scholarly journals Revealing the polarity of actin filaments by cryo-electron tomography

2020 ◽  
Author(s):  
Bruno Martins ◽  
Simona Sorrentino ◽  
Wen-Lu Chung ◽  
Meltem Tatli ◽  
Ohad Medalia ◽  
...  
Keyword(s):  
Actin Filaments ◽  
Electron Tomography ◽  
Signal To Noise Ratio ◽  
Focal Adhesions ◽  
Mechanical Forces ◽  
Structural Imaging ◽  
High Frequencies ◽  
Cellular Processes ◽  
Cryo Electron Tomography ◽  
Adhesion Site

SummaryThe 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.

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 Current data processing strategies for cryo-electron tomography and subtomogram averaging

2021 ◽  
Vol 478 (10) ◽  
pp. 1827-1845
Author(s):  
Euan Pyle ◽  
Giulia Zanetti
Keyword(s):  
Data Processing ◽  
Electron Tomography ◽  
Signal To Noise Ratio ◽  
Three Dimensional ◽  
Current Data ◽  
Processing Strategies ◽  
Cryo Electron Tomography ◽  
Subtomogram Averaging ◽  
The Individual

Cryo-electron tomography (cryo-ET) can be used to reconstruct three-dimensional (3D) volumes, or tomograms, from a series of tilted two-dimensional images of biological objects in their near-native states in situ or in vitro. 3D subvolumes, or subtomograms, containing particles of interest can be extracted from tomograms, aligned, and averaged in a process called subtomogram averaging (STA). STA overcomes the low signal to noise ratio within the individual subtomograms to generate structures of the particle(s) of interest. In recent years, cryo-ET with STA has increasingly been capable of reaching subnanometer resolution due to improvements in microscope hardware and data processing strategies. There has also been an increase in the number and quality of software packages available to process cryo-ET data with STA. In this review, we describe and assess the data processing strategies available for cryo-ET data and highlight the recent software developments which have enabled the extraction of high-resolution information from cryo-ET datasets.

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 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 The elusive actin cytoskeleton of a green alga expressing both conventional and divergent actins

2019 ◽  
Vol 30 (22) ◽  
pp. 2827-2837 ◽  
Author(s):  
Evan W. Craig ◽  
David M. Mueller ◽  
Brae M. Bigge ◽  
Miroslava Schaffer ◽  
Benjamin D. Engel ◽  
...  
Keyword(s):  
Green Alga ◽  
Actin Filaments ◽  
Electron Tomography ◽  
Filamentous Actin ◽  
Cellular Processes ◽  
Cellular Environment ◽  
Actin Structure ◽  
Actin Isoform ◽  
Labeling Method

The green alga Chlamydomonas reinhardtii is a leading model system to study photosynthesis, cilia, and the generation of biological products. The cytoskeleton plays important roles in all of these cellular processes, but to date, the filamentous actin network within Chlamydomonas has remained elusive. By optimizing labeling conditions, we can now visualize distinct linear actin filaments at the posterior of the nucleus in both live and fixed vegetative cells. Using in situ cryo-electron tomography, we confirmed this localization by directly imaging actin filaments within the native cellular environment. The fluorescently labeled structures are sensitive to the depolymerizing agent latrunculin B (Lat B), demonstrating the specificity of our optimized labeling method. Interestingly, Lat B treatment resulted in the formation of a transient ring-like filamentous actin structure around the nucleus. The assembly of this perinuclear ring is dependent upon a second actin isoform, NAP1, which is strongly up-regulated upon Lat B treatment and is insensitive to Lat B–induced depolymerization. Our study combines orthogonal strategies to provide the first detailed visual characterization of filamentous actins in Chlamydomonas, allowing insights into the coordinated functions of two actin isoforms expressed within the same cell.

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 Signal to Noise Ratio Mapping for Cryo-Electron Tomography

2009 ◽  
Vol 15 (S2) ◽  
pp. 568-569
Author(s):  
C Renken
Keyword(s):  
Electron Tomography ◽  
Signal To Noise Ratio ◽  
Signal To Noise ◽  
Cryo Electron Tomography ◽  
Noise Ratio

Extended abstract of a paper presented at Microscopy and Microanalysis 2009 in Richmond, Virginia, USA, July 26 – July 30, 2009

Cryo-electron tomography: gaining insight into cellular processes by structural approaches

2011 ◽  
Vol 21 (5) ◽  
pp. 670-677 ◽  
Author(s):  
Tal Yahav ◽  
Tal Maimon ◽  
Einat Grossman ◽  
Idit Dahan ◽  
Ohad Medalia
Keyword(s):  
Electron Tomography ◽  
Cellular Processes ◽  
Cryo Electron Tomography ◽  
Insight Into ◽  
Gaining Insight

scholarly journals Architecture and mechanism of metazoan retromer:SNX3 tubular coat assembly

2021 ◽  
Vol 7 (13) ◽  
pp. eabf8598 ◽  
Author(s):  
Natalya Leneva ◽  
Oleksiy Kovtun ◽  
Dustin R. Morado ◽  
John A. G. Briggs ◽  
David J. Owen
Keyword(s):  
Electron Tomography ◽  
Structural Basis ◽  
Cellular Transport ◽  
Sorting Nexins ◽  
Membrane Recruitment ◽  
Sorting Nexin ◽  
Cellular Processes ◽  
Cryo Electron Tomography ◽  
Membrane Tubulation ◽  
Membrane Bending

Retromer is a master regulator of cargo retrieval from endosomes, which is critical for many cellular processes including signaling, immunity, neuroprotection, and virus infection. The retromer core (VPS26/VPS29/VPS35) is present on cargo-transporting, tubular carriers along with a range of sorting nexins. Here, we elucidate the structural basis of membrane tubulation and coupled cargo recognition by metazoan and fungal retromer coats assembled with the non–Bin1/Amphiphysin/Rvs (BAR) sorting nexin SNX3 using cryo–electron tomography. The retromer core retains its arched, scaffolding structure but changes its mode of membrane recruitment when assembled with different SNX adaptors, allowing cargo recognition at subunit interfaces. Thus, membrane bending and cargo incorporation can be modulated to allow retromer to traffic cargoes along different cellular transport routes.

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