Towards community-driven visual proteomics with large-scale cryo-electron tomography ofChlamydomonas reinhardtii DOI Creative Commons

Ron Kelley,

Sagar Khavnekar, Ricardo D. Righetto

et al.

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

Published: Dec. 28, 2024

Abstract In situ cryo-electron tomography (cryo-ET) has emerged as the method of choice to investigate structures biomolecules in their native context. However, challenges remain efficient production large-scale cryo-ET datasets, well community sharing this information-rich data. Here, we applied a cryogenic plasma-based focused ion beam (cryo-PFIB) instrument for high-throughput milling green alga Chlamydomonas reinhardtii , useful model organism visualization numerous fundamental cellular processes. Combining cryo-PFIB sample preparation with recent advances data acquisition and processing, generated dataset 1829 reconstructed annotated tomograms, which provide resource drive development inspire biological discovery. To assay quality dataset, performed subtomogram averaging (STA) both soluble membrane-bound complexes ranging size from >3 MDa ∼200 kDa, including 80S ribosomes, Rubisco, nucleosomes, microtubules, clathrin, photosystem II, mitochondrial ATP synthase. The majority these density maps reached sub-nanometer resolution, demonstrating potential C. promise modern workflows open towards visual proteomics.

Language: Английский

Cryo-ET suggests tubulin chaperones form a subset of microtubule lumenal particles with a role in maintaining neuronal microtubules DOI Creative Commons
Saikat Chakraborty, Antonio Martínez-Sánchez, Florian Beck

et al.

Proceedings of the National Academy of Sciences, Journal Year: 2025, Volume and Issue: 122(5)

Published: Jan. 31, 2025

The functional architecture of the long-lived neuronal microtubule (MT) cytoskeleton is maintained by various MT-associated proteins (MAPs), most which are known to bind MT outer surface. However, electron microscopy (EM) has long ago revealed presence particles inside lumens MTs, yet unknown identity and function. Here, we use cryogenic tomography (cryo-ET) analyze three-dimensional (3D) organization structures lumenal in primary hippocampal neurons, human induced pluripotent stem cell-derived differentiated P19 cells. We obtain situ density maps several from respective cells detect common structural features underscoring their potential overarching functions. Mass spectrometry-based proteomics combined with modeling suggest that a subset could be tubulin-binding cofactors (TBCs) bound tubulin monomers. A different smaller particles, remains unidentified, exhibits densities bridge across protofilaments. show increased particle concentration within MTs concomitant differentiation correlates higher curvatures. Enrichment around lattice defects at freshly polymerized open-ends suggests protective role. Together identified resemblance TBCs, these results hint role local proteostasis for maintenance MTs.

Language: Английский

Citations

1
In-cell structure and snapshots of copia retrotransposons in intact tissue by cryoelectron tomography DOI Creative Commons
Sven Klumpe, Kirsten-André Senti, Florian Beck

et al.

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

Published: March 1, 2025

Long terminal repeat (LTR) retrotransposons belong to the transposable elements (TEs), autonomously replicating genetic that integrate into host's genome. Among animals, Drosophila melanogaster serves as an important model organism for TE research and contains several LTR retrotransposons, including Ty1-copia family, which is evolutionarily related retroviruses forms virus-like particles (VLPs). In this study, we use cryo-focused ion beam (FIB) milling lift-out approaches visualize copia VLPs in ovarian cells intact egg chambers, resolving situ capsid structure 7.7 Å resolution by cryoelectron tomography (cryo-ET). Although cytoplasmic vary size, nuclear are homogeneous form densely packed clusters, supporting a import acts size selector. Analyzing flies deficient TE-suppressing PIWI-interacting RNA (piRNA) pathway, observe copia's translocation nucleus during spermatogenesis. Our findings provide insights replication cycle cellular structural biology of active retrotransposon.

Language: Английский

Citations

1
In-cell architecture of the mitochondrial respiratory chain DOI
Florent Waltz, Ricardo D. Righetto, Lorenz Lamm

et al.

Science, Journal Year: 2025, Volume and Issue: 387(6740), P. 1296 - 1301

Published: March 20, 2025

Mitochondria regenerate adenosine triphosphate (ATP) through oxidative phosphorylation. This process is carried out by five membrane-bound complexes collectively known as the respiratory chain, working in concert to transfer electrons and pump protons. The precise organization of these native cells debated. We used situ cryo–electron tomography visualize structures several major mitochondrial Chlamydomonas reinhardtii cells. ATP synthases segregate into curved flat crista membrane domains, respectively. Respiratory I, III, IV assemble a respirasome supercomplex, from which we determined 5-angstrom (Å) resolution structure showing binding electron carrier cytochrome c . Combined with single-particle microscopy at 2.4-Å resolution, model how organize inside mitochondria.

Language: Английский

Citations

0
The postsynaptic density in excitatory synapses is composed of clustered, heterogeneous nanoblocks DOI Creative Commons
Rong Sun, James P. Allen, Zhuqing Mao

et al.

The Journal of Cell Biology, Journal Year: 2025, Volume and Issue: 224(6)

Published: March 27, 2025

The nanoscale organization of proteins within synapses is critical for maintaining and regulating synaptic transmission plasticity. Here, we used cryo-electron tomography (cryo-ET) to directly visualize the three-dimensional architecture supramolecular postsynaptic components in both synaptosomes from cultured neurons. Cryo-ET revealed that density (PSD) composed membrane-associated nanoblocks various sizes. Subtomogram averaging showed two types (type A B) receptor–like particles at resolutions 24 26 Å, respectively. Furthermore, our analysis suggested potential presynaptic release sites are closer with type A/B than without particles. results this study provide a more comprehensive understanding ultrastructure suggest PSD clustering nanoblocks. These heterogeneous size, assembly, distribution, which likely contribute dynamic nature modulating strength.

Language: Английский

Citations

0
A case for community metadata standards in cryo-electron tomography DOI Creative Commons
William Wan

Emerging Topics in Life Sciences, Journal Year: 2025, Volume and Issue: 9(01)

Published: April 29, 2025

In the past decade, cryo-electron microscopy and single particle analysis (SPA) have quickly become key methods in structural biology. particular, increased access to equipment streamlined software has enabled new users successfully carry out SPA projects. At same time, tomography (cryo-ET) also made great technical strides, most notably with cellular cryo-ET. While many challenges remain, developments hardware automation cryo-ET specimen preparation data collection more accessible than ever. There is a growing field of developers, but wide variety biological specimens scientific goals that can be pursued using makes it difficult develop processing workflows analogous those SPA; this becomes major barrier entry for users. perspective, I make case development standardized metadata play role reducing such barriers allow an ecosystem enables enter while retaining diversity approaches.

Language: Английский

Citations

0
Towards community-driven visual proteomics with large-scale cryo-electron tomography ofChlamydomonas reinhardtii DOI Creative Commons

Ron Kelley,

Sagar Khavnekar, Ricardo D. Righetto

et al.

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

Published: Dec. 28, 2024

Abstract In situ cryo-electron tomography (cryo-ET) has emerged as the method of choice to investigate structures biomolecules in their native context. However, challenges remain efficient production large-scale cryo-ET datasets, well community sharing this information-rich data. Here, we applied a cryogenic plasma-based focused ion beam (cryo-PFIB) instrument for high-throughput milling green alga Chlamydomonas reinhardtii , useful model organism visualization numerous fundamental cellular processes. Combining cryo-PFIB sample preparation with recent advances data acquisition and processing, generated dataset 1829 reconstructed annotated tomograms, which provide resource drive development inspire biological discovery. To assay quality dataset, performed subtomogram averaging (STA) both soluble membrane-bound complexes ranging size from >3 MDa ∼200 kDa, including 80S ribosomes, Rubisco, nucleosomes, microtubules, clathrin, photosystem II, mitochondrial ATP synthase. The majority these density maps reached sub-nanometer resolution, demonstrating potential C. promise modern workflows open towards visual proteomics.

Language: Английский

Citations

3