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.

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

2020 ◽  
Author(s):  
Natalya Leneva ◽  
Oleksiy Kovtun ◽  
Dustin R. Morado ◽  
John A. G. Briggs ◽  
David J. Owen
Keyword(s):  
Electron Tomography ◽  
Membrane Curvature ◽  
Structural Basis ◽  
Cellular Transport ◽  
Structural Explanation ◽  
Sorting Nexins ◽  
Membrane Recruitment ◽  
Sorting Nexin ◽  
Cellular Processes ◽  
Membrane Bending

AbstractRetromer is a master regulator of cargo retrieval from endosomes, which is critical for many cellular processes including signalling, immunity, neuroprotection and virus infection. To function in different trafficking routes, retromer core (VPS26/VPS29/VPS35) assembles with a range of sorting nexins to generate tubular carriers and incorporate assorted cargoes. We elucidate the structural basis of membrane remodelling and coupled cargo recognition by assembling metazoan and fungal retromer core trimers on cargo-containing membranes with sorting nexin adaptor SNX3 and determining their structures using cryo-electron tomography. Assembly leads to formation of tubular carriers in the absence of canonical membrane curvature drivers. Interfaces in the retromer coat provide a structural explanation for Parkinson’s disease-linked mutations. We demonstrate that retromer core trimer forms an invariant, evolutionarily-conserved scaffold that can incorporate different auxiliary membrane adaptors by changing its mode of membrane recruitment, so modulating membrane bending and cargo incorporation and thereby allowing retromer to traffic assorted cargoes along different cellular transport routes.

scholarly journals Tubulin glycylation controls axonemal dynein activity, flagellar beat, and male fertility

Science ◽  
2021 ◽  
Vol 371 (6525) ◽  
pp. eabd4914
Author(s):  
Sudarshan Gadadhar ◽  
Gonzalo Alvarez Viar ◽  
Jan Niklas Hansen ◽  
An Gong ◽  
Aleksandr Kostarev ◽  
...  
Keyword(s):  
Posttranslational Modifications ◽  
Male Fertility ◽  
Electron Tomography ◽  
Structural Basis ◽  
Cellular Functions ◽  
Flagellar Beat ◽  
Cryo Electron Tomography ◽  
Axonemal Dynein ◽  
Dynein Arms ◽  
Cilia And Flagella

Posttranslational modifications of the microtubule cytoskeleton have emerged as key regulators of cellular functions, and their perturbations have been linked to a growing number of human pathologies. Tubulin glycylation modifies microtubules specifically in cilia and flagella, but its functional and mechanistic roles remain unclear. In this study, we generated a mouse model entirely lacking tubulin glycylation. Male mice were subfertile owing to aberrant beat patterns of their sperm flagella, which impeded the straight swimming of sperm cells. Using cryo–electron tomography, we showed that lack of glycylation caused abnormal conformations of the dynein arms within sperm axonemes, providing the structural basis for the observed dysfunction. Our findings reveal the importance of microtubule glycylation for controlled flagellar beating, directional sperm swimming, and male fertility.

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 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 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

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 Cryo-electron tomography reveals that dynactin recruits a team of dyneins for processive motility

2017 ◽  
Author(s):  
Danielle A. Grotjahn ◽  
Saikat Chowdhury ◽  
Yiru Xu ◽  
Richard J. McKenney ◽  
Trina A. Schroer ◽  
...  
Keyword(s):  
Structural Information ◽  
Electron Tomography ◽  
Force Production ◽  
Cytoplasmic Dynein ◽  
Dimensional Structure ◽  
Cellular Transport ◽  
3 Dimensional ◽  
Dynein Motor ◽  
Cryo Electron Tomography ◽  
Dynactin Complex

AbstractA key player in the intracellular trafficking network is cytoplasmic dynein, a protein complex that transports molecular cargo along microtubule tracks. It has been shown that vertebrate dynein’s movement becomes strikingly enhanced upon interacting with a cofactor named dynactin and one of several cargo-adapters, such as BicaudalD2. However, the mechanisms responsible for this increase in transport efficiency are not well understood, largely due to a lack of structural information. We used cryo-electron tomography to visualize the first 3-dimensional structure of the intact dynein-dynactin complex bound to microtubules. Our structure reveals that the dynactin-cargo-adapter complex recruits and binds to two dimeric cytoplasmic dyneins. Interestingly, the dynein motor organization closely resembles that of axonemal dynein, suggesting that cytoplasmic dynein and axonemal dyneins may utilize similar mechanisms to coordinate multiple motors. We propose that grouping dyneins onto a single dynactin scaffold promotes collective force production as well as unidirectional processive motility. These findings provide a structural platform that facilitates a deeper biochemical and biophysical understanding of dynein regulation and cellular transport.

scholarly journals SARS-CoV-2 structure and replication characterized by in situ cryo-electron tomography

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Steffen Klein ◽  
Mirko Cortese ◽  
Sophie L. Winter ◽  
Moritz Wachsmuth-Melm ◽  
Christopher J. Neufeldt ◽  
...  
Keyword(s):  
Viral Replication ◽  
Electron Tomography ◽  
Membrane Vesicles ◽  
Membrane Curvature ◽  
Virion Assembly ◽  
Ribonucleoprotein Complexes ◽  
Cryo Electron Tomography ◽  
Membrane Bending ◽  
Subtomogram Averaging

AbstractSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the COVID19 pandemic, is a highly pathogenic β-coronavirus. As other coronaviruses, SARS-CoV-2 is enveloped, replicates in the cytoplasm and assembles at intracellular membranes. Here, we structurally characterize the viral replication compartment and report critical insights into the budding mechanism of the virus, and the structure of extracellular virions close to their native state by in situ cryo-electron tomography and subtomogram averaging. We directly visualize RNA filaments inside the double membrane vesicles, compartments associated with viral replication. The RNA filaments show a diameter consistent with double-stranded RNA and frequent branching likely representing RNA secondary structures. We report that assembled S trimers in lumenal cisternae do not alone induce membrane bending but laterally reorganize on the envelope during virion assembly. The viral ribonucleoprotein complexes (vRNPs) are accumulated at the curved membrane characteristic for budding sites suggesting that vRNP recruitment is enhanced by membrane curvature. Subtomogram averaging shows that vRNPs are distinct cylindrical assemblies. We propose that the genome is packaged around multiple separate vRNP complexes, thereby allowing incorporation of the unusually large coronavirus genome into the virion while maintaining high steric flexibility between the vRNPs.

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