Improved Technology for Focused-Ion-Beam Thinning of Tissue for Cryo-Electron Tomography. Application to Triad Junctions

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.

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The molecular architecture of engulfment during Bacillus subtilis sporulation

eLife ◽

10.7554/elife.45257 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 13

Author(s):

Kanika Khanna ◽

Javier Lopez-Garrido ◽

Ziyi Zhao ◽

Reika Watanabe ◽

Yuan Yuan ◽

...

Keyword(s):

Bacillus Subtilis ◽

Mother Cell ◽

Cell Biology ◽

Focused Ion Beam ◽

High Spatial Resolution ◽

Electron Tomography ◽

Ion Beam ◽

Molecular Architecture ◽

Native State ◽

Cryo Electron Tomography

The study of bacterial cell biology is limited by difficulties in visualizing cellular structures at high spatial resolution within their native milieu. Here, we visualize Bacillus subtilis sporulation using cryo-electron tomography coupled with cryo-focused ion beam milling, allowing the reconstruction of native-state cellular sections at molecular resolution. During sporulation, an asymmetrically-positioned septum generates a larger mother cell and a smaller forespore. Subsequently, the mother cell engulfs the forespore. We show that the septal peptidoglycan is not completely degraded at the onset of engulfment. Instead, the septum is uniformly and only slightly thinned as it curves towards the mother cell. Then, the mother cell membrane migrates around the forespore in tiny finger-like projections, whose formation requires the mother cell SpoIIDMP protein complex. We propose that a limited number of SpoIIDMP complexes tether to and degrade the peptidoglycan ahead of the engulfing membrane, generating an irregular membrane front.

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Decision letter: Fully automated, sequential focused ion beam milling for cryo-electron tomography

10.7554/elife.52286.sa1 ◽

2019 ◽

Keyword(s):

Focused Ion Beam ◽

Electron Tomography ◽

Ion Beam ◽

Cryo Electron Tomography ◽

Focused Ion Beam Milling

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PIE-scope, integrated cryo-correlative light and FIB/SEM microscopy

eLife ◽

10.7554/elife.45919 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 20

Author(s):

Sergey Gorelick ◽

Genevieve Buckley ◽

Gediminas Gervinskas ◽

Travis K Johnson ◽

Ava Handley ◽

...

Keyword(s):

Focused Ion Beam ◽

Fluorescent Proteins ◽

Electron Tomography ◽

Protein Complexes ◽

Ion Beam ◽

Correlative Microscopy ◽

New Approach ◽

Macromolecular Complexes ◽

Cryo Electron Tomography

Cryo-electron tomography (cryo-ET) is emerging as a revolutionary method for resolving the structure of macromolecular complexes in situ. However, sample preparation for in situ Cryo-ET is labour-intensive and can require both cryo-lamella preparation through cryo-focused ion beam (FIB) milling and correlative light microscopy to ensure that the event of interest is present in the lamella. Here, we present an integrated cryo-FIB and light microscope setup called the Photon Ion Electron microscope (PIE-scope) that enables direct and rapid isolation of cellular regions containing protein complexes of interest. Specifically, we demonstrate the versatility of PIE-scope by preparing targeted cryo-lamellae from subcellular compartments of neurons from transgenic Caenorhabditis elegans and Drosophila melanogaster expressing fluorescent proteins. We designed PIE-scope to enable retrofitting of existing microscopes, which will increase the throughput and accuracy on projects requiring correlative microscopy to target protein complexes. This new approach will make cryo-correlative workflow safer and more accessible.

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Multi-Scale 3D Cryo-Correlative Microscopy for Vitrified Cells

10.1101/2020.05.21.107771 ◽

2020 ◽

Author(s):

Gong-Her Wu ◽

Patrick G. Mitchell ◽

Jesus G. Galaz-Montoya ◽

Corey W. Hecksel ◽

Emily M. Sontag ◽

...

Keyword(s):

Large Scale ◽

Focused Ion Beam ◽

Electron Tomography ◽

3D Visualization ◽

Ion Beam ◽

Three Dimensional ◽

Yeast Cells ◽

Multi Scale ◽

Fluorescence Confocal Microscopy ◽

Cryo Electron Tomography

SUMMARYThree-dimensional (3D) visualization of vitrified cells can uncover structures of subcellular complexes without chemical fixation or staining. Here, we present a pipeline integrating three imaging modalities to visualize the same specimen at cryogenic temperature at different scales: cryo-fluorescence confocal microscopy, volume cryo-focused ion beam scanning electron microscopy, and transmission cryo-electron tomography. Our proof-of-concept benchmark revealed the 3D distribution of organelles and subcellular structures in whole heat-shocked yeast cells, including the ultrastructure of protein inclusions that recruit fluorescently-labelled chaperone Hsp104. Since our workflow efficiently integrates imaging at three different scales and can be applied to other types of cells, it could be used for large-scale phenotypic studies of frozen-hydrated specimens in a variety of healthy and diseased conditions with and without treatments.

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Protocol for cryo-focused ion beam lift-out technique

10.21203/rs.2.10392/v1 ◽

2019 ◽

Cited By ~ 1

Author(s):

Miroslava Schaffer ◽

Stefan Pfeffer ◽

Julia Mahamid ◽

Stephan Kleindiek ◽

Tim Laugks ◽

...

Keyword(s):

High Pressure ◽

Structural Biology ◽

Focused Ion Beam ◽

Preparation Method ◽

Electron Tomography ◽

Ion Beam ◽

Cell Interior ◽

Cryo Electron Tomography ◽

Multicellular Organisms

Abstract Cryo-focused ion beam milling of frozen hydrated cells for the production of thin lamellas in combination with cryo-electron tomography (cryo-ET) has yielded unprecedented insights into the cell interior. This method allows access to native structures deep inside cells, enabling structural studies of macromolecules in situ. However, it is only suitable for cells that can be vitrified by plunge freezing (<10 μm). Multicellular organisms and tissues are considerably thicker and high-pressure freezing is required to ensure optimal preservation. Here, we describe a preparation method for extracting lamellas from high pressure frozen samples with a new cryo-gripper tool. This in situ lift-out technique at cryo-temperatures enables cryo-ET to be performed on multicellular organisms and tissue, extending the range of applications for in situ structural biology.

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Opening Windows into the Cell: Focused-Ion-Beam Milling for Cryo-Electron Tomography

Biophysical Journal ◽

10.1016/j.bpj.2013.11.3320 ◽

2014 ◽

Vol 106 (2) ◽

pp. 600a ◽

Cited By ~ 3

Author(s):

Elizabeth Villa ◽

Miroslava Schaffer ◽

Jürgen M. Plitzko ◽

Wolfgang Baumeister

Keyword(s):

Focused Ion Beam ◽

Electron Tomography ◽

Ion Beam ◽

Cryo Electron Tomography ◽

Focused Ion Beam Milling

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In situ 3D comparison of Chlorella pyrenoidosa with nuclear-irradiated mutagenic strains by using focused ion beam milling and cryo-electron tomography

10.1101/2020.11.05.369421 ◽

2020 ◽

Author(s):

Wangbiao Guo ◽

Lingchong Feng ◽

Zhenyi Wang ◽

Jiansheng Guo ◽

Donghyun Park ◽

...

Keyword(s):

Growth Rate ◽

Focused Ion Beam ◽

Electron Tomography ◽

Ion Beam ◽

Chlorella Pyrenoidosa ◽

Biofuel Production ◽

Full Potential ◽

Wide Range ◽

Cryo Electron Tomography ◽

Mutant Cells

AbstractMicroalgae are highly efficient photosynthetic organisms that hold enormous potential as sources of renewable energy. In particular, Chlorella pyrenoidosa displays a rapid growth rate, high tolerance to light, and high lipid content, making it especially valuable for applications such as flue gas CO2 fixation, biofuel production, and nutritional extracts. In order to unveil its full potential, it is necessary to characterize its subcellular architecture. Here, we achieved three-dimensional (3D) visualization of the architectures of C. pyrenoidosa cells, by combining focused ion beam scanning electron microscopy (FIB/SEM), cryo-FIB milling, and cryo-electron tomography (cryo-ET). These high-resolution images bring to light intricate features of intact organelles, including thylakoid membranes, pyrenoid, starch granules, mitochondria, nucleus, lipid droplets and vacuoles, as well as the fine architectures within the chloroplast, including the concave-convex pyrenoid, plastoglobules, thylakoid tips, and convergence zones. Significantly, comparative analysis of wild-type and nuclear-irradiated mutagenic strains determined that cell volume and surface area of mutant cells have increased substantially to 2.2-fold and 1.7-fold, respectively, consistent with up-regulation of the enzyme Rubisco and enhanced photosynthetic metabolic processes. Moreover, quantitative analysis established that the thylakoid membrane width in mutant cells increased to 1.3-fold, while the membrane gap decreased to 0.8-fold, possibly contributing to the higher biomass growth rate of mutant cells. Our work reveals the first 3D subcellular architectures of C. pyrenoidosa cell and provides a structural framework for unlocking the higher growth rate in microalgae relevant to a wide range of industrial applications.

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The molecular architecture of engulfment during Bacillussubtilis sporulation

10.1101/498220 ◽

2018 ◽

Author(s):

Kanika Khanna ◽

Javier Lopez-Garrido ◽

Ziyi Zhao ◽

Reika Watanabe ◽

Yuan Yuan ◽

...

Keyword(s):

Mother Cell ◽

Cell Biology ◽

Focused Ion Beam ◽

Electron Tomography ◽

Molecular Model ◽

Ion Beam ◽

Leading Edge ◽

Cellular Structures ◽

Molecular Architecture ◽

Cryo Electron Tomography

AbstractThe study of cell biology is limited by the difficulty in visualizing cellular structures at high spatial resolution within their native milieu. Here, we have visualized sporulation inBacillus subtilisusing cryo-electron tomography coupled with cryo-focused ion beam milling, a technique that allows the 3D reconstruction of cellular structures in near-native state at molecular resolution. During sporulation, an asymmetrically-positioned septum divides the cell into a larger mother cell and a smaller forespore. Subsequently, the mother cell phagocytoses the forespore in a process called engulfment, which entails a dramatic rearrangement of the peptidoglycan (PG) cell wall around the forespore. By imaging wild-type sporangia, engulfment mutants, and sporangia treated with PG synthesis inhibitors, we show that the initiation of engulfment does not entail the complete dissolution of the septal PG by the mother cell SpoIIDMP complex, as was previously thought. Instead, DMP is required to maintain a flexible septum that is uniformly and only slightly thinned at the onset of engulfment. Then, the mother cell membrane migrates around the forespore by forming tiny finger-like projections, the formation of which requires both SpoIIDMP and new PG synthesized ahead of the leading edge of the engulfing membrane. We propose a molecular model for engulfment membrane migration in which a limited number of SpoIIDMP complexes tether the membrane to and degrade the new PG ahead of the leading edge, thereby generating an irregular engulfing membrane front. Our data also reveal other structures that will provide a valuable resource for future mechanistic studies of endospore formation.

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