Amyloid-like aggregating proteins cause lysosomal defects in neurons via gain-of-function toxicity

The autophagy-lysosomal pathway is impaired in many neurodegenerative diseases characterized by protein aggregation, but the link between aggregation and lysosomal dysfunction remains poorly understood. Here, we combine cryo-electron tomography, proteomics, and cell biology studies to investigate the effects of protein aggregates in primary neurons. We use artificial amyloid-like β-sheet proteins (β proteins) to focus on the gain-of-function aspect of aggregation. These proteins form fibrillar aggregates and cause neurotoxicity. We show that late stages of autophagy are impaired by the aggregates, resulting in lysosomal alterations reminiscent of lysosomal storage disorders. Mechanistically, β proteins interact with and sequester AP-3 μ1, a subunit of the AP-3 adaptor complex involved in protein trafficking to lysosomal organelles. This leads to destabilization of the AP-3 complex, missorting of AP-3 cargo, and lysosomal defects. Restoring AP-3μ1 expression ameliorates neurotoxicity caused by β proteins. Altogether, our results highlight the link between protein aggregation, lysosomal impairments, and neurotoxicity.

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Amyloid-like aggregates cause lysosomal defects in neurons via gain-of-function toxicity

10.1101/2019.12.16.877431 ◽

2019 ◽

Author(s):

Tillman Schaefer ◽

Irene Riera-Tur ◽

Daniel Hornburg ◽

Archana Mishra ◽

Lorena Fernández-Mosquera ◽

...

Keyword(s):

Neurodegenerative Diseases ◽

Protein Aggregation ◽

Protein Trafficking ◽

Electron Tomography ◽

Lysosomal Storage ◽

Gain Of Function ◽

Β Protein ◽

Protein Interactome ◽

A Subunit

AbstractThe autophagy-lysosomal pathway is impaired in many neurodegenerative diseases characterized by protein aggregation, but the link between aggregation and lysosomal dysfunction remains poorly understood. Here, we use artificial amyloid-like β-sheet proteins (β proteins) to investigate the gain-of-function effects of protein aggregation in primary neurons. We show that β proteins form fibrillar aggregates and cause neurotoxicity. Cryo-electron tomography reveals lysosomal alterations reminiscent of lysosomal storage disorders. Mass spectrometry-based analysis of the β protein interactome shows that β proteins sequester AP-3μ1, a subunit of the AP-3 adaptor complex involved in protein trafficking to lysosomal organelles. Importantly, restoring AP-3μ1 expression ameliorates neurotoxicity caused by β proteins. Our results point to lysosomes as particularly vulnerable organelles in neurodegenerative diseases, and emphasize the role of toxic gain-of-function of protein aggregates in lysosomal defects.

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The molecular architecture of engulfment during Bacillus subtilis sporulation

eLife ◽

10.7554/elife.45257 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 13

Author(s):

Kanika Khanna ◽

Javier Lopez-Garrido ◽

Ziyi Zhao ◽

Reika Watanabe ◽

Yuan Yuan ◽

...

Keyword(s):

Bacillus Subtilis ◽

Mother Cell ◽

Cell Biology ◽

Focused Ion Beam ◽

High Spatial Resolution ◽

Electron Tomography ◽

Ion Beam ◽

Molecular Architecture ◽

Native State ◽

Cryo Electron Tomography

The study of bacterial cell biology is limited by difficulties in visualizing cellular structures at high spatial resolution within their native milieu. Here, we visualize Bacillus subtilis sporulation using cryo-electron tomography coupled with cryo-focused ion beam milling, allowing the reconstruction of native-state cellular sections at molecular resolution. During sporulation, an asymmetrically-positioned septum generates a larger mother cell and a smaller forespore. Subsequently, the mother cell engulfs the forespore. We show that the septal peptidoglycan is not completely degraded at the onset of engulfment. Instead, the septum is uniformly and only slightly thinned as it curves towards the mother cell. Then, the mother cell membrane migrates around the forespore in tiny finger-like projections, whose formation requires the mother cell SpoIIDMP protein complex. We propose that a limited number of SpoIIDMP complexes tether to and degrade the peptidoglycan ahead of the engulfing membrane, generating an irregular membrane front.

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The molecular architecture of engulfment during Bacillussubtilis sporulation

10.1101/498220 ◽

2018 ◽

Author(s):

Kanika Khanna ◽

Javier Lopez-Garrido ◽

Ziyi Zhao ◽

Reika Watanabe ◽

Yuan Yuan ◽

...

Keyword(s):

Mother Cell ◽

Cell Biology ◽

Focused Ion Beam ◽

Electron Tomography ◽

Molecular Model ◽

Ion Beam ◽

Leading Edge ◽

Cellular Structures ◽

Molecular Architecture ◽

Cryo Electron Tomography

AbstractThe study of cell biology is limited by the difficulty in visualizing cellular structures at high spatial resolution within their native milieu. Here, we have visualized sporulation inBacillus subtilisusing cryo-electron tomography coupled with cryo-focused ion beam milling, a technique that allows the 3D reconstruction of cellular structures in near-native state at molecular resolution. During sporulation, an asymmetrically-positioned septum divides the cell into a larger mother cell and a smaller forespore. Subsequently, the mother cell phagocytoses the forespore in a process called engulfment, which entails a dramatic rearrangement of the peptidoglycan (PG) cell wall around the forespore. By imaging wild-type sporangia, engulfment mutants, and sporangia treated with PG synthesis inhibitors, we show that the initiation of engulfment does not entail the complete dissolution of the septal PG by the mother cell SpoIIDMP complex, as was previously thought. Instead, DMP is required to maintain a flexible septum that is uniformly and only slightly thinned at the onset of engulfment. Then, the mother cell membrane migrates around the forespore by forming tiny finger-like projections, the formation of which requires both SpoIIDMP and new PG synthesized ahead of the leading edge of the engulfing membrane. We propose a molecular model for engulfment membrane migration in which a limited number of SpoIIDMP complexes tether the membrane to and degrade the new PG ahead of the leading edge, thereby generating an irregular engulfing membrane front. Our data also reveal other structures that will provide a valuable resource for future mechanistic studies of endospore formation.

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Fully automated, sequential focused ion beam milling for cryo-electron tomography

10.1101/797514 ◽

2019 ◽

Author(s):

Tobias Zachs ◽

João M. Medeiros ◽

Andreas Schertel ◽

Gregor L. Weiss ◽

Jannik Hugener ◽

...

Keyword(s):

Cell Biology ◽

Focused Ion Beam ◽

Electron Tomography ◽

Ion Beam ◽

Model Organisms ◽

User Input ◽

Cryo Electron Tomography ◽

Cellular Context ◽

Rough Milling ◽

Time Required

AbstractCryo-electron tomography (cryoET) has become a powerful technique at the interface of structural biology and cell biology, with the unique ability to determine structures of macromolecular complexes in their cellular context. A major limitation of cryoET is its restriction to relatively thin samples. Sample thinning by cryo-focused ion beam (cryoFIB) milling has significantly expanded the range of samples that can be analyzed by cryoET. Unfortunately, cryoFIB milling is low-throughput, time-consuming and manual. Here we report a method for fully automated sequential cryoFIB preparation of high-quality lamellae, including rough milling and polishing. We reproducibly applied this method to eukaryotic and bacterial model organisms, and show that the resulting lamellae are suitable for cryoET imaging and subtomogram averaging. Since our method reduces the time required for lamella preparation and minimizes the need for user input, we envision the technique will render previously inaccessible projects feasible.

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Structural Cell Biology: Preparing Specimens for Cryo-Electron Tomography Using Focused-Ion-Beam Milling

Microscopy and Microanalysis ◽

10.1017/s1431927614007843 ◽

2014 ◽

Vol 20 (S3) ◽

pp. 1222-1223

Author(s):

Elizabeth Villa ◽

Miroslava Schaffer ◽

Ben Engel ◽

Jürgen Plitzko ◽

Wolfgang Baumeister

Keyword(s):

Cell Biology ◽

Focused Ion Beam ◽

Electron Tomography ◽

Ion Beam ◽

Structural Cell ◽

Cryo Electron Tomography ◽

Focused Ion Beam Milling

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PilY1 and minor pilins form a complex priming the type IVa pilus in Myxococcus xanthus

Nature Communications ◽

10.1038/s41467-020-18803-z ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Anke Treuner-Lange ◽

Yi-Wei Chang ◽

Timo Glatter ◽

Marco Herfurth ◽

Steffi Lindow ◽

...

Keyword(s):

Cell Surface ◽

Bacterial Cell ◽

Cell Biology ◽

Electron Tomography ◽

Cell Envelope ◽

Myxococcus Xanthus ◽

Core Proteins ◽

Complex Functions ◽

Cryo Electron Tomography ◽

Conserved Core

Abstract Type IVa pili are ubiquitous and versatile bacterial cell surface filaments that undergo cycles of extension, adhesion and retraction powered by the cell-envelope spanning type IVa pilus machine (T4aPM). The overall architecture of the T4aPM and the location of 10 conserved core proteins within this architecture have been elucidated. Here, using genetics, cell biology, proteomics and cryo-electron tomography, we demonstrate that the PilY1 protein and four minor pilins, which are widely conserved in T4aP systems, are essential for pilus extension in Myxococcus xanthus and form a complex that is an integral part of the T4aPM. Moreover, these proteins are part of the extended pilus. Our data support a model whereby the PilY1/minor pilin complex functions as a priming complex in T4aPM for pilus extension, a tip complex in the extended pilus for adhesion, and a cork for terminating retraction to maintain a priming complex for the next round of extension.

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Fully automated, sequential focused ion beam milling for cryo-electron tomography

eLife ◽

10.7554/elife.52286 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 14

Author(s):

Tobias Zachs ◽

Andreas Schertel ◽

João Medeiros ◽

Gregor L Weiss ◽

Jannik Hugener ◽

...

Keyword(s):

Cell Biology ◽

Focused Ion Beam ◽

Electron Tomography ◽

Ion Beam ◽

Model Organisms ◽

User Input ◽

Cryo Electron Tomography ◽

Cellular Context ◽

Rough Milling ◽

Time Required

Cryo-electron tomography (cryoET) has become a powerful technique at the interface of structural biology and cell biology, due to its unique ability for imaging cells in their native state and determining structures of macromolecular complexes in their cellular context. A limitation of cryoET is its restriction to relatively thin samples. Sample thinning by cryo-focused ion beam (cryoFIB) milling has significantly expanded the range of samples that can be analyzed by cryoET. Unfortunately, cryoFIB milling is low-throughput, time-consuming and manual. Here, we report a method for fully automated sequential cryoFIB preparation of high-quality lamellae, including rough milling and polishing. We reproducibly applied this method to eukaryotic and bacterial model organisms, and show that the resulting lamellae are suitable for cryoET imaging and subtomogram averaging. Since our method reduces the time required for lamella preparation and minimizes the need for user input, we envision the technique will render previously inaccessible projects feasible.

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Cryo-electron tomography-the cell biology that came in from the cold

FEBS Letters ◽

10.1002/1873-3468.12757 ◽

2017 ◽

Vol 591 (17) ◽

pp. 2520-2533 ◽

Cited By ~ 29

Author(s):

Jonathan Wagner ◽

Miroslava Schaffer ◽

Rubén Fernández-Busnadiego

Keyword(s):

Cell Biology ◽

Electron Tomography ◽

Cryo Electron Tomography

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Gel-like inclusions of C-terminal fragments of TDP-43 sequester and inhibit proteasomes in neurons

10.1101/2021.03.15.435268 ◽

2021 ◽

Author(s):

Henrick Riemenschneider ◽

Qiang Guo ◽

Jakob Bader ◽

Frederic Frottin ◽

Daniel Farny ◽

...

Keyword(s):

Amyotrophic Lateral Sclerosis ◽

Frontotemporal Dementia ◽

Electron Tomography ◽

Biophysical Properties ◽

Gain Of Function ◽

Functional Assays ◽

Pathogenic Mutations ◽

Cryo Electron Tomography ◽

Substrate Processing ◽

Lateral Sclerosis

TDP-43 inclusions enriched in C-terminal fragments of ~25kDa ("TDP-25") are associated with neurodegeneration in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Here, we analyzed gain-of-function mechanisms of TDP-25 combining cryo-electron tomography, proteomics and functional assays. TDP-25 inclusions are amorphous with gel-like biophysical properties and sequester proteasomes adopting exclusively substrate-processing conformations. This leads to proteostasis impairment, further enhanced by pathogenic mutations. These findings bolster the importance of proteasome dysfunction in ALS/FTD.

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The pore conformation of lymphocyte perforin

10.1101/2021.07.03.450947 ◽

2021 ◽

Author(s):

Marina Ivanova ◽

Natalya Lukoyanova ◽

Sony Malhotra ◽

Maya Topf ◽

Joseph A Trapani ◽

...

Keyword(s):

Conformational Changes ◽

Target Cell ◽

Pore Formation ◽

Electron Tomography ◽

Membrane Surface ◽

Membrane Insertion ◽

Cytotoxic Lymphocytes ◽

Cryo Electron Tomography ◽

Cancerous Cells ◽

Β Sheet

Perforin is a pore-forming protein that facilitates rapid killing of pathogen-infected or cancerous cells by the immune system. Perforin is released from cytotoxic lymphocytes, together with pro-apoptotic granzymes, to bind to the plasma membrane of the target cell where it oligomerises and forms pores. The pores allow granzyme entry, which rapidly triggers the apoptotic death of the target cell. Here we present a 4 Å resolution cryo-EM structure of the perforin pore, revealing new inter- and intra-molecular interactions stabilising the pore. During assembly and pore formation, the helix-turn-helix motif at the bend in the central β-sheet moves away from the bend to form an intermolecular contact. Cryo-electron tomography shows that prepores form on the membrane surface with minimal conformational changes. Our findings suggest the sequence of conformational changes underlying oligomerisation and membrane insertion, and explain how several pathogenic mutations affect function.

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