Towards community-driven visual proteomics with large-scale cryo-electron tomography of Chlamydomonas reinhardtii
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Abstract
In situ cryo-electron tomography (cryo-ET) has emerged as the method of choice to investigate structures of biomolecules in their native context. However, challenges remain in the efficient production of large-scale cryo-ET datasets, as well as the community sharing of this information-rich data. Here, we applied a cryogenic plasma-based focused ion beam (cryo-PFIB) instrument for high-throughput milling of the green alga Chlamydomonas reinhardtii , a useful model organism for in situ visualization of numerous fundamental cellular processes. Combining cryo-PFIB sample preparation with recent advances in cryo-ET data acquisition and processing, we generated a dataset of 1829 reconstructed and annotated tomograms, which we provide as a community resource to drive method development and inspire biological discovery. To assay the quality of this dataset, we performed subtomogram averaging (STA) of both soluble and membrane-bound complexes ranging in size from >3 MDa to ∼200 kDa, including 80S ribosomes, Rubisco, nucleosomes, microtubules, clathrin, photosystem II, and mitochondrial ATP synthase. The majority of these density maps reached sub-nanometer resolution, demonstrating the potential of this C. reinhardtii dataset, as well as the promise of modern cryo-ET workflows and open data sharing towards visual proteomics.
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Data sharing and exploration for community-driven visual proteomics
How exciting! I can't wait to dig around in this dataset!
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The potential for localizing and resolving different components of the clathrin pathway in action within the cell remains to be explored, potentially using this C. reinhardtii dataset.
This is so cool! Many of the components of the clathrin pathway in mammals are at least present in Chlamydomonas (with varying degrees of similarity/identity) so that could be a good place to start. And in addition to looking at those components, I would love to see the maturation of the vesicles from bud to mature vesicle. Do you actually see cases in your dataset where there's budding at the membrane that could be called endocytosis?
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One thing I think is not explicitly stated here but which I think is really great is that the cells were grown in constant light in TAP media, meaning that they won't be synchronized/as homogenous, and therefore will likely capture a wider range of biology in the dataset than might otherwise be observed.
Another note is the use of the mat3-4 mutant strain, which as stated has the experimental benefit of a smaller size for vitrification (almost half the diameter of wild-type strains). If I recall correctly when we were looking for actin filaments in mat3-4 tomograms, they seemed much more infrequent than we would have expected based on how abundant filaments are when phalloidin staining CC-125 or CC-124 wild-type strains (https://doi.org/10.1091/mbc.E19-03-0141). And I recall we tried phalloidin staining the mat3-4s and had trouble doing …
One thing I think is not explicitly stated here but which I think is really great is that the cells were grown in constant light in TAP media, meaning that they won't be synchronized/as homogenous, and therefore will likely capture a wider range of biology in the dataset than might otherwise be observed.
Another note is the use of the mat3-4 mutant strain, which as stated has the experimental benefit of a smaller size for vitrification (almost half the diameter of wild-type strains). If I recall correctly when we were looking for actin filaments in mat3-4 tomograms, they seemed much more infrequent than we would have expected based on how abundant filaments are when phalloidin staining CC-125 or CC-124 wild-type strains (https://doi.org/10.1091/mbc.E19-03-0141). And I recall we tried phalloidin staining the mat3-4s and had trouble doing so. So my suspicion is that there might be a few key differences in macromolecular composition in these mutant strains that might prevent some structures from resolving or being abundant enough to detect readily. It's not a criticism, but just worth noting I think for those trying to extrapolate from this extremely helpful resource.
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The cytoplasmic microtubules (MTs) of C. reinhardtii are nucleated near the centrioles and extend around the periphery of the cell.
Did you notice any acentriolar microtubule organizing centers in your dataset? Even if rarely occurring?
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