Periodic Reporting for period 1 - cryoSPARTAN (in situ Structure Determination of Actin-binding Proteins Through a Novel Cryo-electron Microscopy Workflow)
Reporting period: 2023-04-01 to 2025-03-31
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.
The project has two main objectives: the first is the development of a hybrid imaging cryo-EM method, and the second is the determination of ABP structures in situ. For the first objective, I have completed testing different grid types to assess their suitability for cell imaging in cryo-EM. Have ruled out which grids likely are not suitable and which grids are too fragile to use. I have explored the possibility of graphene oxide layer and we now have a working protocol to coat grids with graphene oxide. Most recently have begun exploring grids with silicon dioxide support film. Regarding the hybrid cryo-EM data collection I have met with our ISTA EM facility staff and together we have discussed the scripts and strategy we will use. I have completed a python course which has improved my coding skills substantially. For the second objective I have carried out an in-depth literature review of cortactin and fascin in order to foresee possible problems. I have also begun discussions with other labs in order to acquire different types of cells which may be more amenable to structure determination in the lamellipodia. I have met with the virus facility at our institute and have developed a strategy to tranduce cells with actin binding proteins in order for them to be overexpressed. We have completed the development of geometric classification for actin networks, and the software is ready for application in real data once collection begins. As a test dataset I recently collected single particle cryo-EM data on B16 lamellipodia and was able to resolve the structure of tropomyosin in situ to 6 Å resolution. Lastly, as a further test of feasibility to study actin-binding proteins in situ I transfected cells with F-tractin, an actin-binding probe, and collected a single particle dataset. In this dataset we could not resolve F-tractin structure but we have plans to create a stable cell line using viral transduction which will permit better expression.
This project is on track to yield an impact as laid out in the original proposal, however it is too soon to show final results. The methods development objective is on track, and we are very optimistic that it will succeed. The success of this part of the project will be a helpful tool for other cryo-EM labs who seek to determine in situ structure. This will be useful for labs studying basic biological questions, but also for industry labs aiming to determine drug-bound structures within cells.
The impact regarding structures of cortactin and fascin has not changed since the start of the project. Progress here will continue, and we hope will result in the first ever high resolution structures of these key cell migration proteins. This will provide fundamental insight into how cells move, but also may open opportunities for therapeutic targeting of these proteins in disease such as cancer. This will be of enormous benefit to society and will have economic impact as well.
The impact regarding structures of cortactin and fascin has not changed since the start of the project. Progress here will continue, and we hope will result in the first ever high resolution structures of these key cell migration proteins. This will provide fundamental insight into how cells move, but also may open opportunities for therapeutic targeting of these proteins in disease such as cancer. This will be of enormous benefit to society and will have economic impact as well.