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Ohad Medalia - One of the best experts on this subject based on the ideXlab platform.

  • Unveiling the polarity of actin filaments by Cryo-Electron Tomography
    Structure (London England : 1993), 2021
    Co-Authors: Bruno Martins, Ohad Medalia, Simona Sorrentino, Wen-lu Chung, Meltem Tatli, Matthias Eibauer
    Abstract:

    Summary The actin cytoskeleton plays a fundamental role in numerous cellular processes, such as cell motility, cytokinesis, and adhesion to the extracellular matrix. Revealing the polarity of individual actin filaments in intact cells would foster an unprecedented understanding of cytoskeletal processes and their associated mechanical forces. Cryo-Electron Tomography provides the means for high-resolution structural imaging of cells. However, the low signal-to-noise ratio of cryo-tomograms obscures the high frequencies, and therefore the polarity of actin filaments cannot be directly measured. Here, we developed a method that enables us to determine the polarity of actin filaments in cellular cryo-tomograms. We applied it to reveal the actin polarity distribution in focal adhesions, and show a linear relation between actin polarity and distance from the apical boundary of the adhesion site.

  • Revealing the polarity of actin filaments by Cryo-Electron Tomography
    2020
    Co-Authors: Bruno Martins, Ohad Medalia, Simona Sorrentino, Wen-lu Chung, Meltem Tatli, Matthias Eibauer
    Abstract:

    The actin cytoskeleton plays a fundamental role in numerous cellular processes, such as cell motility, cytokinesis, and adhesion to the extracellular matrix. Revealing the polarity of individual actin filaments in cells, would foster an unprecedented understanding of cytoskeletal processes and their associated mechanical forces. Cryo-Electron Tomography provides the means for high-resolution structural imaging of cells. However, the low signal-to-noise ratio of cryo-tomograms obscures the high frequencies and therefore the polarity of actin filaments cannot be directly measured. Here, we developed an approach that enables to determine the polarity of actin filaments in cellular cryo-tomograms. We applied it to reveal the actin polarity distribution in focal adhesions, and show a linear relation between actin polarity and distance from the apical boundary of the adhesion site. ### Competing Interest Statement The authors have declared no competing interest.

  • Cellular structural biology as revealed by Cryo-Electron Tomography
    Journal of Cell Science, 2016
    Co-Authors: Rossitza N. Irobalieva, Bruno Martins, Ohad Medalia
    Abstract:

    Understanding the function of cellular machines requires a thorough analysis of the structural elements that underline their function. Electron microscopy (EM) has been pivotal in providing information about cellular ultrastructure, as well as macromolecular organization. Biological materials can be physically fixed by vitrification and imaged with Cryo-Electron Tomography (cryo-ET) in a close-to-native condition. Using this technique, one can acquire three-dimensional (3D) information about the macromolecular architecture of cells, depict unique cellular states and reconstruct molecular networks. Technical advances over the last few years, such as improved sample preparation and electron detection methods, have been instrumental in obtaining data with unprecedented structural details. This presents an exciting opportunity to explore the molecular architecture of both individual cells and multicellular organisms at nanometer to subnanometer resolution. In this Commentary, we focus on the recent developments and in situ applications of cryo-ET to cell and structural biology.

  • Structural analysis of multicellular organisms with Cryo-Electron Tomography
    Nature Methods, 2015
    Co-Authors: Jan Harapin, K. Tanuj Sapra, Mandy Börmel, Damian Brunner, Andres Kaech, Ohad Medalia
    Abstract:

    We developed a method for visualizing tissues from multicellular organisms using Cryo-Electron Tomography. Our protocol involves vitrifying samples with high-pressure freezing, thinning them with cryo-FIB-SEM (focused-ion-beam scanning electron microscopy) and applying fiducial gold markers under cryogenic conditions to the lamellae post-milling. We applied this protocol to acquire tomograms of vitrified Caenorhabditis elegans embryos and worms, which showed the intracellular organization of selected tissues at particular developmental stages in otherwise intact specimens.

  • Developments in Cryo-Electron Tomography for in situ structural analysis.
    Archives of Biochemistry and Biophysics, 2015
    Co-Authors: Anna Dubrovsky, Simona Sorrentino, Jan Harapin, K. Tanuj Sapra, Ohad Medalia
    Abstract:

    Structural analysis of macromolecular assemblies and their remodeling during physiological processes is instrumental to defining the fundament of cellular and molecular biology. Recent advances in computational and analytical tools for Cryo-Electron Tomography have enabled the study of macromolecular structures in their native environment, providing unprecedented insights into cell function. Moreover, the recent implementation of direct electron detectors has progressed Cryo-Electron Tomography to a stage where it can now be applied to the reconstruction of macromolecular structures at high resolutions. Here, we discuss some of the recent technical developments in Cryo-Electron Tomography to reveal structures of macromolecular complexes in their physiological medium, focusing mainly on eukaryotic cells.

Wolfgang Baumeister - One of the best experts on this subject based on the ideXlab platform.

  • The promise and the challenges of Cryo-Electron Tomography.
    FEBS letters, 2020
    Co-Authors: Martin Turk, Wolfgang Baumeister
    Abstract:

    Structural biologists have traditionally approached cellular complexity in a reductionist manner in which the cellular molecular components are fractionated and purified before being studied individually. This 'divide and conquer' approach has been highly successful. However, awareness has grown in recent years that biological functions can rarely be attributed to individual macromolecules. Most cellular functions arise from their concerted action, and there is thus a need for methods enabling structural studies performed in situ, ideally in unperturbed cellular environments. Cryo-Electron Tomography (Cryo-ET) combines the power of 3D molecular-level imaging with the best structural preservation that is physically possible to achieve. Thus, it has a unique potential to reveal the supramolecular architecture or 'molecular sociology' of cells and to discover the unexpected. Here, we review state-of-the-art Cryo-ET workflows, provide examples of biological applications, and discuss what is needed to realize the full potential of Cryo-ET.

  • The Promise and the Challenges of Cryo-Electron Tomography.
    FEBS Letters, 2020
    Co-Authors: Martin Turk, Wolfgang Baumeister
    Abstract:

    Structural biologists have traditionally approached cellular complexity in a reductionist manner in which the cellular molecular components are fractionated and purified before being studied individually. This 'divide and conquer' approach has been highly successful. However, awareness has grown in recent years that biological functions can rarely be attributed to individual macromolecules. Most cellular functions arise from their concerted action and there is thus a need for methods enabling structural studies performed in situ, ideally in unperturbed cellular environments. Cryo-Electron Tomography combines the power of three-dimensional molecular level imaging with the best structural preservation that is physically possible to achieve. Thus, it has a unique potential to reveal the supramolecular architecture or 'molecular sociology' of cells and to discover the unexpected. Here we review state-of-the-art Cryo-Electron Tomography workflows, provide examples of biological applications and discuss what is needed to realize the full potential of Cryo-Electron Tomography.

  • Three‐dimensional organization of the cytoskeleton: A cryo‐electron Tomography perspective
    Protein Science, 2020
    Co-Authors: Saikat Chakraborty, Marion Jasnin, Wolfgang Baumeister
    Abstract:

    Traditionally, structures of cytoskeletal components have been studied ex situ, that is, with biochemically purified materials. There are compelling reasons to develop approaches to study them in situ in their native functional context. In recent years, Cryo-Electron Tomography emerged as a powerful method for visualizing the molecular organization of unperturbed cellular landscapes with the potential to attain near-atomic resolution. Here, we review recent works on the cytoskeleton using Cryo-Electron Tomography, demonstrating the power of in situ studies. We also highlight the potential of this method in addressing important questions pertinent to the field of cytoskeletal biomechanics.

  • Cryo-Electron Tomography
    Springer Handbook of Microscopy, 2019
    Co-Authors: Jürgen M. Plitzko, Wolfgang Baumeister
    Abstract:

    Classical structural biology approaches rely on highly purified molecules which are isolated from their neighbors far from the complex macromolecular interaction network of the cell (ex situ). We have seen breathtaking results of such isolated molecular structures at atomic or near-atomic resolution obtained by single-particle Cryo-Electron microscopy (cryo-EM). However, many supra- and macromolecular complexes involved in key cellular processes cannot be studied in isolation; their function is so deeply rooted in their cellular context that it is impossible to isolate them without compromising their structural integrity. The challenge now is to apply cryo-EM to protein complexes and other biological objects in their natural environment, namely cells. Cryo-Electron Tomography (cryo-ET) offers this opportunity, and in this chapter we provide an overview of recent advances in sample preparation, data acquisition and data processing, including technology for focused ion beam milling, correlative light and electron microscopy, phase-plate imaging and direct electron detection. We show that these developments can be used synergistically to generate 3-D images of cells of unprecedented quality, enabling direct visualization of macromolecular complexes and their spatial coordination in undisturbed eukaryotic cell environments (in situ).

  • Estimation of Membrane Curvature for Cryo-Electron Tomography
    2019
    Co-Authors: Maria Kalemanov, Wolfgang Baumeister, Rubén Fernández-busnadiego, Javier Collado, Antonio Martinez-sanchez
    Abstract:

    Abstract Curvature is an important morphological descriptor of cellular membranes. Cryo-Electron Tomography (cryo-ET) is particularly well-suited to visualize and analyze membrane morphology in a close-to-native state and high resolution. However, current curvature estimation methods cannot be applied directly to membrane segmentations in cryo-ET. Additionally, a reliable estimation requires to cope with quantization noise. Here, we developed and implemented a method for membrane curvature estimation from tomogram segmentations. From a membrane segmentation, a signed surface (triangle mesh) is first extracted. The triangle mesh is then represented by a graph (vertices and edges), which facilitates finding neighboring triangles and the calculation of geodesic distances necessary for local curvature estimation. Here, we present several approaches for accurate curvature estimation based on tensor voting. Beside curvatures, these methods also provide robust estimations of surface normals and principal directions. We tested the different methods on benchmark surfaces with known curvature, demonstrating the validity of these methods and their robustness to quantization noise. We also applied two of these approaches to biological cryo-ET data. The results allowed us to determine the best approach to estimate membrane curvature in cellular cryo-ET data.

Kuniaki Nagayama - One of the best experts on this subject based on the ideXlab platform.

  • Zernike phase contrast Cryo-Electron Tomography of whole mounted frozen cells.
    Journal of Structural Biology, 2012
    Co-Authors: Y. Fukuda, Kuniaki Nagayama
    Abstract:

    Cryo-Electron Tomography of frozen hydrated cells has provided cell biologists with an indispensable tool for delineating three-dimensional arrangements of cellular ultrastructure. To avoid the damage induced by electron irradiation, images of frozen hydrated biological specimens are generally acquired under low-dose conditions, resulting in weakly contrasted images that are difficult to interpret, and in which ultrastructural details remain ambiguous. Zernike phase contrast transmission electron microscopy can improve contrast, and can also fix a fatal problem related to the inherent low contrast of conventional electron microscopy, namely, image modulation due to the unavoidable setting of deep defocus. In this study, we applied Cryo-Electron Tomography enhanced with a Zernike phase plate, which avoids image modulation by allowing in-focus setting. The Zernike phase contrast Cryo-Electron Tomography has a potential to suppress grainy background generation. Due to the smoother background in comparison with defocus phase contrast Cryo-Electron Tomography, Zernike phase contrast Cryo-Electron Tomography could yield higher visibility for particulate or filamentous ultrastructure inside the cells, and allowed us to clearly recognize membrane protein structures.

  • Zernike phase contrast Cryo-Electron Tomography
    Journal of Structural Biology, 2010
    Co-Authors: Radostin Danev, Shuji Kanamaru, Michael Marko, Kuniaki Nagayama
    Abstract:

    Cryo-Tomography in the electron microscope is unique in its ability to provide high-resolution, three-dimensional structural information about cells, organelles and macromolecules in a nearly native, frozen-hydrated state. However, the phase-contrast imaging method used in conventional Cryo-Electron Tomography fails to faithfully represent the full range of structural features in such specimens. Only certain features are recorded with adequate contrast, and overall contrast is low. The recently developed Zernike phase contrast method has the potential to solve this problem, and here we apply it for the first time to Cryo-Electron Tomography. The new method has uniform transfer characteristics for a wide range of spatial frequencies, leading to improved overall signal-to-noise ratio and raising the prospects of higher resolution and quantitative representation of specimen densities in the reconstructed tomograms.

Daniela Nicastro - One of the best experts on this subject based on the ideXlab platform.

  • Cryo-Electron Tomography reveals ciliary defects underlying human RSPH1 primary ciliary dyskinesia
    Nature Communications, 2014
    Co-Authors: Jianfeng Lin, Weining Yin, Maria C. Smith, Kangkang Song, Margaret W. Leigh, Maimoona A. Zariwala, Michael R. Knowles, Lawrence E. Ostrowski, Daniela Nicastro
    Abstract:

    Our current understanding of cilia biology and ciliary diseases is incomplete, in part because cilia are hard to visualize. Here, the authors use Cryo-Electron Tomography to image the structure of human cilia with high resolution and uncover the elusive ciliary defects in Primary Ciliary Dyskinesia patients.

  • cryo electron Tomography reveals ciliary defects underlying human rsph1 primary ciliary dyskinesia
    Nature Communications, 2014
    Co-Authors: Jianfeng Lin, Weining Yin, Maria C. Smith, Kangkang Song, Margaret W. Leigh, Maimoona A. Zariwala, Michael R. Knowles, Lawrence E. Ostrowski, Daniela Nicastro
    Abstract:

    Cilia play essential roles in normal human development and health; cilia dysfunction results in diseases such as primary ciliary dyskinesia (PCD). Despite their importance, the native structure of human cilia is unknown, and structural defects in the cilia of patients are often undetectable or remain elusive because of heterogeneity. Here we develop an approach that enables visualization of human (patient) cilia at high-resolution using Cryo-Electron Tomography of samples obtained noninvasively by nasal scrape biopsy. We present the native 3D structures of normal and PCD-causing RSPH1-mutant human respiratory cilia in unprecedented detail; this allows comparisons of cilia structure across evolutionarily distant species and reveals the previously unknown primary defect and the heterogeneous secondary defects in RSPH1-mutant cilia. Our data provide evidence for structural and functional heterogeneity in radial spokes, suggest a mechanism for the milder RSPH1 PCD phenotype and demonstrate that Cryo-Electron Tomography can be applied to human disease by directly imaging patient samples.

  • 3D structure of eukaryotic flagella in a quiescent state revealed by Cryo-Electron Tomography
    Proceedings of the National Academy of Sciences, 2005
    Co-Authors: Daniela Nicastro, J. R. Mcintosh, Wolfgang Baumeister
    Abstract:

    We have used Cryo-Electron Tomography to investigate the 3D structure and macromolecular organization of intact, frozen-hydrated sea urchin sperm flagella in a quiescent state. The tomographic reconstructions provide information at a resolution better than 6 nm about the in situ arrangements of macromolecules that are key for flagellar motility. We have visualized the heptameric rings of the motor domains in the outer dynein arm complex and determined that they lie parallel to the plane that contains the axes of neighboring flagellar microtubules. Both the material associated with the central pair of microtubules and the radial spokes display a plane of symmetry that helps to explain the planar beat pattern of these flagella. Cryo-Electron Tomography has proven to be a powerful technique for helping us understand the relationships between flagellar structure and function and the design of macromolecular machines in situ.

  • Cryo-Electron Tomography of neurospora mitochondria.
    Journal of structural biology, 2000
    Co-Authors: Daniela Nicastro, Achilleas S Frangakis, Dieter Typke, Wolfgang Baumeister
    Abstract:

    Cryo-Electron Tomography was used to study the structural organization of whole frozen-hydrated mitochondria from Neurospora crassa. Unlike mitochondria from many other species and tissues, in this case the cristae form a three-dimensional network of interconnected lamellae. Basically, the three-dimensional structure of ice-embedded mitochondria from this species is consistent with previous descriptions of mitochondria prepared by chemical fixation and resin embedding. Nonetheless, ice-embedded mitochondria display some important differences: the outer surface of the mitochondria was found to be rather smooth, the intermembrane space was constant in width, and distinct contact sites between the membranes were clearly revealed. Furthermore ATP synthase particles on the outer surface of an "inside-out vesicle" were visible in 3-D reconstructions. Thus, Cryo-Electron Tomography can provide detailed insights into these organelles with minimal perturbations of the physiological state. This indicates that it is a realistic goal to achieve "molecular resolution" with rather large biological specimens in the near future, ultimately allowing the identification and localization of macromolecules in their cellular context.

Jianfeng Lin - One of the best experts on this subject based on the ideXlab platform.

  • Cryo-Electron Tomography reveals ciliary defects underlying human RSPH1 primary ciliary dyskinesia
    Nature Communications, 2014
    Co-Authors: Jianfeng Lin, Weining Yin, Maria C. Smith, Kangkang Song, Margaret W. Leigh, Maimoona A. Zariwala, Michael R. Knowles, Lawrence E. Ostrowski, Daniela Nicastro
    Abstract:

    Our current understanding of cilia biology and ciliary diseases is incomplete, in part because cilia are hard to visualize. Here, the authors use Cryo-Electron Tomography to image the structure of human cilia with high resolution and uncover the elusive ciliary defects in Primary Ciliary Dyskinesia patients.

  • cryo electron Tomography reveals ciliary defects underlying human rsph1 primary ciliary dyskinesia
    Nature Communications, 2014
    Co-Authors: Jianfeng Lin, Weining Yin, Maria C. Smith, Kangkang Song, Margaret W. Leigh, Maimoona A. Zariwala, Michael R. Knowles, Lawrence E. Ostrowski, Daniela Nicastro
    Abstract:

    Cilia play essential roles in normal human development and health; cilia dysfunction results in diseases such as primary ciliary dyskinesia (PCD). Despite their importance, the native structure of human cilia is unknown, and structural defects in the cilia of patients are often undetectable or remain elusive because of heterogeneity. Here we develop an approach that enables visualization of human (patient) cilia at high-resolution using Cryo-Electron Tomography of samples obtained noninvasively by nasal scrape biopsy. We present the native 3D structures of normal and PCD-causing RSPH1-mutant human respiratory cilia in unprecedented detail; this allows comparisons of cilia structure across evolutionarily distant species and reveals the previously unknown primary defect and the heterogeneous secondary defects in RSPH1-mutant cilia. Our data provide evidence for structural and functional heterogeneity in radial spokes, suggest a mechanism for the milder RSPH1 PCD phenotype and demonstrate that Cryo-Electron Tomography can be applied to human disease by directly imaging patient samples.