scholarly journals Cellular and Structural Studies of Eukaryotic Cells by Cryo-Electron Tomography

Cells ◽  
2019 ◽  
Vol 8 (1) ◽  
pp. 57 ◽  
Author(s):  
Miriam Weber ◽  
Matthias Wojtynek ◽  
Ohad Medalia
Keyword(s):  
High Resolution ◽  
Sample Preparation ◽  
Electron Tomography ◽  
Eukaryotic Cell ◽  
Electron Microscopic ◽  
Macromolecular Assemblies ◽  
Cellular Processes ◽  
Physiological Processes ◽  
Technological Advances ◽  
Cryo Electron Tomography

The architecture of protein assemblies and their remodeling during physiological processes is fundamental to cells. Therefore, providing high-resolution snapshots of macromolecular complexes in their native environment is of major importance for understanding the molecular biology of the cell. Cellular structural biology by means of cryo-electron tomography (cryo-ET) offers unique insights into cellular processes at an unprecedented resolution. Recent technological advances have enabled the detection of single impinging electrons and improved the contrast of electron microscopic imaging, thereby significantly increasing the sensitivity and resolution. Moreover, various sample preparation approaches have paved the way to observe every part of a eukaryotic cell, and even multicellular specimens, under the electron beam. Imaging of macromolecular machineries at high resolution directly within their native environment is thereby becoming reality. In this review, we discuss several sample preparation and labeling techniques that allow the visualization and identification of macromolecular assemblies in situ, and demonstrate how these methods have been used to study eukaryotic cellular landscapes.

scholarly journals A guided approach for subtomogram averaging of challenging macromolecular assemblies

2020 ◽  
Author(s):  
Danielle Grotjahn ◽  
Saikat Chowdhury ◽  
Gabriel C. Lander
Keyword(s):  
Electron Tomography ◽  
A Priori ◽  
Three Dimensional ◽  
Structural Heterogeneity ◽  
Dimensional Structure ◽  
Diverse Range ◽  
Macromolecular Assemblies ◽  
Cryo Electron Tomography ◽  
Subtomogram Averaging

AbstractCryo-electron tomography is a powerful biophysical technique enabling three-dimensional visualization of complex biological systems. Macromolecular targets of interest identified within cryo-tomograms can be computationally extracted, aligned, and averaged to produce a better-resolved structure through a process called subtomogram averaging (STA). However, accurate alignment of macromolecular machines that exhibit extreme structural heterogeneity and conformational flexibility remains a significant challenge with conventional STA approaches. To expand the applicability of STA to a broader range of pleomorphic complexes, we developed a user-guided, focused refinement approach that can be incorporated into the standard STA workflow to facilitate the robust alignment of particularly challenging samples. We demonstrate that it is possible to align visually recognizable portions of multi-subunit complexes by providing a priori information regarding their relative orientations within cryo-tomograms, and describe how this strategy was applied to successfully elucidate the first three-dimensional structure of the dynein-dynactin motor protein complex bound to microtubules. Our approach expands the application of STA for solving a more diverse range of heterogeneous biological structures, and establishes a conceptual framework for the development of automated strategies to deconvolve the complexity of crowded cellular environments and improve in situ structure determination technologies.

scholarly journals An Economical, Portable Manual Cryogenic Plunge Freezer for the Preparation of Vitrified Biological Samples for Cryogenic Electron Microscopy

2020 ◽  
Vol 26 (3) ◽  
pp. 413-418
Author(s):  
Jamie S. Depelteau ◽  
Gert Koning ◽  
Wen Yang ◽  
Ariane Briegel
Keyword(s):  
Electron Microscopy ◽  
Electron Tomography ◽  
Particle Analysis ◽  
Bacterial Cells ◽  
Cellular Processes ◽  
Cryo Electron Tomography ◽  
Specialized Equipment ◽  
Improved Design ◽  
Commercial Equipment ◽  
Local Machine

AbstractVisualizing biological structures and cellular processes in their native state is a major goal of many scientific laboratories. In the past 20 years, the technique of preserving samples by vitrification has greatly expanded, specifically for use in cryogenic electron microscopy (cryo-EM). Here, we report on improvements in the design and use of a portable manual cryogenic plunge freezer that is intended for use in laboratories that are not equipped for the cryopreservation of samples. The construction of the instrument is economical, can be produced by a local machine shop without specialized equipment, and lowers the entry barriers for newcomers with a reliable alternative to costly commercial equipment. The improved design allows for successful freezing of isolated proteins for single particle analysis as well as bacterial cells for cryo-electron tomography. With this instrument, groups will be able to prepare vitreous samples whenever and wherever necessary, which can then be imaged at local or national cryo-EM facilities.

scholarly journals In situ cryo-electron tomography reveals filamentous actin within the microtubule lumen

2020 ◽  
Vol 219 (9) ◽  
Author(s):  
Danielle M. Paul ◽  
Judith Mantell ◽  
Ufuk Borucu ◽  
Jennifer Coombs ◽  
Katherine J. Surridge ◽  
...  
Keyword(s):  
Experimental Approach ◽  
Electron Tomography ◽  
Filamentous Actin ◽  
Significant Development ◽  
Cellular Processes ◽  
In Situ Study ◽  
Complex Interactions ◽  
Cryo Electron Tomography ◽  
And Migration

Microtubules and filamentous (F-) actin engage in complex interactions to drive many cellular processes from subcellular organization to cell division and migration. This is thought to be largely controlled by proteins that interface between the two structurally distinct cytoskeletal components. Here, we use cryo-electron tomography to demonstrate that the microtubule lumen can be occupied by extended segments of F-actin in small molecule–induced, microtubule-based, cellular projections. We uncover an unexpected versatility in cytoskeletal form that may prompt a significant development of our current models of cellular architecture and offer a new experimental approach for the in situ study of microtubule structure and contents.

scholarly journals In situ cryo-electron tomography reveals filamentous actin within the microtubule lumen

2019 ◽  
Author(s):  
Danielle M Paul ◽  
Judith Mantell ◽  
Ufuk Borucu ◽  
Jennifer Coombs ◽  
Katherine J Surridge ◽  
...  
Keyword(s):  
Experimental Approach ◽  
Electron Tomography ◽  
Filamentous Actin ◽  
Significant Development ◽  
Cellular Processes ◽  
Complex Interactions ◽  
Cryo Electron Tomography ◽  
And Migration ◽  
Microtubule Structure

AbstractMicrotubules and filamentous (F-) actin engage in complex interactions to drive many cellular processes from subcellular organisation to cell division and migration. This is thought to be largely controlled by proteins that interface between the two structurally distinct cytoskeletal components. Here, we use cryo-electron tomography to demonstrate that the microtubule lumen can be occupied by extended segments of F-actin in small-molecule induced, microtubule-based cellular projections. We uncover an unexpected versatility in cytoskeletal form that may prompt a significant development of our current models of cellular architecture and offer a new experimental approach for the in-situ study of microtubule structure and contents.

scholarly journals Visualizing cellular processes at the molecular level by cryo-electron tomography

2009 ◽  
Vol 123 (1) ◽  
pp. 7-12 ◽  
Author(s):  
K. Ben-Harush ◽  
T. Maimon ◽  
I. Patla ◽  
E. Villa ◽  
O. Medalia
Keyword(s):  
Molecular Level ◽  
Electron Tomography ◽  
Cellular Processes ◽  
Cryo Electron Tomography

scholarly journals Development of an Intact Mammalian System for High-resolution Imaging by Cryo-Electron Tomography

2019 ◽  
Vol 25 (S2) ◽  
pp. 1306-1307
Author(s):  
Xia Li ◽  
Donghyun Park ◽  
Yunjie Chang ◽  
Abhijith Radhakrishnan ◽  
Hangjun Wu ◽  
...  
Keyword(s):  
High Resolution ◽  
Electron Tomography ◽  
High Resolution Imaging ◽  
Cryo Electron Tomography ◽  
Resolution Imaging ◽  
Mammalian System

scholarly journals Three-Dimensional Electron Microscopic Imaging of Membrane Invaginations in Escherichia coli Overproducing the Chemotaxis Receptor Tsr

2004 ◽  
Vol 186 (15) ◽  
pp. 5052-5061 ◽  
Author(s):  
Jonathan Lefman ◽  
Peijun Zhang ◽  
Teruhisa Hirai ◽  
Robert M. Weis ◽  
Jemma Juliani ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Bacterial Chemotaxis ◽  
Molecular Architecture ◽  
Electron Microscopic ◽  
E Coli ◽  
Macromolecular Assemblies ◽  
Membrane Networks ◽  
Receptor Clusters

ABSTRACT Electron tomography is a powerful method for determining the three-dimensional structures of large macromolecular assemblies, such as cells, organelles, and multiprotein complexes, when crystallographic averaging methods are not applicable. Here we used electron tomographic imaging to determine the molecular architecture of Escherichia coli cells engineered to overproduce the bacterial chemotaxis receptor Tsr. Tomograms constructed from fixed, cryosectioned cells revealed that overproduction of Tsr led to formation of an extended internal membrane network composed of stacks and extended tubular structures. We present an interpretation of the tomogram in terms of the packing arrangement of Tsr using constraints derived from previous X-ray and electron-crystallographic studies of receptor clusters. Our results imply that the interaction between the cytoplasmic ends of Tsr is likely to stabilize the presence of the membrane networks in cells overproducing Tsr. We propose that membrane invaginations that are potentially capable of supporting axial interactions between receptor clusters in apposing membranes could also be present in wild-type E. coli and that such receptor aggregates could play an important role in signal transduction during bacterial chemotaxis.

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