scholarly journals In-cell structures of a conserved supramolecular array at the mitochondria-cytoskeleton interface in mammalian sperm

2021 ◽  
Author(s):  
Miguel Ricardo Leung ◽  
Riccardo Zenezini Chiozzi ◽  
Marc C. Roelofs ◽  
Johannes F. Hevler ◽  
Ravi Teja Ravi ◽  
...  
Keyword(s):  
Focused Ion Beam ◽  
Structural Information ◽  
Electron Tomography ◽  
Ion Beam ◽  
Mammalian Species ◽  
Cell Types ◽  
Mammalian Sperm ◽  
Cell Structures ◽  
Voltage Dependent ◽  
Subtomogram Averaging

SummaryMitochondria-cytoskeleton interactions modulate cellular physiology by regulating mitochondrial transport, positioning, and immobilization. However, there is very little structural information defining mitochondria-cytoskeleton interfaces in any cell type. Here, we use cryo-focused ion beam milling-enabled cryo-electron tomography to image mammalian sperm, where mitochondria wrap around the ciliary cytoskeleton. We find that mitochondria are tethered to their neighbors through inter-mitochondrial linkers and are anchored to the cytoskeleton through ordered arrays on the outer mitochondrial membrane. We use subtomogram averaging to resolve in-cell structures of these arrays from three mammalian species, revealing they are conserved across species despite variations in mitochondrial dimensions and cristae organization. We find that the arrays consist of boat-shaped particles anchored on a network of membrane pores whose arrangement and dimensions are consistent with voltage dependent anion channels. Proteomics and in-cell cross-linking mass spectrometry suggest that the conserved arrays are composed of glycerol kinase-like proteins. Ordered supramolecular assemblies may serve to stabilize similar contact sites in other cell types where mitochondria need to be immobilized in specific subcellular environments, such as in muscles and neurons.

scholarly journals In-cell structures of conserved supramolecular protein arrays at the mitochondria–cytoskeleton interface in mammalian sperm

2021 ◽  
Vol 118 (45) ◽  
pp. e2110996118
Author(s):  
Miguel Ricardo Leung ◽  
Riccardo Zenezini Chiozzi ◽  
Marc C. Roelofs ◽  
Johannes F. Hevler ◽  
Ravi Teja Ravi ◽  
...  
Keyword(s):  
Structural Information ◽  
Ion Beam ◽  
Mammalian Species ◽  
Cell Types ◽  
Anion Channels ◽  
Membrane Pores ◽  
Mammalian Sperm ◽  
Cell Structures ◽  
Voltage Dependent ◽  
Subtomogram Averaging

Mitochondria–cytoskeleton interactions modulate cellular physiology by regulating mitochondrial transport, positioning, and immobilization. However, there is very little structural information defining mitochondria–cytoskeleton interfaces in any cell type. Here, we use cryofocused ion beam milling-enabled cryoelectron tomography to image mammalian sperm, where mitochondria wrap around the flagellar cytoskeleton. We find that mitochondria are tethered to their neighbors through intermitochondrial linkers and are anchored to the cytoskeleton through ordered arrays on the outer mitochondrial membrane. We use subtomogram averaging to resolve in-cell structures of these arrays from three mammalian species, revealing they are conserved across species despite variations in mitochondrial dimensions and cristae organization. We find that the arrays consist of boat-shaped particles anchored on a network of membrane pores whose arrangement and dimensions are consistent with voltage-dependent anion channels. Proteomics and in-cell cross-linking mass spectrometry suggest that the conserved arrays are composed of glycerol kinase-like proteins. Ordered supramolecular assemblies may serve to stabilize similar contact sites in other cell types in which mitochondria need to be immobilized in specific subcellular environments, such as in muscles and neurons.

scholarly journals The multi-scale architecture of mammalian sperm flagella and implications for ciliary motility

2020 ◽  
Author(s):  
Miguel Ricardo Leung ◽  
Marc C. Roelofs ◽  
Ravi Teja Ravi ◽  
Paula Maitan ◽  
Min Zhang ◽  
...  
Keyword(s):  
Focused Ion Beam ◽  
Electron Tomography ◽  
Ion Beam ◽  
Mammalian Species ◽  
Cell Types ◽  
Molecular Structures ◽  
Ciliary Motility ◽  
Mammalian Sperm ◽  
Cell Structures ◽  
Flagellar Beat

SummaryMotile cilia are molecular machines used by a myriad of eukaryotic cells to swim through fluid environments. However, available molecular structures represent only a handful of cell types, limiting our understanding of how cilia are modified to support motility in diverse media. Here, we use cryo-focused ion beam milling-enabled cryo-electron tomography to image sperm flagella from three mammalian species. We resolve in-cell structures of centrioles, axonemal doublets, central pair apparatus, and endpiece singlets, revealing novel protofilament-bridging microtubule inner proteins throughout the flagellum. We present native structures of the flagellar base, which is crucial for shaping the flagellar beat. We show that outer dense fibers are directly coupled to microtubule doublets in the principal piece but not in the midpiece. Thus, mammalian sperm flagella are ornamented across scales, from protofilament-bracing structures rein-forcing microtubules at the nano-scale to accessory structures that impose micron-scale asymmetries on the entire assembly. Our structures provide vital foundations for linking molecular structure to ciliary motility and evolution.

scholarly journals The structure of the COPI coat determined within the cell

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Yury S Bykov ◽  
Miroslava Schaffer ◽  
Svetlana O Dodonova ◽  
Sahradha Albert ◽  
Jürgen M Plitzko ◽  
...  
Keyword(s):  
Focused Ion Beam ◽  
Structural Model ◽  
Electron Tomography ◽  
Ion Beam ◽  
Membrane Thickness ◽  
In Vitro Reconstitution ◽  
Cryo Electron Tomography ◽  
Subtomogram Averaging

COPI-coated vesicles mediate trafficking within the Golgi apparatus and from the Golgi to the endoplasmic reticulum. The structures of membrane protein coats, including COPI, have been extensively studied with in vitro reconstitution systems using purified components. Previously we have determined a complete structural model of the in vitro reconstituted COPI coat (Dodonova et al., 2017). Here, we applied cryo-focused ion beam milling, cryo-electron tomography and subtomogram averaging to determine the native structure of the COPI coat within vitrified Chlamydomonas reinhardtii cells. The native algal structure resembles the in vitro mammalian structure, but additionally reveals cargo bound beneath β’–COP. We find that all coat components disassemble simultaneously and relatively rapidly after budding. Structural analysis in situ, maintaining Golgi topology, shows that vesicles change their size, membrane thickness, and cargo content as they progress from cis to trans, but the structure of the coat machinery remains constant.

scholarly journals Native architecture of the Chlamydomonas chloroplast revealed by in situ cryo-electron tomography

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Benjamin D Engel ◽  
Miroslava Schaffer ◽  
Luis Kuhn Cuellar ◽  
Elizabeth Villa ◽  
Jürgen M Plitzko ◽  
...  
Keyword(s):  
Focused Ion Beam ◽  
Carbon Fixation ◽  
Nearest Neighbor ◽  
Electron Tomography ◽  
Close Association ◽  
Ion Beam ◽  
Three Dimensional ◽  
Chloroplast Envelope ◽  
Cryo Electron Tomography ◽  
Subtomogram Averaging

Chloroplast function is orchestrated by the organelle's intricate architecture. By combining cryo-focused ion beam milling of vitreous Chlamydomonas cells with cryo-electron tomography, we acquired three-dimensional structures of the chloroplast in its native state within the cell. Chloroplast envelope inner membrane invaginations were frequently found in close association with thylakoid tips, and the tips of multiple thylakoid stacks converged at dynamic sites on the chloroplast envelope, implicating lipid transport in thylakoid biogenesis. Subtomogram averaging and nearest neighbor analysis revealed that RuBisCO complexes were hexagonally packed within the pyrenoid, with ∼15 nm between their centers. Thylakoid stacks and the pyrenoid were connected by cylindrical pyrenoid tubules, physically bridging the sites of light-dependent photosynthesis and light-independent carbon fixation. Multiple parallel minitubules were bundled within each pyrenoid tubule, possibly serving as conduits for the targeted one-dimensional diffusion of small molecules such as ATP and sugars between the chloroplast stroma and the pyrenoid matrix.

scholarly journals Correlative cryogenic montage electron tomography for comprehensive in-situ whole-cell structural studies

2022 ◽  
Author(s):  
Jie E Yang ◽  
Matthew R Larson ◽  
Bryan S Sibert ◽  
Joseph Y Kim ◽  
Daniel Parrell ◽  
...  
Keyword(s):  
Focused Ion Beam ◽  
Low Dose ◽  
Structural Information ◽  
Electron Tomography ◽  
Ion Beam ◽  
Three Dimensional ◽  
Structural Studies ◽  
Cryo Electron Tomography ◽  
Efficient Data

Imaging large fields of view while preserving high-resolution structural information remains a challenge in low-dose cryo-electron tomography. Here, we present robust tools for montage electron tomography tailored for vitrified specimens. The integration of correlative cryo-fluorescence microscopy, focused-ion beam milling, and micropatterning produces contextual three-dimensional architecture of cells. Montage tilt series may be processed in their entirety or as individual tiles suitable for sub-tomogram averaging, enabling efficient data processing and analysis.

scholarly journals In situ structural analysis of Golgi intracisternal protein arrays

2015 ◽  
Vol 112 (36) ◽  
pp. 11264-11269 ◽  
Author(s):  
Benjamin D. Engel ◽  
Miroslava Schaffer ◽  
Sahradha Albert ◽  
Shoh Asano ◽  
Jürgen M. Plitzko ◽  
...  
Keyword(s):  
Focused Ion Beam ◽  
Electron Tomography ◽  
Ion Beam ◽  
Asymmetric Membrane ◽  
Protein Arrays ◽  
Cellular Environment ◽  
Filament Bundles ◽  
Subtomogram Averaging ◽  
And Function

We acquired molecular-resolution structures of the Golgi within its native cellular environment. Vitreous Chlamydomonas cells were thinned by cryo-focused ion beam milling and then visualized by cryo-electron tomography. These tomograms revealed structures within the Golgi cisternae that have not been seen before. Narrow trans-Golgi lumina were spanned by asymmetric membrane-associated protein arrays that had ∼6-nm lateral periodicity. Subtomogram averaging showed that the arrays may determine the narrow central spacing of the trans-Golgi cisternae through zipper-like interactions, thereby forcing cargo to the trans-Golgi periphery. Additionally, we observed dense granular aggregates within cisternae and intracisternal filament bundles associated with trans-Golgi buds. These native in situ structures provide new molecular insights into Golgi architecture and function.

scholarly journals Author response: Fully automated, sequential focused ion beam milling for cryo-electron tomography

2020 ◽  
Author(s):  
Tobias Zachs ◽  
Andreas Schertel ◽  
João Medeiros ◽  
Gregor L Weiss ◽  
Jannik Hugener ◽  
...  
Keyword(s):  
Focused Ion Beam ◽  
Electron Tomography ◽  
Ion Beam ◽  
Author Response ◽  
Cryo Electron Tomography ◽  
Focused Ion Beam Milling

scholarly journals Quantitative method for estimating stain density in electron microscopy of conventionally prepared biological specimens

2019 ◽  
Author(s):  
Andrea Fera ◽  
Qianping He ◽  
Guofeng Zhang ◽  
Richard D. Leapman
Keyword(s):  
Electron Microscopy ◽  
Focused Ion Beam ◽  
Electron Tomography ◽  
Blood Platelets ◽  
Ion Beam ◽  
Simple Expression ◽  
Heavy Atoms ◽  
3D Electron Microscopy ◽  
Block Face ◽  
Microscopy Techniques

SummaryStain density is an important parameter for optimizing the quality of ultrastructural data obtained from several types of 3D electron microscopy techniques, including serial block-face electron microscopy (SBEM), and focused ion beam scanning electron microscopy (FIB-SEM). Here, we show how some straightforward measurements in the TEM can be used to determine the stain density based on a simple expression that we derive. Numbers of stain atoms per unit volume are determined from the measured ratio of the bright-field intensities from regions of the specimen that contain both pure embedding material and the embedded biological structures of interest. The determination only requires knowledge of the section thickness, which can either be estimated from the microtome setting, or from low-dose electron tomography, and the elastic scattering cross section for the heavy atoms used to stain the specimen. The method is tested on specimens of embedded blood platelets, brain tissue, and liver tissue.

scholarly journals Automated cryo-lamella preparation for high-throughput in-situ structural biology

2019 ◽  
Author(s):  
Genevieve Buckley ◽  
Gediminas Gervinskas ◽  
Cyntia Taveneau ◽  
Hari Venugopal ◽  
James C. Whisstock ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Structural Biology ◽  
Focused Ion Beam ◽  
Electron Tomography ◽  
Ion Beam ◽  
Automated Method ◽  
Cellular Environment ◽  
Transmission Electron

AbstractCryo-transmission electron tomography (cryo-ET) in association with cryo-focused ion beam (cryo-FIB) milling enables structural biology studies to be performed directly within the cellular environment. Cryo-preserved cells are milled and a lamella with a thickness of 200-300 nm provides an electron transparent window suitable for cryo-ET imaging. Cryo-FIB milling is an effective method, but it is a tedious and time-consuming process, which typically results in ~10 lamellae per day. Here, we introduce an automated method to reproducibly prepare cryo-lamellae on a grid and reduce the amount of human supervision. We tested the routine on cryo-preserved Saccharomyces cerevisiae and demonstrate that this method allows an increased throughput, achieving a rate of 5 lamellae/hour without the need to supervise the FIB milling. We demonstrate that the quality of the lamellae is consistent throughout the preparation and their compatibility with cryo-ET analyses.

scholarly journals A modular platform for automated cryo-FIB workflows

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Sven Klumpe ◽  
Herman K H Fung ◽  
Sara K Goetz ◽  
Ievgeniia Zagoriy ◽  
Bernhard Hampoelz ◽  
...  
Keyword(s):  
Focused Ion Beam ◽  
Structural Integrity ◽  
Electron Tomography ◽  
Ion Beam ◽  
Light And Electron Microscopy ◽  
Software Tool ◽  
3 Dimensional ◽  
Improved Stability ◽  
Modular Platform ◽  
Beam Currents

Lamella micromachining by focused ion beam milling at cryogenic temperature (cryo-FIB) has matured into a preparation method widely used for cellular cryo-electron tomography. Due to the limited ablation rates of low Ga+ ion beam currents required to maintain the structural integrity of vitreous specimens, common preparation protocols are time-consuming and labor intensive. The improved stability of new generation cryo-FIB instruments now enables automated operations. Here, we present an open-source software tool, SerialFIB, for creating automated and customizable cryo-FIB preparation protocols. The software encompasses a graphical user interface for easy execution of routine lamellae preparations, a scripting module compatible with available Python packages, and interfaces with 3-dimensional correlative light and electron microscopy (CLEM) tools. SerialFIB enables the streamlining of advanced cryo-FIB protocols such as multi-modal imaging, CLEM-guided lamella preparation and in situ lamella lift-out procedures. Our software therefore provides a foundation for further development of advanced cryogenic imaging and sample preparation protocols.

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