cryoSPARTAN · in situ Structure Determination of Actin-binding Proteins Through a Novel Cryo-electron Microscopy Workflow
Horizon Europe — Marie Skłodowska-Curie Actions
- Duration
- 2023-04-01 → 2025-03-31
- EU contribution
- €199,441
- Participants
- 1
- Scheme
- HORIZON-TMA-MSCA-PF-EF
Lines connect the coordinator with its partners.
Results in brief
in situ Structure Determination of Actin-binding Proteins Through a Novel Cryo-electron Microscopy Workflow
Cell motility is an essential process in all of life, and it is driven by a complex network of force-generating biological machinery. The key component in this machinery is the dynamic network of filamentous actin (F-actin) and actin binding proteins (ABPs) which maintain and regulate the network. ABP-actin interactions lead to networks with straight, curved, branched, and bundled morphologies, but it is unclear how such diversity can co-exist in the same space. This has hindered a complete understanding of actin network regulation in cell migration and other relevant actin- dependent processes. Addressing this important question requires understanding exactly how ABPs select F-actin, and conversely how F-actin geometry recruits specific ABPs. However, the structural information for many ABP-actin complexes, as well as their in situ spatial distribution and quantification, remain elusive. This is because ABPs cannot be easily studied in isolation, often exhibiting structural stability only when embedded in a complex filamentous network found within cells. Here, I propose developing a novel hybrid single particle cryo-electron tomography approach in order to reveal high-resolution structures and contextual information of ABPs bound to F-actin directly within cellular protrusions. We will initially target structures of ARP2/3 complex in branch junctions in wild type and isoform knockout cells. We will subsequently target two ABPs for which there are only very low resolution structures: the ABP cortactin, and the bundling protein fascin. The outcome of this work is the generation of tools for in situ high resolution structure determination and will contribute towards a more holistic description of the proteome at the leading edge of migrating cells.
Data: CORDIS, © European Union
Project objective
Cell motility is driven by a complex network of force-generating biological machinery. The key component in this machinery is the dynamic network of filamentous actin (F-actin) and actin binding proteins (ABPs) which maintain and regulate the network. However, structural information for many ABP-actin complexes, as well as their in situ spatial distribution remain elusive. This is because ABPs cannot be easily studied in isolation, often exhibiting structural stability only when embedded in a complex filamentous network found within cells. This has hindered a complete understanding of actin network regulation in cell migration. Addressing this important question requires understanding exactly how ABPs select F-actin, and conversely how F-actin geometry recruits specific ABPs. Cryo-electron tomography (cryo-ET) can reveal both cellular ultrastructure and molecular details, but often is lower resolution than a single particle cryo-EM approach. Thus, innovative methods remain key to drive advancement in understanding in situ structures. In this fellowship I will combine my expertise of single particle cryo-electron microscopy with expertise of cryo-ET in the Schur lab to develop a novel hybrid single particle cryo-ET approach in order to reveal high-resolution structures and contextual information of ABPs bound to F-actin directly within cellular protrusions. ISTA is the ideal research institute due to abundant access to high-end electron microscopes necessary for methods development. The outcome of this action are tools for high resolution in situ structure determination and a better understanding of cell migration, a process deeply rooted in malignant metastasis. Results will be disseminated through key research conferences and high-impact open-access publications. Communication activities will be achieved through 3D rendered visual scientific illustrations targeting social media platforms and institute-organized public outreach events.
Original text from CORDIS.
Participants
- INSTITUTE OF SCIENCE AND TECHNOLOGY AUSTRIA · KlosterneuburgCoordinatorAustria
Links
- View on CORDIS
- DOI: 10.3030/101108395
- https://ec.europa.eu/research/participants/documents/downloadPublic?documentIds=080166e501725fb0&appId=PPGMS
- https://ec.europa.eu/research/participants/documents/downloadPublic?documentIds=080166e501726dcf&appId=PPGMS
Data: CORDIS, © European Union