In situ cryo-electron tomography of the T. pseudonana diatom PyShell
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ABSTRACT:
SUBMITTER: Ricardo Diogo Righetto
PROVIDER: EMPIAR-11747 | biostudies-other |
REPOSITORIES: biostudies-other
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Project description: Not available
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Project description:We present a microfluidic platform for studying structure-function relationships at the cellular level by connecting video rate live cell imaging with in situ microfluidic cryofixation and cryo-electron tomography of near natively preserved, unstained specimens. Correlative light and electron microscopy (CLEM) has been limited by the time required to transfer live cells from the light microscope to dedicated cryofixation instruments, such as a plunge freezer or high-pressure freezer. We recently demonstrated a microfluidic based approach that enables sample cryofixation directly in the light microscope with millisecond time resolution, a speed improvement of up to three orders of magnitude. Here we show that this cryofixation method can be combined with cryo-electron tomography (cryo-ET) by using Focused Ion Beam milling at cryogenic temperatures (cryo-FIB) to prepare frozen hydrated electron transparent sections. To make cryo-FIB sectioning of rapidly frozen microfluidic channels achievable, we developed a sacrificial layer technique to fabricate microfluidic devices with a PDMS bottom wall <5?µm thick. We demonstrate the complete workflow by rapidly cryo-freezing Caenorhabditis elegans roundworms L1 larvae during live imaging in the light microscope, followed by cryo-FIB milling and lift out to produce thin, electron transparent sections for cryo-ET imaging. Cryo-ET analysis of initial results show that the structural preservation of the cryofixed C. elegans was suitable for high resolution cryo-ET work. The combination of cryofixation during live imaging enabled by microfluidic cryofixation with the molecular resolution capabilities of cryo-ET offers an exciting avenue to further advance space-time correlative light and electron microscopy (st-CLEM) for investigation of biological processes at high resolution in four dimensions.
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Project description:Understanding molecular mechanisms of cellular pathways regularly requires knowledge of the identities of participating proteins, their cellular localization and their 3D structures. Contemporary workflows typically require multiple techniques to identify target proteins, track their localization using fluorescence microscopy, followed by in vitro structure determination. To identify mammal-specific sperm proteins and understand their functions, we developed a visual proteomics workflow to directly address these challenges. Our in situ cryo-electron tomography analyses of mouse and human sperm revealed key microtubule structures at 6.0 Å resolution via subtomogram averaging. The well-resolved secondary and tertiary structures allowed us to unbiasedly match novel densities discovered in our 3D reconstruction maps with 21615 protein models from the library of the mouse proteome generated by AlphaFold2. Additional biochemical and mass spectrometry analyses helped validate potential candidates. Not only was it possible to de novo identify novel mammal-specific sperm proteins using this label-free structural approach, but their cellular localizations and molecular interactions could be determined in situ. Specifically, the novel sperm proteins form an extensive interaction network crosslinking the lumen of microtubules, suggesting they could modulate the mechanical and elastic properties of the microtubule filaments required for the vigorous beating motions of flagella. This project includes global protein abundance data from five biochemical fractionations of mouse sperm cells.
2024-05-21 | PXD036885 | Pride
Project description:The flagella of mammalian sperm display non-planar, asymmetric beating, in contrast to the planar, symmetric beating of flagella from sea urchin sperm and unicellular organisms. The molecular basis of this difference is unclear. Here, we perform in situ cryo-electron tomography of mouse and human sperm, providing the highest-resolution structural information to date. Our subtomogram averages reveal mammalian sperm-specific protein complexes within the microtubules, the radial spokes and nexin-dynein regulatory complexes. The locations and structures of these complexes suggest potential roles in enhancing the mechanical strength of mammalian sperm axonemes and regulating dynein-based axonemal bending. Intriguingly, we find that each of the nine outer microtubule doublets is decorated with a distinct combination of sperm-specific complexes. We propose that this asymmetric distribution of proteins differentially regulates the sliding of each microtubule doublet and may underlie the asymmetric beating of mammalian sperm.
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Project description:The bacterial flagellum is a sophisticated self-assembling nanomachine responsible for motility in many bacterial pathogens, including Pseudomonas aeruginosa, Vibrio spp., and Salmonella enterica The bacterial flagellum has been studied extensively in the model systems Escherichia coli and Salmonella enterica serovar Typhimurium, yet the range of variation in flagellar structure and assembly remains incompletely understood. Here, we used cryo-electron tomography and subtomogram averaging to determine in situ structures of polar flagella in P. aeruginosa and peritrichous flagella in S Typhimurium, revealing notable differences between these two flagellar systems. Furthermore, we observed flagellar outer membrane complexes as well as many incomplete flagellar subassemblies, which provide additional insight into mechanisms underlying flagellar assembly and loss in both P. aeruginosa and S Typhimurium.IMPORTANCE The bacterial flagellum has evolved as one of the most sophisticated self-assembled molecular machines, which confers locomotion and is often associated with virulence of bacterial pathogens. Variation in species-specific features of the flagellum, as well as in flagellar number and placement, results in structurally distinct flagella that appear to be adapted to the specific environments that bacteria encounter. Here, we used cutting-edge imaging techniques to determine high-resolution in situ structures of polar flagella in Pseudomonas aeruginosa and peritrichous flagella in Salmonella enterica serovar Typhimurium, demonstrating substantial variation between flagella in these organisms. Importantly, we observed novel flagellar subassemblies and provided additional insight into the structural basis of flagellar assembly and loss in both P. aeruginosa and S Typhimurium.
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