of all kinesins to study how their activities contribute to replication throughout the parasite life cycle. In vivo studies will also enable us to look for regulatory mechanisms and interaction partners for these motors during different replicative phases and thereby provide insight into a poorly understood aspect of parasite biology.
Planned Impact
The work described will lead to a greater understanding of essential mechanisms involved in proliferation/replication and cell division in parasites affecting animal and human health. Kinesin motors could be potential targets for novel anti-parasite agents that block parasite replication and our research will aid development of these. Beneficiaries from this aspect of the research would be pharmaceutical and veterinary medicine companies, as greater understanding from academic studies such as ours could lead to more effective generation of improved anti-parasite drugs and, therefore, improved sales. We would aim to engage these beneficiaries as soon as possible. This work would also ultimately lead to benefits for livestock farmers whose output could be increased by control of livestock health. In the future, novel pharmaceuticals would also benefit human health.
Science and technology is a key facet of global economy, and we will liase with UCL Business (UCLB), who work with Birkbeck researchers on technology development and intellectual property matters, and Nottingham University's IP Commercialisation Office, to maximise the impact of our discoveries. This will ultimately have benefits for the economic competitiveness of the UK. It is essential to retain talented young researchers in the UK, and the proposed research programme will provide an attractive research opportunity for excellent young scientists looking for multi-disciplinary and collaborative areas of discovery. In addition, transferable skills - such as time- and project management, presentation and collaboration - that can be applied in all employment sectors - will be acquired, particularly through transferable skills training within the Institute of Structural and Molecular Biology and at Nottingham University.
We will aim to make our research available not only to the academic community, but also to the general public. The PIs have proven track-records of public communication of science: Prof Moores was the 2006 winner of the prestigious DeMontfort medal for science communication (SET for Britain), has attended a BBSRC Media Training Day, and has presented her research to a general audience for example as part of Birkbeck Science Week 2012. Prof Tewari liaises regularly with the Press and Media office at Nottingham University to communicate her research findings to the wider community. She has presented her research on the malaria parasite at the Community Day of the University of Nottingham, to which the general public is invited, and regularly trains summer students through the Nuffield fellowship and Erasmus student programmes. The appointed PDRAs and PIs will undertake to design lab web-pages which are accessible to the general public and will seek to participate in other public understanding of science activities, for example by inviting sixth-form students to visit the lab and experience the day-to-day life of scientists. During the project period, the PIs will arrange school visits to inspire future scientists, and will continue to be involved in advancing gender equality in science, engineering and technology through involvement with the Athena SWAN programme. Prof Moores is coordinator of her Department's self-assessment team and is actively involved in mentoring and career development activities.
Science and technology is a key facet of global economy, and we will liase with UCL Business (UCLB), who work with Birkbeck researchers on technology development and intellectual property matters, and Nottingham University's IP Commercialisation Office, to maximise the impact of our discoveries. This will ultimately have benefits for the economic competitiveness of the UK. It is essential to retain talented young researchers in the UK, and the proposed research programme will provide an attractive research opportunity for excellent young scientists looking for multi-disciplinary and collaborative areas of discovery. In addition, transferable skills - such as time- and project management, presentation and collaboration - that can be applied in all employment sectors - will be acquired, particularly through transferable skills training within the Institute of Structural and Molecular Biology and at Nottingham University.
We will aim to make our research available not only to the academic community, but also to the general public. The PIs have proven track-records of public communication of science: Prof Moores was the 2006 winner of the prestigious DeMontfort medal for science communication (SET for Britain), has attended a BBSRC Media Training Day, and has presented her research to a general audience for example as part of Birkbeck Science Week 2012. Prof Tewari liaises regularly with the Press and Media office at Nottingham University to communicate her research findings to the wider community. She has presented her research on the malaria parasite at the Community Day of the University of Nottingham, to which the general public is invited, and regularly trains summer students through the Nuffield fellowship and Erasmus student programmes. The appointed PDRAs and PIs will undertake to design lab web-pages which are accessible to the general public and will seek to participate in other public understanding of science activities, for example by inviting sixth-form students to visit the lab and experience the day-to-day life of scientists. During the project period, the PIs will arrange school visits to inspire future scientists, and will continue to be involved in advancing gender equality in science, engineering and technology through involvement with the Athena SWAN programme. Prof Moores is coordinator of her Department's self-assessment team and is actively involved in mentoring and career development activities.
Publications
Bunnik EM
(2019)
Comparative 3D genome organization in apicomplexan parasites.
in Proceedings of the National Academy of Sciences of the United States of America
Cook AD
(2021)
Cryo-EM structure of a microtubule-bound parasite kinesin motor and implications for its mechanism and inhibition.
in The Journal of biological chemistry
Green JL
(2017)
Compositional and expression analyses of the glideosome during the Plasmodium life cycle reveal an additional myosin light chain required for maximum motility.
in The Journal of biological chemistry
Guttery DS
(2022)
Division and Transmission: Malaria Parasite Development in the Mosquito.
in Annual review of microbiology
Guttery DS
(2020)
Plasmodium DEH is ER-localized and crucial for oocyst mitotic division during malaria transmission.
in Life science alliance
Description | We have finished the Plasmodium kinesin functional screen and now submitted for publication. We found that out of 9 plasmodium kinesin only one of them is essential for blood stage development and the other eight are required in the parasite development with mosquito host. |
Exploitation Route | This is too early to implicate but in future can be used for drug discovery programme. We have submitted frther grants for funding based on these finding to understand the spindle dynamics in parasite cell. |
Sectors | Creative Economy,Education,Healthcare |
URL | https://www.biorxiv.org/content/10.1101/2021.05.26.445751v2.full |
Title | Live cell imagine for anaysing spindle dynamics |
Description | T study rapid spindle dynamics and basal body formation in male gametocyte of malaria parasite |
Type Of Material | Model of mechanisms or symptoms - in vitro |
Year Produced | 2019 |
Provided To Others? | Yes |
Impact | This has been shared with many lab nows |
Title | Protein interaction map |
Description | We have developed the protocol for parasite gametocyte purified using GFP transgenic lines and studying the protein interaction map. THis is mainly to understand the chrosome segregation in this divergent parasite and could be used for blocking cell proliferation |
Type Of Material | Technology assay or reagent |
Year Produced | 2020 |
Provided To Others? | Yes |
Impact | It is useful to study the kinetochore and spindle dynamics of the unique parasite cell that is necessary for male gamete formation and can be information for transmission blocking. |
Title | transgenic parasite lines |
Description | THese are transgenic parasite lines for functional of kinesin in Plasmodium |
Type Of Material | Technology assay or reagent |
Provided To Others? | No |
Impact | No |
Title | PRIDE |
Description | This is the research data base for Mass spec data produced during the protein interaction studies |
Type Of Material | Database/Collection of data |
Year Produced | 2020 |
Provided To Others? | Yes |
Impact | It is open data base and can be used by anyone |
Title | Rodent Malaria Data base |
Description | This is reasearch data base where all the malaria transgenic parasite information is provided |
Type Of Material | Database/Collection of data |
Year Produced | 2010 |
Provided To Others? | Yes |
Impact | Many of the mutant are distributed to various researcher across europe and USA |
URL | http://www.pberghei.eu/. |
Title | Rodent malaria data base |
Description | All the information for phosphatase mutants for malaria model is deposited. It is a rodent malaria transgenic data base |
Type Of Material | Database/Collection of data |
Year Produced | 2014 |
Provided To Others? | Yes |
Impact | The parasites are provided to different group in France and USA |
URL | http://www.pberghei.eu/. |
Description | Atypical celldivision in male gametogony |
Organisation | University of Geneva |
Country | Switzerland |
Sector | Academic/University |
PI Contribution | We have been contributing the resources in the lab in terms of tagged and knockdown parasites to his group |
Collaborator Contribution | He has been able to help us with phosphoproteome |
Impact | in process |
Start Year | 2018 |
Description | Condensin in Plasmodium |
Organisation | University of California, Riverside |
Country | United States |
Sector | Academic/University |
PI Contribution | We have provided the transgenic parasite and ptoteomics approaches for the condensin copmplex subunit and their charecterisation during parasite life cycle mainly in the mosquito stages |
Collaborator Contribution | Prof Karine Le Roch has provided the support fr genome wide approaches like Chipseq and RNa seq for this project |
Impact | We have published two paper together in Cell Reports and Plos Pathogens. This collaboration is multidiscplinary. PMID: 32501284;J Cell Sci. 2020 Jun 30;134(5):jcs245753. doi: 10.1242/jcs.245753. PMID: 32049018; Cell Rep. 2020 Feb 11;30(6):1883-1897.e6. doi: 10.1016/j.celrep.2020.01.033. PMID: 31600347; PLoS Pathog. 2019 Oct 10;15(10):e1008048. doi: 10.1371/journal.ppat.1008048. eCollection 2019 Oct. |
Start Year | 2018 |
Description | Kinesin in Plasmodium |
Organisation | Birkbeck, University of London |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | We colloborate with Prof Carolyn Moores who works with biochemostry and molecular dissection of kinesin motor using CryoEm |
Collaborator Contribution | Prof Carolyn will provide the biochemistry and structual side of the project to this study |
Impact | no |
Start Year | 2015 |
Description | Kinesin in Plasmodium |
Organisation | Birkbeck, University of London |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | I am collaborating with Prof Carolyn Moores who is an expert on Kinesin in mammalian system and is a reputed Structural Biologist |
Collaborator Contribution | She will provide us the biochemistry of of the Kinesin 5 and Kininesin 13 |
Impact | We will be writing the paper at the end of the year. |
Start Year | 2014 |
Description | Phosphorylation |
Organisation | University of Leicester |
Department | Department of Cell Physiology and Pharmacology |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | significant intellectual input |
Collaborator Contribution | To provide technical help with Global Phosphorylationtraining of staff |
Impact | Two manuscript in preparation |
Start Year | 2010 |
Description | Phosphorylation |
Organisation | University of Leicester |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | significant intellectual input |
Collaborator Contribution | To provide technical help with Global Phosphorylationtraining of staff |
Impact | Two manuscript in preparation |
Start Year | 2010 |
Description | A seminar at Mredical Erasmus University |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Study participants or study members |
Results and Impact | This helped me to meet scientist who are working with Cohesin in mammalian system. |
Year(s) Of Engagement Activity | 2017 |
Description | Interview with BBC Radio nottingham |
Form Of Engagement Activity | A press release, press conference or response to a media enquiry/interview |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Media (as a channel to the public) |
Results and Impact | This was radio interview where I was interviewed about our work malaria cell division and how this research help in wider context. |
Year(s) Of Engagement Activity | 2019 |
Description | Organised a Midland Cel Cycle Club 2017 for the East Midland |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Postgraduate students |
Results and Impact | This meeting made good network of scientist working in the area of Cel Cycle and Cytoskeleton in various model system like human, drosophila, yeat parasites etc. |
Year(s) Of Engagement Activity | 2017 |
Description | Organising the London Molecular Parasitology Club |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Study participants or study members |
Results and Impact | Three meeting of London Molecular Parasitology are organised every year. The speakers range from any where in Uk or Europe. It benefots the scientist working in London and adjoining areas. |
Year(s) Of Engagement Activity | 2007,2008,2009,2010,2011,2012,2013,2014,2015,2016,2017,2018 |
Description | School student demonstration |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Schools |
Results and Impact | This was mainly organised for school student around Nottinghamshire to make them aware of research studies and mainly to teach them about cell biology and microscopy detection of malaria parasite |
Year(s) Of Engagement Activity | 2019 |