Heterogeneity of radial spokes structural components and associated enzymes inTetrahymenacilia DOI Creative Commons
Marta Bicka, Avrin Ghanaeian, Corbin Black

et al.

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

Published: Aug. 12, 2023

Summary Radial spokes, RS1-RS2-RS3, are T-shaped, multiprotein complexes that transmit regulatory signals from central apparatus to outer doublet dyneins. RSs, especially RS3, differ in morphology, protein composition, and RS base-docked IDAs. Spokes defects alter cilia beating frequency, waveform, amplitude leading, humans, primary ciliary dyskinesia infertility. In contrast RS1 RS2, the composition of RS3 is partly resolved. Moreover, role particular spokes unclear. Ciliate Tetrahymena thermophila has three Rsp3 paralogs two or some other RSPs. Using multiple complementary approaches, we showed forms RS2 subtypes having core composed various one type Rsp3-less RS3. We elucidated proteomes identified novel RS-associated proteins, including enzymatic proteins involved local regulation ADP/ATP levels phosphorylation, whose presence further diversifies RSs properties likely functions. brief their architecture. Studies a ciliate revealed paralogs-dependent subtypes, diversity RSP3 mutants’ phenotype. Known components newly structural were assigned RSs.

Language: Английский

Integrating cellular electron microscopy with multimodal data to explore biology across space and time DOI Creative Commons
Caitlyn L McCafferty, Sven Klumpe, Rommie E. Amaro

et al.

Cell, Journal Year: 2024, Volume and Issue: 187(3), P. 563 - 584

Published: Feb. 1, 2024

Biology spans a continuum of length and time scales. Individual experimental methods only glimpse discrete pieces this spectrum but can be combined to construct more holistic view. In Review, we detail the latest advancements in volume electron microscopy (vEM) cryo-electron tomography (cryo-ET), which together visualize biological complexity across scales from organization cells large tissues molecular details inside native cellular environments. addition, discuss emerging methodologies for integrating three-dimensional (3DEM) imaging with multimodal data, including fluorescence microscopy, mass spectrometry, single-particle analysis, AI-based structure prediction. This multifaceted approach fills gaps continuum, providing functional context, spatial organization, identity, interactions. We conclude perspective on incorporating diverse data into computational simulations that further bridge extend while dimension time.

Language: Английский

Citations

43
Advancing structure modeling from cryo-EM maps with deep learning DOI
Shu Li, Genki Terashi, Zicong Zhang

et al.

Biochemical Society Transactions, Journal Year: 2025, Volume and Issue: 53(01)

Published: Feb. 7, 2025

Cryo-electron microscopy (cryo-EM) has revolutionized structural biology by enabling the determination of biomolecular structures that are challenging to resolve using conventional methods. Interpreting a cryo-EM map requires accurate modeling underlying biomolecules. Here, we concisely discuss evolution and current state automatic structure from density maps. We classify methods into two categories: de novo high-resolution maps (better than 5 Å) model fitting individual component proteins at lower resolution (worse Å). Special attention is given role deep learning in process, highlighting how AI-driven approaches transformative modeling. conclude discussing future directions field.

Language: Английский

Citations

0
A ternary complex of MIPs in the A-tubule of basal bodies and axonemes depends on RIB22 and the EF-hand domain of RIB72A in Tetrahymena cilia DOI
Rachel A. Howard-Till, Sam Li,

Usha Pallabi Kar

et al.

Molecular Biology of the Cell, Journal Year: 2025, Volume and Issue: 36(4)

Published: Feb. 12, 2025

The lumens of the highly stable microtubules that make up core basal bodies, cilia, and flagella are coated with a network proteins known as MIPs, or microtubule inner proteins. MIPs hypothesized to enhance rigidity stability these microtubules, but how they assemble contribute cilia function is poorly understood. Here we describe ciliate specific MIP, RIB22, in Tetrahymena thermophila. RIB22 calmodulin-like protein found A-tubule doublet triplet bodies. Its localization dependent on conserved MIP RIB72. use cryogenic electron tomography (cryoET) examine its interacting partners axonemes forms ternary complex C-terminal EF-hand domain RIB72A another FAM166A. strains lacking showed impaired function. CryoET from demonstrated an interdependence three for stabilization within structure. Deletion resulted apparent loss multiple region. These findings emphasize intricacy importance understanding MIPs’ functions during cilium assembly regulation.

Language: Английский

Citations

0
Techniques for Selective Labeling of Molecules and Subcellular Structures for Cryo-Electron Tomography DOI

Eugene P Kazakov,

Igor I. Kireev, S. A. Golyshev

et al.

Biochemistry (Moscow), Journal Year: 2025, Volume and Issue: 90(2), P. 173 - 187

Published: Feb. 1, 2025

Language: Английский

Citations

0
Structures of Native Doublet Microtubules from Trichomonas vaginalis Reveal Parasite-Specific Proteins DOI Creative Commons
Alexander Stevens, S. K. Kashyap,

Ethan Crofut

et al.

Nature Communications, Journal Year: 2025, Volume and Issue: 16(1)

Published: April 29, 2025

Abstract Doublet microtubules (DMTs) are flagellar components required for the protist Trichomonas vaginalis ( Tv ) to swim through human genitourinary tract cause trichomoniasis, most common non-viral sexually transmitted disease. Lack of structures ’s DMT -DMT) has prevented structure-guided drug design manage infection. Here, we determine 16 nm, 32 48 nm and 96 nm-repeat native Tv- at resolution ranging from 3.4 4.4 Å by cryogenic electron microscopy (cryoEM) built an atomic model entire -DMT. These show that -DMT is composed 30 different proteins, including α- β-tubulin, 19 microtubule inner proteins (MIPs) 9 outer proteins. While A-tubule simplistic compared DMTs other organisms, B-tubule features parasite-specific such as FAP40 FAP35. Notably, FAP35 form filaments near junctions, respectively, interface with stabilizing MIPs. This highlights diversity eukaryotic motility machineries provides a structural framework inform rational therapeutics against trichomoniasis.

Language: Английский

Citations

0
The structure of basal body inner junctions from Tetrahymena revealed by electron cryo-tomography DOI Creative Commons
Sam Li, José‐Jesús Fernández, Marisa D. Ruehle

et al.

The EMBO Journal, Journal Year: 2025, Volume and Issue: unknown

Published: Feb. 24, 2025

Abstract The cilium is a microtubule-based eukaryotic organelle critical for many cellular functions. Its assembly initiates at basal body and continues as an axoneme that projects out of the cell to form functional cilium. This process tightly regulated. However, our knowledge molecular architecture mechanism limited. By applying cryo-electron tomography, we obtained structures inner junction in three regions from Tetrahymena : proximal, central core body, axoneme. We identified several protein components body. While few proteins are distributed throughout entire length organelle, restricted specific regions, forming intricate local interaction networks bolstering structural stability. examining POC1 knockout mutant, found triplet microtubule was destabilized, resulting defective structure. Surprisingly, axoneme-specific were “infiltrate” into mutant Our findings provide insight junctions, underscoring its precise spatial regulation.

Language: Английский

Citations

0
Structures of Native Doublet Microtubules from Trichomonas vaginalis Reveal Parasite-Specific Proteins as Potential Drug Targets DOI Open Access
Alexander Stevens, S. K. Kashyap,

Ethan Crofut

et al.

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

Published: June 11, 2024

Doublet microtubules (DMTs) are flagellar components required for the protist

Language: Английский

Citations

2
The Structure of Cilium Inner Junctions Revealed by Electron Cryo-tomography DOI Creative Commons
Sam Li, José‐Jesús Fernández, Marisa D. Ruehle

et al.

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

Published: Sept. 9, 2024

The cilium is a microtubule-based organelle critical for many cellular functions. Its assembly initiates at basal body and continues as an axoneme that projects out of the cell to form functional cilium. This process tightly regulated. However, our knowledge molecular architecture mechanism limited. By applying electron cryo-tomography subtomogram averaging, we obtained subnanometer resolution structures inner junction in three distinct regions cilium: proximal region body, central core flagellar axoneme. allowed us identify several components. While few proteins are distributed throughout entire length organelle, restricted particular cilium, forming intricate local interaction networks bolstering structural stability. Finally, by knocking component Poc1, found triplet MT was destabilized, resulting defective structure. Surprisingly, axoneme-specific components were "infiltrate" into mutant body. Our findings provide insight its junctions, underscoring precise spatial regulation.

Language: Английский

Citations

1
Structure of the ciliary tip central pair reveals the unique role of the microtubule-seam binding protein SPEF1 DOI Creative Commons
Thibault Legal, Ewa Joachimiak, M. Parra

et al.

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

Published: Dec. 4, 2024

Motile cilia are unique organelles with the ability to autonomously move. Force generated by beating propels cells and moves fluids. The ciliary skeleton is made of peripheral doublet microtubules a central pair (CP) distinct structure at tip. In this study, we present high-resolution CP in tip ciliate

Language: Английский

Citations

1
In-cell structural insight into the asymmetric assembly of central apparatus in mammalian sperm axoneme DOI Open Access
Yun Zhu,

Guoliang Yin,

Linhua Tai

et al.

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

Published: Aug. 6, 2024

Abstract In motile cilia or flagella, the axoneme typically exhibits a “9+2” configuration, with central apparatus (CA) consisting of extensively modified microtubules C1 and C2 to regulate ciliary motility. How are interconnected asymmetrically remains unknown due lack complete structural model CA. Here, we utilized in situ cryo-electron tomography approach solve in-cell structure intact mouse sperm CA at sub-nanometer resolution built its near-complete atomic aid AlphaFold2. We identified 39 different CA-associated proteins 8 never reported from isolated specimen. assigned long chain-like ASH-containing CFAP47 HYDIN responsible for connecting C2. Sperm Cfap47 -knockout mice displayed hollowing bridge structure, correlating reduced progressive Our findings elucidate molecular mechanisms components motility, implications their mutations human ciliopathies.

Language: Английский

Citations

0