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

  • Cryoelectron Tomography Reveals Nanoscale Organization of the Cytoskeleton and Its Relation to Microtubule Curvature Inside Cells
    Structure (London England : 1993), 2020
    Co-Authors: Saikat Chakraborty, Julia Mahamid, Wolfgang Baumeister
    Abstract:

    Summary Microtubules (MTs) are the most rigid elements of the cytoskeleton with in vitro persistence lengths (Lp) in the range of 1–6 mm. In cellular environments, however, MTs often appear strongly curved. This has been attributed to the forces acting upon them in situ where they are embedded in composite networks of different cytoskeletal elements. Hitherto, the nanoscale organization of these networks has remained largely uncharacterized. Cryo-electron Tomography (cryo-ET) allowed to visualize and analyze the in situ structure of cytoskeletal networks in pristinely preserved cellular environments and at high resolution. Here, we studied the molecular organization of MTs and their interactions with the composite cytoskeleton in frozen-hydrated HeLa and P19 cells at different cell-cycle stages. We describe modulation of MT curvature correlated with the surrounding molecular architecture, and show that nanoscale defects occur in curved MTs. The data presented here contribute to constructing realistic models of cytoskeletal biomechanics.

  • 70 Three-dimensional arrangements of ribosomes inside fast growing E. coli cells
    Journal of Biomolecular Structure and Dynamics, 2015
    Co-Authors: Thomas Hoffmann, F. Ulrich Hartl, Julio O. Ortiz, Wolfgang Baumeister
    Abstract:

    Translating bacterial ribosomes can form structurally ordered polysomes. The three-dimensional (3D) organization of such polysomes has been described in solution using Cryoelectron Tomography (CET)...

  • JCB: REPORT Luminal particles within cellular microtubules
    2013
    Co-Authors: Boyan K. Garvalov, Martin Beck, Benoît Zuber, Mikhail Kudryashev, Andrew Leis, Friedrich Frischknecht, Cédric Bouchet-marquis, Manuela Gruska, Frank Bradke, Wolfgang Baumeister
    Abstract:

    The regulation of microtubule dynamics is attributed to microtubule-associated proteins that bind to the microtubule outer surface, but little is known about cellular components that may associate with the internal side of microtubules. We used Cryoelectron Tomography to investigate in a quantitative manner the three dimensional structure of microtubules in intact mammalian cells. We show that the lumen of microtubules in this native state is filled with discrete, globular particles with a diameter o

  • focused ion beam micromachining of eukaryotic cells for Cryoelectron Tomography
    Proceedings of the National Academy of Sciences of the United States of America, 2012
    Co-Authors: Alexander Rigort, Wolfgang Baumeister, Felix J.b. Bäuerlein, Elizabeth Villa, Matthias Eibauer, Tim Laugks, Jürgen M. Plitzko
    Abstract:

    Cryoelectron Tomography provides unprecedented insights into the macromolecular and supramolecular organization of cells in a close-to-living state. However because of the limited thickness range (< 0.5–1 μm) that is accessible with today’s intermediate voltage electron microscopes only small prokaryotic cells or peripheral regions of eukaryotic cells can be examined directly. Key to overcoming this limitation is the ability to prepare sufficiently thin samples. Cryosectioning can be used to prepare thin enough sections but suffers from severe artefacts, such as substantial compression. Here we describe a procedure, based upon focused ion beam (FIB) milling for the preparation of thin (200–500 nm) lamellae from vitrified cells grown on electron microscopy (EM) grids. The self-supporting lamellae are apparently free of distortions or other artefacts and open up large windows into the cell’s interior allowing tomographic studies to be performed on any chosen part of the cell. We illustrate the quality of sample preservation with a structure of the nuclear pore complex obtained from a single tomogram.

  • Focused ion beam micromachining of eukaryotic cells for Cryoelectron Tomography
    Proceedings of the National Academy of Sciences of the United States of America, 2012
    Co-Authors: Alexander Rigort, Wolfgang Baumeister, Felix J.b. Bäuerlein, Elizabeth Villa, Matthias Eibauer, Tim Laugks, Jürgen M. Plitzko
    Abstract:

    Cryoelectron Tomography provides unprecedented insights into the macromolecular and supramolecular organization of cells in a close-to-living state. However because of the limited thickness range (

Jun Liu - One of the best experts on this subject based on the ideXlab platform.

  • Analysis of Dot/Icm Type IVB Secretion System Subassemblies by Cryoelectron Tomography Reveals Conformational Changes Induced by DotB Binding
    mBio, 2020
    Co-Authors: Donghyun Park, Craig R Roy, David Chetrit, Jun Liu
    Abstract:

    ABSTRACT Type IV secretion systems (T4SSs) are sophisticated nanomachines used by many bacterial pathogens to translocate protein and DNA substrates across a host cell membrane. Although T4SSs have important roles in promoting bacterial infections, little is known about the biogenesis of the apparatus and the mechanism of substrate transfer. Here, high-throughput Cryoelectron Tomography (cryo-ET) was used to visualize Legionella pneumophila T4SSs (also known as Dot/Icm secretion machines) in both the whole-cell context and at the cell pole. These data revealed the distribution patterns of individual Dot/Icm machines in the bacterial cell and identified five distinct subassembled intermediates. High-resolution in situ structures of the Dot/Icm machine derived from subtomogram averaging revealed that docking of the cytoplasmic DotB (VirB11-related) ATPase complex onto the DotO (VirB4-related) ATPase complex promotes a conformational change in the secretion system that results in the opening of a channel in the bacterial inner membrane. A model is presented for how the Dot/Icm apparatus is assembled and for how this machine may initiate the transport of cytoplasmic substrates across the inner membrane. IMPORTANCE Many bacteria use type IV secretion systems (T4SSs) to translocate proteins and nucleic acids into target cells, which promotes DNA transfer and host infection. The Dot/Icm T4SS in Legionella pneumophila is a multiprotein nanomachine that is known to translocate over 300 different protein effectors into eukaryotic host cells. Here, advanced Cryoelectron Tomography and subtomogram analysis were used to visualize the Dot/Icm machine assembly and distribution in a single L. pneumophila cell. Extensive classification and averaging revealed five distinct intermediates of the Dot/Icm machine at high resolution. Comparative analysis of the Dot/Icm machine and subassemblies derived from wild-type cells and several mutants provided a structural basis for understanding mechanisms that underlie the assembly and activation of the Dot/Icm machine.

  • defining the layers of a sensory cilium with storm and Cryoelectron nanoscopy
    Proceedings of the National Academy of Sciences of the United States of America, 2019
    Co-Authors: Michael A Robichaux, Jun Liu, Valencia Potter, Zhixian Zhang, Michael Schmid, Theodore G Wensel
    Abstract:

    Primary cilia carry out numerous signaling and sensory functions, and defects in them, “ciliopathies,” cause a range of symptoms, including blindness. Understanding of their nanometer-scale ciliary substructures and their disruptions in ciliopathies has been hindered by limitations of conventional microscopic techniques. We have combined Cryoelectron Tomography, enhanced by subtomogram averaging, with superresolution stochastic optical reconstruction microscopy (STORM) to define subdomains within the light-sensing rod sensory cilium of mouse retinas and reveal previously unknown substructures formed by resident proteins. Domains are demarcated by structural features such as the axoneme and its connections to the ciliary membrane, and are correlated with molecular markers of subcompartments, including the lumen and walls of the axoneme, the membrane glycocalyx, and the intervening cytoplasm. Within this framework, we report spatial distributions of key proteins in wild-type (WT) mice and the effects on them of genetic deficiencies in 3 models of Bardet–Biedl syndrome.

  • Integrin αIIbβ3 in a Membrane Environment Remains the Same Height after Mn2+ Activation when Observed by Cryo-electron Tomography
    2015
    Co-Authors: Jun Liu, Hanspeter Winkler, Kenneth A. Taylor
    Abstract:

    Integrins perform the critical function of signalling cell attachment to the extracellular matrix or to other cells. This signalling is done through a structural change propagated bidirectionally across the plasma membrane. Integrin activation has been extensively studied with ectodomain constructs, but the structural change within intact, membrane-bound molecules remains a subject of live debate. Using Cryoelectron Tomography, we examined the simplest predication of the different integrin activation models, i.e. the change in height of the molecules. Analysis using techniques that compensate for the missing wedge during alignment and averaging and that search for patterns in the structure of the aligned molecular subvolumes extracted from the tomogram reveals that the vast majority of molecules show no dramatic height change upon Mn2+ induced activation of membrane bound integrins when compared with an inactive integrin control group. Thus, the result is inconsistent with the switchblade activation model

  • The Unique Paradigm of Spirochete Motility and Chemotaxis
    Annual review of microbiology, 2012
    Co-Authors: Nyles W. Charon, A. Motaleb, Kelly A. Miller, Jun Liu, Andrew Cockburn, Michael R. Miller, Charles W. Wolgemuth
    Abstract:

    Spirochete motility is enigmatic: It differs from the motility of most other bacteria in that the entire bacterium is involved in translocation in the absence of external appendages. Using the Lyme disease spirochete Borrelia burgdorferi (Bb) as a model system, we explore the current research on spirochete motility and chemotaxis. Bb has periplasmic flagella (PFs) subterminally attached to each end of the protoplasmic cell cylinder, and surrounding the cell is an outer membrane. These internal helix-shaped PFs allow the spirochete to swim by generating backward-moving waves by rotation. Exciting advances using Cryoelectron Tomography are presented with respect to in situ analysis of cell, PF, and motor structure. In addition, advances in the dynamics of motility, chemotaxis, gene regulation, and the role of motility and chemotaxis in the life cycle of Bb are summarized. The results indicate that the motility paradigms of flagellated bacteria do not apply to these unique bacteria.

  • Structural Comparison of HIV-1 Envelope Spikes with and without the V1/V2 Loop
    Journal of virology, 2010
    Co-Authors: Jun Liu, Kenneth A. Taylor, Kenneth H. Roux
    Abstract:

    We have used Cryoelectron Tomography of vitreous-ice-embedded HIV-1 virions to compare the envelope (Env) spikes of a wild-type strain with those of a mutant strain in which the V1/V2 loop has been deleted. Deletion of V1/V2 results in a spike with far more structural heterogeneity than is observed in the wild type, likely reflecting greatly enhanced gp120 protomer flexibility. A major difference between the two forms is a pronounced loss of mass from the "peak" of the native Env spike. The apparent loss of contact among three gp120 protomers likely accounts for the more open structure, heterogeneity in configuration, and previous observations that broadly neutralizing epitopes and reactive sites on other structural elements are more exposed in such constructs.

Ju Liu - One of the best experts on this subject based on the ideXlab platform.

  • analysis of dot icm type ivb secretion system subassemblies by Cryoelectron Tomography reveals conformational changes induced by dotb binding
    Mbio, 2020
    Co-Authors: Donghyu Park, David Chetri, Craig R Roy, Ju Liu
    Abstract:

    ABSTRACT Type IV secretion systems (T4SSs) are sophisticated nanomachines used by many bacterial pathogens to translocate protein and DNA substrates across a host cell membrane. Although T4SSs have important roles in promoting bacterial infections, little is known about the biogenesis of the apparatus and the mechanism of substrate transfer. Here, high-throughput Cryoelectron Tomography (cryo-ET) was used to visualize Legionella pneumophila T4SSs (also known as Dot/Icm secretion machines) in both the whole-cell context and at the cell pole. These data revealed the distribution patterns of individual Dot/Icm machines in the bacterial cell and identified five distinct subassembled intermediates. High-resolution in situ structures of the Dot/Icm machine derived from subtomogram averaging revealed that docking of the cytoplasmic DotB (VirB11-related) ATPase complex onto the DotO (VirB4-related) ATPase complex promotes a conformational change in the secretion system that results in the opening of a channel in the bacterial inner membrane. A model is presented for how the Dot/Icm apparatus is assembled and for how this machine may initiate the transport of cytoplasmic substrates across the inner membrane. IMPORTANCE Many bacteria use type IV secretion systems (T4SSs) to translocate proteins and nucleic acids into target cells, which promotes DNA transfer and host infection. The Dot/Icm T4SS in Legionella pneumophila is a multiprotein nanomachine that is known to translocate over 300 different protein effectors into eukaryotic host cells. Here, advanced Cryoelectron Tomography and subtomogram analysis were used to visualize the Dot/Icm machine assembly and distribution in a single L. pneumophila cell. Extensive classification and averaging revealed five distinct intermediates of the Dot/Icm machine at high resolution. Comparative analysis of the Dot/Icm machine and subassemblies derived from wild-type cells and several mutants provided a structural basis for understanding mechanisms that underlie the assembly and activation of the Dot/Icm machine.

Kenneth A. Taylor - One of the best experts on this subject based on the ideXlab platform.

  • Integrin αIIbβ3 in a Membrane Environment Remains the Same Height after Mn2+ Activation when Observed by Cryo-electron Tomography
    2015
    Co-Authors: Jun Liu, Hanspeter Winkler, Kenneth A. Taylor
    Abstract:

    Integrins perform the critical function of signalling cell attachment to the extracellular matrix or to other cells. This signalling is done through a structural change propagated bidirectionally across the plasma membrane. Integrin activation has been extensively studied with ectodomain constructs, but the structural change within intact, membrane-bound molecules remains a subject of live debate. Using Cryoelectron Tomography, we examined the simplest predication of the different integrin activation models, i.e. the change in height of the molecules. Analysis using techniques that compensate for the missing wedge during alignment and averaging and that search for patterns in the structure of the aligned molecular subvolumes extracted from the tomogram reveals that the vast majority of molecules show no dramatic height change upon Mn2+ induced activation of membrane bound integrins when compared with an inactive integrin control group. Thus, the result is inconsistent with the switchblade activation model

  • Structural Comparison of HIV-1 Envelope Spikes with and without the V1/V2 Loop
    Journal of virology, 2010
    Co-Authors: Jun Liu, Kenneth A. Taylor, Kenneth H. Roux
    Abstract:

    We have used Cryoelectron Tomography of vitreous-ice-embedded HIV-1 virions to compare the envelope (Env) spikes of a wild-type strain with those of a mutant strain in which the V1/V2 loop has been deleted. Deletion of V1/V2 results in a spike with far more structural heterogeneity than is observed in the wild type, likely reflecting greatly enhanced gp120 protomer flexibility. A major difference between the two forms is a pronounced loss of mass from the "peak" of the native Env spike. The apparent loss of contact among three gp120 protomers likely accounts for the more open structure, heterogeneity in configuration, and previous observations that broadly neutralizing epitopes and reactive sites on other structural elements are more exposed in such constructs.

  • Cryoelectron Tomography of HIV-1 envelope spikes: further evidence for tripod-like legs.
    PLoS pathogens, 2008
    Co-Authors: Ping Zhu, Kenneth A. Taylor, Hanspeter Winkler, Elena Chertova, Kenneth H. Roux
    Abstract:

    A detailed understanding of the morphology of the HIV-1 envelope (Env) spike is key to understanding viral pathogenesis and for informed vaccine design. We have previously presented a Cryoelectron microscopic tomogram (cryoET) of the Env spikes on SIV virions. Several structural features were noted in the gp120 head and gp41 stalk regions. Perhaps most notable was the presence of three splayed legs projecting obliquely from the base of the spike head toward the viral membrane. Subsequently, a second 3D image of SIV spikes, also obtained by cryoET, was published by another group which featured a compact vertical stalk. We now report the cryoET analysis of HIV-1 virion-associated Env spikes using enhanced analytical cryoET procedures. More than 2,000 Env spike volumes were initially selected, aligned, and sorted into structural classes using algorithms that compensate for the ‘‘missing wedge’’ and do not impose any symmetry. The results show varying morphologies between structural classes: some classes showed trimers in the head domains; nearly all showed two or three legs, though unambiguous three-fold symmetry was not observed either in the heads or the legs. Subsequently, clearer evidence of trimeric head domains and three splayed legs emerged when head and leg volumes were independently aligned and classified. These data show that HIV-1, like SIV, also displays the tripod-like leg configuration, and, unexpectedly, shows considerable gp41 leg flexibility/heteromorphology. The tripod-like model for gp41 is consistent with, and helps explain, many of the unique biophysical and immunological features of this region.

  • Integrin αIIbβ3 in a Membrane Environment Remains the Same Height after Mn2+ Activation when Observed by Cryoelectron Tomography
    Journal of molecular biology, 2008
    Co-Authors: Jun Liu, Hanspeter Winkler, Kenneth A. Taylor
    Abstract:

    Abstract Integrins perform the critical function of signalling cell attachment to the extracellular matrix or to other cells. This signalling is done through a structural change propagated bidirectionally across the plasma membrane. Integrin activation has been extensively studied with ectodomain constructs, but the structural change within intact, membrane-bound molecules remains a subject of live debate. Using Cryoelectron Tomography, we examined the simplest predication of the different integrin activation models, i.e., the change in height of the molecules. Analysis using techniques that compensate for the missing wedge during alignment and averaging and that search for patterns in the structure of the aligned molecular subvolumes extracted from the tomogram reveals that the vast majority of molecules show no dramatic height change upon Mn 2+ -induced activation of membrane-bound integrins when compared with an inactive integrin control group. Thus, the result is inconsistent with the switchblade activation model.

  • Regulation and recycling of myosin V.
    Current Opinion in Cell Biology, 2007
    Co-Authors: Kenneth A. Taylor
    Abstract:

    Recently there has been considerable progress in our understanding of regulation for unconventional myosin-V through elucidation of the structure of its inactive conformation and the factors that affect stability of this conformation. The inactive conformation is a folded compact structure characterized by interactions between the myosin head and the C-terminal cargo binding domain. Concentrations of Ca2+ greater than 10 μM disrupt folding. The 3-D structure determined by Cryoelectron Tomography of 2-D arrays in one study and electron micrographs of isolated molecules reported in another reveal similar features, but suggest different F-actin affinities for the inactive conformation. This has raised the question of how inactive myosin-V is recycled to other sites for additional rounds of cargo transport.

Friedrich Förster - One of the best experts on this subject based on the ideXlab platform.

  • Functions and Mechanisms of the Human Ribosome-Translocon Complex.
    Sub-cellular biochemistry, 2019
    Co-Authors: Sven Lang, Friedrich Förster, Stefan Pfeffer, Duy Nguyen, Volkhard Helms, Richard Zimmermann
    Abstract:

    The membrane of the endoplasmic reticulum (ER) in human cells harbors the protein translocon, which facilitates membrane insertion and translocation of almost every newly synthesized polypeptide targeted to organelles of the secretory pathway. The translocon comprises the polypeptide-conducting Sec61 channel and several additional proteins, which are associated with the heterotrimeric Sec61 complex. This ensemble of proteins facilitates ER targeting of precursor polypeptides, Sec61 channel opening and closing, and modification of precursor polypeptides in transit through the Sec61 complex. Recently, Cryoelectron Tomography of translocons in native ER membranes has given unprecedented insights into the architecture and dynamics of the native, ribosome-associated translocon and the Sec61 channel. These structural data are discussed in light of different Sec61 channel activities including ribosome receptor function, membrane insertion or translocation of newly synthesized polypeptides as well as the possible roles of the Sec61 channel as a passive ER calcium leak channel and regulator of ATP/ADP exchange between cytosol and ER.

  • Structure of the Human Mitochondrial Ribosome Studied In Situ by Cryoelectron Tomography
    Structure (London England : 1993), 2017
    Co-Authors: Robert Englmeier, Stefan Pfeffer, Friedrich Förster
    Abstract:

    Summary Mitochondria maintain their own genome and its corresponding protein synthesis machine, the mitochondrial ribosome (mitoribosome). Mitoribosomes primarily synthesize highly hydrophobic proteins of the inner mitochondrial membrane. Recent studies revealed the complete structure of the isolated mammalian mitoribosome, but its mode of membrane association remained hypothetical. In this study, we used Cryoelectron Tomography to visualize human mitoribosomes in isolated mitochondria. The subtomogram average of the membrane-associated human mitoribosome reveals a single major contact site with the inner membrane, mediated by the mitochondria-specific protein mL45. A second rRNA-mediated contact site that is present in yeast is absent in humans, resulting in a more variable association of the human mitoribosome with the inner membrane. Despite extensive structural differences of mammalian and fungal mitoribosomal structure, the principal organization of peptide exit tunnel and the mL45 homolog remains invariant, presumably to align the mitoribosome with the membrane-embedded insertion machinery.

  • Proteintranslation und Prozessierung in physiologischer Umgebung abgebildet
    BIOspektrum, 2015
    Co-Authors: Stefan Pfeffer, Friedrich Förster
    Abstract:

    Cryoelectron Tomography allows 3D imaging of crowded pleiomorphic environments at molecular resolution and is consequently an excellent method for studying the structure and organization of large molecules in their natural context. Using this approach for the analysis of ribosomal complexes in different cellular compartments, we obtained detailed insights into the supramolecular organization of the cytosolic and mitochondrial translation machineries and their association to membranes for co-translational protein transport.

  • Organization of the mitochondrial translation machinery studied in situ by Cryoelectron Tomography
    Nature communications, 2015
    Co-Authors: Stefan Pfeffer, Michael W. Woellhaf, Johannes M. Herrmann, Friedrich Förster
    Abstract:

    Whereas the structure and function of cytosolic ribosomes have been studied in great detail, we know surprisingly little about the structural basis of mitochondrial protein synthesis. Here we used Cryoelectron Tomography and subtomogram analysis to visualize mitoribosomes in isolated yeast mitochondria, avoiding perturbations during ribosomal purification. Most mitoribosomes reside in immediate proximity to the inner mitochondrial membrane, in line with their specialization in the synthesis of hydrophobic membrane proteins. The subtomogram average of membrane-associated mitoribosomes reveals two distinct membrane contact sites, formed by the 21S rRNA expansion segment 96-ES1 and the inner membrane protein Mba1. On the basis of our data, we further hypothesize that Mba1 is not just a passive mitoribosome receptor on the inner membrane, but that it spatially aligns mitoribosomes with the membrane insertion machinery. This study reveals detailed insights into the supramolecular organization of the mitochondrial translation machinery and its association with the inner membrane in translation-competent mitochondria.

  • automated detection of polysomes in Cryoelectron Tomography
    International Conference on Image Processing, 2014
    Co-Authors: Luis Kuhn Cuellar, Stefan Pfeffer, Yuxiang Chen, Friedrich Förster
    Abstract:

    Ribosomes and messenger RNA assemble to polysomes during protein synthesis. Cryoelectron Tomography enables detection and identification of large macromolecular complexes under physiological conditions making the method uniquely suitable to study the supercomplexes that govern translation of mRNA into proteins. Here, we describe a method for automated assignment of polysomes in Cryoelectron tomograms using the positions and orientations of ribosomes, as localized by template matching on tomographic data, as input. On the basis of a training dataset of expert-curated polysomes in Cryoelectron tomograms, we define the relative 3D arrangements of neighboring ribosomes in polysomes. This prior distribution is used in a probabilistic framework for polysome assignment: the localized ribosomes from a tomogram are represented as a graph of which the edge weights are defined by the prior distribution. A Markov Random Field is embedded on the graph structure, and a message-passing algorithm is used to infer a polysome-label for each ribosome, i.e., to cluster ribosomes into polysomes. The performance of the method is assessed based on simulated tomograms and experimental tomograms indicating that polysome detection is reliable for typical signal-to-noise ratios of Cryoelectron tomograms.