Correlative microscopy of vitreous sections provides insights into BAR-domain organisation in situ
Electron microscopy imaging of macromolecular complexes in their native cellular context is limited by the inherent difficulty to acquire high-resolution tomographic data from thick cells and to specifically identify elusive structures within crowded cellular environments. Here, we combined cryo-flu...
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| Format: | Journal article |
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Cell Press
2018
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| _version_ | 1797064225048231936 |
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| author | Bharat, T Hoffnann, P Kukulski, W |
| author_facet | Bharat, T Hoffnann, P Kukulski, W |
| author_sort | Bharat, T |
| collection | OXFORD |
| description | Electron microscopy imaging of macromolecular complexes in their native cellular context is limited by the inherent difficulty to acquire high-resolution tomographic data from thick cells and to specifically identify elusive structures within crowded cellular environments. Here, we combined cryo-fluorescence microscopy with electron cryo-tomography of vitreous sections into a coherent correlative microscopy workflow, ideal for detection and structural analysis of elusive protein assemblies in situ. We used this workflow to address an open question on BAR-domain coating of yeast plasma membrane compartments known as eisosomes. BAR domains can sense or induce membrane curvature, and form scaffold-like membrane coats in vitro. Our results demonstrate that in cells, the BAR protein Pil1 localizes to eisosomes of varying membrane curvature. Sub-tomogram analysis revealed a dense protein coat on curved eisosomes, which was not present on shallow eisosomes, indicating that while BAR domains can assemble at shallow membranes in vivo, scaffold formation is tightly coupled to curvature generation. |
| first_indexed | 2024-03-06T21:11:12Z |
| format | Journal article |
| id | oxford-uuid:3e3983ae-ab27-4790-b1ca-ed6a25469407 |
| institution | University of Oxford |
| last_indexed | 2024-03-06T21:11:12Z |
| publishDate | 2018 |
| publisher | Cell Press |
| record_format | dspace |
| spelling | oxford-uuid:3e3983ae-ab27-4790-b1ca-ed6a254694072022-03-26T14:24:18ZCorrelative microscopy of vitreous sections provides insights into BAR-domain organisation in situJournal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:3e3983ae-ab27-4790-b1ca-ed6a25469407Symplectic Elements at OxfordCell Press2018Bharat, THoffnann, PKukulski, WElectron microscopy imaging of macromolecular complexes in their native cellular context is limited by the inherent difficulty to acquire high-resolution tomographic data from thick cells and to specifically identify elusive structures within crowded cellular environments. Here, we combined cryo-fluorescence microscopy with electron cryo-tomography of vitreous sections into a coherent correlative microscopy workflow, ideal for detection and structural analysis of elusive protein assemblies in situ. We used this workflow to address an open question on BAR-domain coating of yeast plasma membrane compartments known as eisosomes. BAR domains can sense or induce membrane curvature, and form scaffold-like membrane coats in vitro. Our results demonstrate that in cells, the BAR protein Pil1 localizes to eisosomes of varying membrane curvature. Sub-tomogram analysis revealed a dense protein coat on curved eisosomes, which was not present on shallow eisosomes, indicating that while BAR domains can assemble at shallow membranes in vivo, scaffold formation is tightly coupled to curvature generation. |
| spellingShingle | Bharat, T Hoffnann, P Kukulski, W Correlative microscopy of vitreous sections provides insights into BAR-domain organisation in situ |
| title | Correlative microscopy of vitreous sections provides insights into BAR-domain organisation in situ |
| title_full | Correlative microscopy of vitreous sections provides insights into BAR-domain organisation in situ |
| title_fullStr | Correlative microscopy of vitreous sections provides insights into BAR-domain organisation in situ |
| title_full_unstemmed | Correlative microscopy of vitreous sections provides insights into BAR-domain organisation in situ |
| title_short | Correlative microscopy of vitreous sections provides insights into BAR-domain organisation in situ |
| title_sort | correlative microscopy of vitreous sections provides insights into bar domain organisation in situ |
| work_keys_str_mv | AT bharatt correlativemicroscopyofvitreoussectionsprovidesinsightsintobardomainorganisationinsitu AT hoffnannp correlativemicroscopyofvitreoussectionsprovidesinsightsintobardomainorganisationinsitu AT kukulskiw correlativemicroscopyofvitreoussectionsprovidesinsightsintobardomainorganisationinsitu |