scholarly journals In situ architecture of neuronal α-Synuclein inclusions

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Victoria A. Trinkaus ◽  
Irene Riera-Tur ◽  
Antonio Martínez-Sánchez ◽  
Felix J. B. Bäuerlein ◽  
Qiang Guo ◽  
...  
Keyword(s):  
Detailed Analysis ◽  
Neurodegenerative Disorders ◽  
Electron Tomography ◽  
Cellular Interaction ◽  
Molecular Architecture ◽  
Membrane Interactions ◽  
Intracellular Membranes ◽  
Cryo Electron Tomography ◽  
Cellular Organelles

AbstractThe molecular architecture of α-Synuclein (α-Syn) inclusions, pathognomonic of various neurodegenerative disorders, remains unclear. α-Syn inclusions were long thought to consist mainly of α-Syn fibrils, but recent reports pointed to intracellular membranes as the major inclusion component. Here, we use cryo-electron tomography (cryo-ET) to image neuronal α-Syn inclusions in situ at molecular resolution. We show that inclusions seeded by α-Syn aggregates produced recombinantly or purified from patient brain consist of α-Syn fibrils crisscrossing a variety of cellular organelles. Using gold-labeled seeds, we find that aggregate seeding is predominantly mediated by small α-Syn fibrils, from which cytoplasmic fibrils grow unidirectionally. Detailed analysis of membrane interactions revealed that α-Syn fibrils do not contact membranes directly, and that α-Syn does not drive membrane clustering. Altogether, we conclusively demonstrate that neuronal α-Syn inclusions consist of α-Syn fibrils intermixed with membranous organelles, and illuminate the mechanism of aggregate seeding and cellular interaction.

scholarly journals Molecular architecture of inner dynein arms in situ in Chlamydomonas reinhardtii flagella

2008 ◽  
Vol 183 (5) ◽  
pp. 923-932 ◽  
Author(s):  
Khanh Huy Bui ◽  
Hitoshi Sakakibara ◽  
Tandis Movassagh ◽  
Kazuhiro Oiwa ◽  
Takashi Ishikawa
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Molecular Architecture ◽  
Heavy Chains ◽  
Distal Side ◽  
Cryo Electron Tomography ◽  
Dynein Arms

The inner dynein arm regulates axonemal bending motion in eukaryotes. We used cryo-electron tomography to reconstruct the three-dimensional structure of inner dynein arms from Chlamydomonas reinhardtii. All the eight different heavy chains were identified in one 96-nm periodic repeat, as expected from previous biochemical studies. Based on mutants, we identified the positions of the AAA rings and the N-terminal tails of all the eight heavy chains. The dynein f dimer is located close to the surface of the A-microtubule, whereas the other six heavy chain rings are roughly colinear at a larger distance to form three dyads. Each dyad consists of two heavy chains and has a corresponding radial spoke or a similar feature. In each of the six heavy chains (dynein a, b, c, d, e, and g), the N-terminal tail extends from the distal side of the ring. To interact with the B-microtubule through stalks, the inner-arm dyneins must have either different handedness or, more probably, the opposite orientation of the AAA rings compared with the outer-arm dyneins.

scholarly journals In situ architecture of neuronal α-Synuclein inclusions

2020 ◽  
Author(s):  
Victoria A. Trinkaus ◽  
Irene Riera-Tur ◽  
Antonio Martínez-Sánchez ◽  
Felix J.B. Bäuerlein ◽  
Qiang Guo ◽  
...  
Keyword(s):  
Amyloid Fibrils ◽  
Electron Tomography ◽  
Structural Flexibility ◽  
Multiple Systems ◽  
Cryo Electron Tomography ◽  
Multiple Systems Atrophy ◽  
The Brain ◽  
Cellular Organelles

Summaryα-Synuclein (α-Syn) aggregation is a hallmark of devastating neurodegenerative disorders including Parkinson’s disease (PD) and multiple systems atrophy (MSA)1,2. α-Syn aggregates spread throughout the brain during disease progression2, suggesting mechanisms of intercellular seeding. Formation of α-Syn amyloid fibrils is observed in vitro3,4 and fibrillar α-Syn has been purified from patient brains5,6, but recent reports questioned whether disease-relevant α-Syn aggregates are fibrillar in structure7-9. Here we use cryo-electron tomography (cryo-ET) to image neuronal Lewy body-like α-Syn inclusions in situ at molecular resolution. We show that the inclusions consist of α-Syn fibrils crisscrossing a variety of cellular organelles such as the endoplasmic reticulum (ER), mitochondria and autophagic structures, without interacting with membranes directly. Neuronal inclusions seeded by recombinant or MSA patient-derived α-Syn aggregates have overall similar architecture, although MSA-seeded fibrils show higher structural flexibility. Using gold-labeled seeds we find that aggregate nucleation is predominantly mediated by α-Syn oligomers, with fibrils growing unidirectionally from the seed. Our results conclusively demonstrate that neuronal α-Syn inclusions contain α-Syn fibrils intermixed with cellular membranes, and illuminate the mechanism of aggregate nucleation.

scholarly journals High Throughput Cryo-Electron Tomography: Towards Molecular Architecture of Spirochetal Flagellar Motor in situ

2010 ◽  
Vol 16 (S2) ◽  
pp. 1070-1071
Author(s):  
J Liu ◽  
S Norris
Keyword(s):  
High Throughput ◽  
Electron Tomography ◽  
Flagellar Motor ◽  
Molecular Architecture ◽  
Cryo Electron Tomography

Extended abstract of a paper presented at Microscopy and Microanalysis 2010 in Portland, Oregon, USA, August 1 – August 5, 2010.

scholarly journals Integrated Cryo-Correlative Microscopy for Targeted Structural Investigation In Situ

2021 ◽  
Vol 29 (6) ◽  
pp. 20-25
Author(s):  
Marit Smeets ◽  
Anna Bieber ◽  
Cristina Capitanio ◽  
Oda Schioetz ◽  
Thomas van der Heijden ◽  
...  
Keyword(s):  
Focused Ion Beam ◽  
Electron Tomography ◽  
Ion Beam ◽  
Building Blocks ◽  
Quality Data ◽  
Cellular Interaction ◽  
High Quality Data ◽  
Cryo Electron Tomography ◽  
Sample Transfer

Abstract:Cryo-electron tomography (cryo-ET) has the potential to revolutionize our understanding of the building blocks of life since it provides the unique opportunity to study molecules and membrane architectures in the context of cellular interaction. In particular, the combination of fluorescence imaging with focused ion beam (FIB) milling allows the targeting of specific structures in thick cellular samples by preparing thin lamellae that contain a specific fluorescence marker. This technique has conventionally been time-consuming, as it requires sample transfer to multiple microscopes and presents several technical challenges that currently limit its success. Here we describe METEOR, a FIB-integrated microscopy solution that streamlines the correlative cryo-ET workflow. It protects the sample from ice contamination by minimizing handling steps, thus increasing the likelihood of high-quality data. It also allows for monitoring of the milling procedure to ensure the molecule of interest is captured and can then be imaged by cryo-ET.

Cryo electron tomography of vitrified fibroblasts: Microtubule plus ends in situ

2008 ◽  
Vol 161 (3) ◽  
pp. 459-468 ◽  
Author(s):  
Roman I. Koning ◽  
Sandra Zovko ◽  
Montserrat Bárcena ◽  
Gert T. Oostergetel ◽  
Henk K. Koerten ◽  
...  
Keyword(s):  
Electron Tomography ◽  
Cryo Electron Tomography

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 Structural insights into flagellar stator–rotor interactions

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Yunjie Chang ◽  
Ki Hwan Moon ◽  
Xiaowei Zhao ◽  
Steven J Norris ◽  
MD A Motaleb ◽  
...  
Keyword(s):  
Conformational Change ◽  
High Speed ◽  
Electron Tomography ◽  
Deletion Mutants ◽  
Flagellar Motor ◽  
Wild Type ◽  
Cryo Electron Tomography ◽  
Flagellar Filament ◽  
Structural Insights

The bacterial flagellar motor is a molecular machine that can rotate the flagellar filament at high speed. The rotation is generated by the stator–rotor interaction, coupled with an ion flux through the torque-generating stator. Here we employed cryo-electron tomography to visualize the intact flagellar motor in the Lyme disease spirochete, Borrelia burgdorferi. By analyzing the motor structures of wild-type and stator-deletion mutants, we not only localized the stator complex in situ, but also revealed the stator–rotor interaction at an unprecedented detail. Importantly, the stator–rotor interaction induces a conformational change in the flagella C-ring. Given our observation that a non-motile mutant, in which proton flux is blocked, cannot generate the similar conformational change, we propose that the proton-driven torque is responsible for the conformational change required for flagellar rotation.

In situ Structure of Viral RNA by Cryo Electron Tomography with Volta Phase Plate, Energy Filtering and Direct Electron Counting

2016 ◽  
Vol 22 (S3) ◽  
pp. 74-75
Author(s):  
Z. Hong Zhou ◽  
Wong H. Hui ◽  
Jiayan Zhang ◽  
Ivo Atanasov ◽  
Cristina C. Celma ◽  
...  
Keyword(s):  
Electron Tomography ◽  
Viral Rna ◽  
Phase Plate ◽  
Energy Filtering ◽  
Electron Counting ◽  
Cryo Electron Tomography
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