Vesicle Picker: A tool for efficient identification of membrane protein complexes in vesicles DOI
Ryan Karimi, Claire E. Coupland, John L. Rubinstein

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: July 16, 2024

Abstract Electron cryomicroscopy (cryo-EM) has recently allowed determination of near-atomic resolution structures membrane proteins and protein complexes embedded in lipid vesicles. However, particle selection from electron micrographs these vesicles can be challenging due to the strong signal contributed bilayer. This challenge often requires iterative laborious workflows generate a dataset high-quality images for subsequent analysis. Here we present Vesicle Picker, an open-source program built on Segment Anything model. Picker enables automatic identification cryo-EM with high recall precision. It then exhaustively selects all potential locations, either at perimeter or uniformly over surface projection vesicle. The is designed interface cryoSPARC, which performs both upstream micrograph processing downstream single image We demonstrate Picker’s utility by determining high-resolution map vacuolar-type ATPase native synaptic (SVs) identifying additional complex SV membrane.

Language: Английский

Cell Membranes Sustain Phospholipid Imbalance Via Cholesterol Asymmetry DOI Creative Commons
Milka Doktorova, Jessica L. Symons,

Xiaoxuan Zhang

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2023, Volume and Issue: unknown

Published: July 31, 2023

Membranes are molecular interfaces that compartmentalize cells to control the flow of nutrients and information. These functions facilitated by diverse collections lipids, nearly all which distributed asymmetrically between two bilayer leaflets. Most models biomembrane structure function often include implicit assumption these leaflets have similar abundances phospholipids. Here, we show this is generally invalid investigate consequences lipid abundance imbalances in mammalian plasma membranes (PM). Using quantitative lipidomics, discovered cytoplasmic human erythrocyte >50% overabundance phospholipids compared exoplasmic This imbalance enabled an asymmetric interleaflet distribution cholesterol, regulates cellular cholesterol homeostasis. features produce unique functional characteristics, including low PM permeability resting tension leaflet protein localization. largely overlooked aspects membrane asymmetry represent evolution classic paradigms physiology.

Language: Английский

Citations

29
Structure and topography of the synaptic V-ATPase–synaptophysin complex DOI Creative Commons
Chuchu Wang,

Wenhong Jiang,

Jeremy Leitz

et al.

Nature, Journal Year: 2024, Volume and Issue: 631(8022), P. 899 - 904

Published: June 5, 2024

Synaptic vesicles are organelles with a precisely defined protein and lipid composition

Language: Английский

Citations

13
Structure of the flotillin complex in a native membrane environment DOI Creative Commons
Ziao Fu, Roderick MacKinnon

Proceedings of the National Academy of Sciences, Journal Year: 2024, Volume and Issue: 121(29)

Published: July 10, 2024

In this study, we used cryoelectron microscopy to determine the structures of Flotillin protein complex, part Stomatin, Prohibitin, Flotillin, and HflK/C (SPFH) superfamily, from cell-derived vesicles without detergents. It forms a right-handed helical barrel consisting 22 pairs Flotillin1 Flotillin2 subunits, with diameter 32 nm at its wider end 19 narrower end. Oligomerization is stabilized by C terminus, which two layers linked β-strand, coiled-coil domains that enable strong charge–charge intersubunit interactions. interacts membranes both ends; through SPFH1 wide terminus narrow end, facilitated hydrophobic interactions lipidation. The inward tilting SPFH domain, likely triggered phosphorylation, suggests role in membrane curvature induction, could be connected proposed clathrin-independent endocytosis. structure shared architecture across family proteins will promote further research into Flotillin’s roles cell biology.

Language: Английский

Citations

12
Cell membranes sustain phospholipid imbalance via cholesterol asymmetry DOI
Milka Doktorova, Jessica L. Symons,

Xiaoxuan Zhang

et al.

Cell, Journal Year: 2025, Volume and Issue: unknown

Published: April 1, 2025

Language: Английский

Citations

1
V-ATPase Dysfunction in the Brain: Genetic Insights and Therapeutic Opportunities DOI Creative Commons
Antonio Falace, Greta Volpedo, Marcello Scala

et al.

Cells, Journal Year: 2024, Volume and Issue: 13(17), P. 1441 - 1441

Published: Aug. 28, 2024

Vacuolar-type ATPase (v-ATPase) is a multimeric protein complex that regulates H

Language: Английский

Citations

4
Molecular architecture of synaptic vesicles DOI Creative Commons
Uljana Kravčenko, Max Ruwolt, Jana Kroll

et al.

Proceedings of the National Academy of Sciences, Journal Year: 2024, Volume and Issue: 121(49)

Published: Nov. 27, 2024

Synaptic vesicles (SVs) store and transport neurotransmitters to the presynaptic active zone for release by exocytosis. After release, SV proteins excess membrane are recycled via endocytosis, new SVs can be formed in a clathrin-dependent manner. This process maintains complex molecular composition of through multiple recycling rounds. Previous studies explored proteomic analysis fluorescent microscopy, proposing model an average ( 1 ). However, structural heterogeneity architecture individual not well described. Here, we used cryoelectron tomography visualize details isolated from mouse brains inside cultured neurons. We describe several classes small on surface long proteinaceous densities SVs. identified V-ATPases, determined structure using subtomogram averaging, showed them forming with membrane-embedded protein synaptophysin (Syp). Our bioluminescence assay revealed pairwise interactions between vesicle-associated 2 Syp V-ATPase Voe1 domains. Interestingly, V-ATPases were randomly distributed irrespective vesicle size. A subpopulation neurons contained partially assembled clathrin coat icosahedral symmetry. observed under cages clathrin-coated (CCVs). Additionally, preparations within hippocampal baskets without vesicles. their CCVs preferential location proximity cell membrane. advances understanding SVs' diversity architecture.

Language: Английский

Citations

4
ATG8 in Single Membranes: Fresh Players of Endocytosis and Acidic Organelle Quality Control in Cancer, Neurodegeneration, and Inflammation DOI Creative Commons

Wang B Ben,

Apaja M Pirjo

Biochemical and Biophysical Research Communications, Journal Year: 2025, Volume and Issue: 749, P. 151384 - 151384

Published: Jan. 23, 2025

Ubiquitin-like autophagy-related gene ATG8 proteins are typically associated with degradative quality control via canonical double-membrane macro-autophagosomes in the cell. have now stepped forward non-canonical pathways single membrane organelles. The growing interest roles has been stimulated by recent links to human conditions, especially regulation of inflammation, neurodegeneration and cancers. Here, we summarize evidence linking ATG8s pathologies acidic V-ATPase-regulated organelles

Language: Английский

Citations

0
CryoVIA: An image analysis toolkit for the quantification of membrane structures from cryo-EM micrographs DOI Creative Commons
Philipp Schönnenbeck, Benedikt Junglas, Carsten Sachse

et al.

Structure, Journal Year: 2025, Volume and Issue: unknown

Published: Feb. 1, 2025

Highlights•A software suite for automated analysis of lipid membranes in electron micrographs•Includes segmentation, shape identification, and membrane properties•Applied to datasets with different lipids protein-induced changes•Features an intuitive GUI batch micrograph analysisSummaryImaging structures associated protein complexes using cryoelectron microscopy (cryo-EM) is a common visualization structure determination technique. The quantitative the structures, however, not routine time consuming particular when large amounts data are involved. Here, we introduce image-processing cryo-vesicle image analyzer (CryoVIA) that parametrizes from cryo-EM images. This toolkit combines identification methods automatically perform large-scale local global properties such as bilayer thickness, size, curvature including classifications. We included analyses exemplary compositions changes through endosomal sorting required transport III (ESCRT-III) remodeling protein. opens new possibilities systematically study structural their modifications images.Graphical abstract

Language: Английский

Citations

0
Cryo-EM of native membranes reveals an intimate connection between the Krebs cycle and aerobic respiration in mycobacteria DOI Creative Commons
Justin M. Di Trani, Jiacheng Yu, Gautier M. Courbon

et al.

Proceedings of the National Academy of Sciences, Journal Year: 2025, Volume and Issue: 122(8)

Published: Feb. 19, 2025

To investigate the structure of mycobacterial oxidative phosphorylation machinery, we prepared inverted membrane vesicles from Mycobacterium smegmatis , enriched for containing complexes interest, and imaged with electron cryomicroscopy. We show that this analysis allows determination both ATP synthase supercomplex respiratory III IV in their native membrane. The latter reveals enzyme malate:quinone oxidoreductase (Mqo) physically associates supercomplex, an interaction is lost on extraction proteins lipid bilayer. Mqo catalyzes essential reaction Krebs cycle, vivo survival pathogens compromised when its activity absent. high-speed spectroscopy Mqo:supercomplex enables rapid transfer malate to supercomplex. Further, necessary malate-driven, but not NADH-driven, transport chain oxygen consumption. Together, these findings indicate a connection between cycle aerobic respiration directs electrons along single branch chain.

Language: Английский

Citations

0
A correlative workflow streamlines synaptic imaging by cryo-electron tomography DOI Creative Commons
Thanh Thao, Antje Siegert, Florelle Domart

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2025, Volume and Issue: unknown

Published: March 3, 2025

Abstract Despite decades of intense research, the molecular organization synapse is not well understood. To address this issue, we sought to develop a method for systematic imaging synapses by cryo-electron tomography (cryo-ET), technology capable mapping cellular architecture at resolution. Thinning samples cryo-focused ion beam milling prerequisite high-quality cryo-ET imaging, but process needs be guided structures interest. allow robust synaptic targeting, established correlative cryo-light/electron microscopy approach which are fluorescently labeled in minimally invasive manner, using synthetic binder postsynaptic scaffold PSD-95 and antibodies against presynaptic protein Synaptotagmin-1. Cryo-ET sites colocalization consistently revealed excitatory synapses. Our allows structural studies complexes situ , facilitating investigations

Language: Английский

Citations

0