scholarly journals In situ and high-resolution Cryo-EM structure of the Type VI secretion membrane complex

2018 ◽  
Author(s):  
Chiara Rapisarda ◽  
Yassine Cherrak ◽  
Romain Kooger ◽  
Victoria Schmidt ◽  
Riccardo Pellarin ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Electron Tomography ◽  
Type Vi Secretion System ◽  
Extracellular Medium ◽  
Bacterial Membranes ◽  
Membrane Complex ◽  
Type Vi Secretion ◽  
And Function

AbstractBacteria have evolved macromolecular machineries that secrete effectors and toxins to survive and thrive in diverse environments. The type VI secretion system (T6SS) is a contractile machine that is related to Myoviridae phages. The T6SS is composed of a baseplate that contains a spike onto which an inner tube is built, surrounded by a contractile sheath. Unlike phages that are released to and act in the extracellular medium, the T6SS is an intracellular machine inserted in the bacterial membranes by a trans-envelope complex. This membrane complex (MC) comprises three proteins: TssJ, TssL and TssM. We previously reported the low-resolution negative stain electron microscopy structure of the enteroaggregative Escherichia coli MC and proposed a rotational 5-fold symmetry with a TssJ:TssL:TssM stoichiometry of 2:2:2. Here, cryo-electron tomography analysis of the T6SS MC confirmed the 5-fold symmetry in situ and identified the regions of the structure that insert into the bacterial membranes. A high resolution model obtained by single particle cryo-electron microscopy reveals its global architecture and highlights new features: five additional copies of TssJ, yielding a TssJ:TssL:TssM stoichiometry of 3:2:2, a 11-residue loop in TssM, protruding inside the lumen of the MC and constituting a functionally important periplasmic gate, and hinge regions. Based on these data, we revisit the model on the mechanism of action of the MC during T6SS assembly and function.

scholarly journals In situ and high‐resolution cryo‐ EM structure of a bacterial type VI secretion system membrane complex

2019 ◽  
Vol 38 (10) ◽  
Author(s):  
Chiara Rapisarda ◽  
Yassine Cherrak ◽  
Romain Kooger ◽  
Victoria Schmidt ◽  
Riccardo Pellarin ◽  
...  
Keyword(s):  
High Resolution ◽  
Secretion System ◽  
Type Vi Secretion System ◽  
Membrane Complex ◽  
Type Vi Secretion ◽  
Bacterial Type

scholarly journals In situ Microfluidic Cryofixation for Cryo Focused Ion Beam Milling and Cryo Electron Tomography

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Marie Fuest ◽  
Miroslava Schaffer ◽  
Giovanni Marco Nocera ◽  
Rodrigo I. Galilea-Kleinsteuber ◽  
Jan-Erik Messling ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Light Microscope ◽  
Focused Ion Beam ◽  
Electron Tomography ◽  
Ion Beam ◽  
Light And Electron Microscopy ◽  
Live Imaging ◽  
Cryo Electron Tomography

AbstractWe present a microfluidic platform for studying structure-function relationships at the cellular level by connecting video rate live cell imaging with in situ microfluidic cryofixation and cryo-electron tomography of near natively preserved, unstained specimens. Correlative light and electron microscopy (CLEM) has been limited by the time required to transfer live cells from the light microscope to dedicated cryofixation instruments, such as a plunge freezer or high-pressure freezer. We recently demonstrated a microfluidic based approach that enables sample cryofixation directly in the light microscope with millisecond time resolution, a speed improvement of up to three orders of magnitude. Here we show that this cryofixation method can be combined with cryo-electron tomography (cryo-ET) by using Focused Ion Beam milling at cryogenic temperatures (cryo-FIB) to prepare frozen hydrated electron transparent sections. To make cryo-FIB sectioning of rapidly frozen microfluidic channels achievable, we developed a sacrificial layer technique to fabricate microfluidic devices with a PDMS bottom wall <5 µm thick. We demonstrate the complete workflow by rapidly cryo-freezing Caenorhabditis elegans roundworms L1 larvae during live imaging in the light microscope, followed by cryo-FIB milling and lift out to produce thin, electron transparent sections for cryo-ET imaging. Cryo-ET analysis of initial results show that the structural preservation of the cryofixed C. elegans was suitable for high resolution cryo-ET work. The combination of cryofixation during live imaging enabled by microfluidic cryofixation with the molecular resolution capabilities of cryo-ET offers an exciting avenue to further advance space-time correlative light and electron microscopy (st-CLEM) for investigation of biological processes at high resolution in four dimensions.

mRNA localization by Electron microscopic in situ hybridization

1991 ◽  
Vol 49 ◽  
pp. 430-431
Author(s):  
J. A. Pollock ◽  
M. Martone ◽  
T. Deerinck ◽  
M. H. Ellisman
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Nucleic Acids ◽  
Recombinant Dna ◽  
Light And Electron Microscopy ◽  
Electron Microscopic ◽  
High Voltage Electron Microscopy ◽  
Functional Sites ◽  
Voltage Electron

Localization of specific proteins in cells by both light and electron microscopy has been facilitate by the availability of antibodies that recognize unique features of these proteins. High resolution localization studies conducted over the last 25 years have allowed biologists to study the synthesis, translocation and ultimate functional sites for many important classes of proteins. Recently, recombinant DNA techniques in molecular biology have allowed the production of specific probes for localization of nucleic acids by “in situ” hybridization. The availability of these probes potentially opens a new set of questions to experimental investigation regarding the subcellular distribution of specific DNA's and RNA's. Nucleic acids have a much lower “copy number” per cell than a typical protein, ranging from one copy to perhaps several thousand. Therefore, sensitive, high resolution techniques are required. There are several reasons why Intermediate Voltage Electron Microscopy (IVEM) and High Voltage Electron Microscopy (HVEM) are most useful for localization of nucleic acids in situ.

Ultrastructural analysis of the spatial distribution of MRNA

1992 ◽  
Vol 50 (1) ◽  
pp. 552-553
Author(s):  
Gary Bassell ◽  
Robert H. Singer
Keyword(s):  
Electron Microscopy ◽  
Spatial Distribution ◽  
In Situ Hybridization ◽  
High Resolution ◽  
Nucleic Acids ◽  
Colloidal Gold ◽  
Dna Probe ◽  
Nucleotide Analogs ◽  
Gold Particles

We have been investigating the spatial distribution of nucleic acids intracellularly using in situ hybridization. The use of non-isotopic nucleotide analogs incorporated into the DNA probe allows the detection of the probe at its site of hybridization within the cell. This approach therefore is compatible with the high resolution available by electron microscopy. Biotinated or digoxigenated probe can be detected by antibodies conjugated to colloidal gold. Because mRNA serves as a template for the probe fragments, the colloidal gold particles are detected as arrays which allow it to be unequivocally distinguished from background.

Crystal Structures of Fragments D and Double-D from Fibrinogen and Fibrin

1999 ◽  
Vol 82 (08) ◽  
pp. 271-276 ◽  
Author(s):  
Glen Spraggon ◽  
Stephen Everse ◽  
Russell Doolittle
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Crystal Structures ◽  
Structure And Function ◽  
X Ray ◽  
Long Period ◽  
And Function

IntroductionAfter a long period of anticipation,1 the last two years have witnessed the first high-resolution x-ray structures of fragments from fibrinogen and fibrin.2-7 The results confirmed many aspects of fibrinogen structure and function that had previously been inferred from electron microscopy and biochemistry and revealed some unexpected features. Several matters have remained stubbornly unsettled, however, and much more work remains to be done. Here, we review several of the most significant findings that have accompanied the new x-ray structures and discuss some of the problems of the fibrinogen-fibrin conversion that remain unresolved. * Abbreviations: GPR—Gly-Pro-Arg-derivatives; GPRPam—Gly-Pro-Arg-Pro-amide; GHRPam—Gly-His-Arg-Pro-amide

High-Resolution Resistance Mapping in SEM

2018 ◽  
Author(s):  
Grigore Moldovan ◽  
Wolfgang Joachimi ◽  
Guillaume Boetsch ◽  
Jörg Jatzkowski ◽  
Frank Altman
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
High Resolution ◽  
Reference Structure ◽  
Total Resistance ◽  
Embedded Electronics ◽  
Improved Performance ◽  
Mapping Techniques ◽  
Scanning Electron

Abstract This work presents advanced resistance mapping techniques based on Scanning Electron Microscopy (SEM) with nanoprobing systems and the related embedded electronics. Focus is placed on recent advances to reduce noise and increase speed, such as integration of dedicated in situ electronics into the nanoprobing platform, as well as an important transition from current-sensitive to voltagesensitive amplification. We show that it is now possible to record resistance maps with a resistance sensitivity in the 10W range, even when the total resistance of the mapped structures is in the range of 100W. A reference structure is used to illustrate the improved performance, and a lowresistance failure case is presented as an example of analysis made possible by these developments.

scholarly journals T6SS Mediated Stress Responses for Bacterial Environmental Survival and Host Adaptation

2021 ◽  
Vol 22 (2) ◽  
pp. 478
Author(s):  
Kai-Wei Yu ◽  
Peng Xue ◽  
Yang Fu ◽  
Liang Yang
Keyword(s):  
Protein Secretion ◽  
Stress Responses ◽  
Current Knowledge ◽  
Bacterial Species ◽  
Host Cells ◽  
Interspecies Interactions ◽  
Type Vi Secretion System ◽  
Membrane Complex ◽  
Type Vi Secretion ◽  
Bacterial Type

The bacterial type VI secretion system (T6SS) is a protein secretion apparatus widely distributed in Gram-negative bacterial species. Many bacterial pathogens employ T6SS to compete with the host and to coordinate the invasion process. The T6SS apparatus consists of a membrane complex and an inner tail tube-like structure that is surrounded by a contractile sheath and capped with a spike complex. A series of antibacterial or antieukaryotic effectors is delivered by the puncturing device consisting of a Hcp tube decorated by the VgrG/PAAR complex into the target following the contraction of the TssB/C sheath, which often leads to damage and death of the competitor and/or host cells. As a tool for protein secretion and interspecies interactions, T6SS can be triggered by many different mechanisms to respond to various physiological conditions. This review summarizes our current knowledge of T6SS in coordinating bacterial stress responses against the unfavorable environmental and host conditions.

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