Three-dimensional structure of the radial spokes reveals heterogeneity and interactions with dyneins in Chlamydomonas flagella

Radial spokes (RSs) play an essential role in the regulation of axonemal dynein activity and thus of ciliary and flagellar motility. However, few details are known about the complexes involved. Using cryo–electron tomography and subtomogram averaging, we visualized the three-dimensional structure of the radial spokes in Chlamydomonas flagella in unprecedented detail. Unlike many other species, Chlamydomonas has only two spokes per axonemal repeat, RS1 and RS2. Our data revealed previously uncharacterized features, including two-pronged spoke bases that facilitate docking to the doublet microtubules, and that inner dyneins connect directly to the spokes. Structures of wild type and the headless spoke mutant pf17 were compared to define the morphology and boundaries of the head, including a direct RS1-to-RS2 interaction. Although the overall structures of the spokes are very similar, we also observed some differences, corroborating recent findings about heterogeneity in the docking of RS1 and RS2. In place of a third radial spoke we found an uncharacterized, shorter electron density named “radial spoke 3 stand-in,” which structurally bears no resemblance to RS1 and RS2 and is unaltered in the pf17 mutant. These findings demonstrate that radial spokes are heterogeneous in structure and may play functionally distinct roles in axoneme regulation.

Download Full-text

A guided approach for subtomogram averaging of challenging macromolecular assemblies

10.1101/2020.02.01.930297 ◽

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.

Download Full-text

Structural insights into the cause of human RSPH4A primary ciliary dyskinesia

Molecular Biology of the Cell ◽

10.1091/mbc.e20-12-0806 ◽

2021 ◽

pp. mbc.E20-12-0806

Author(s):

Yanhe Zhao ◽

Justine Pinskey ◽

Jianfeng Lin ◽

Weining Yin ◽

Patrick R. Sears ◽

...

Keyword(s):

Primary Ciliary Dyskinesia ◽

Electron Tomography ◽

Three Dimensional ◽

Dimensional Structure ◽

Structural Basis ◽

Radial Spoke ◽

Radial Spokes ◽

Cilia And Flagella ◽

Tract Infections ◽

Ciliary Dyskinesia

Cilia and flagella are evolutionarily conserved eukaryotic organelles involved in cell motility and signaling. In humans, mutations in Radial Spoke Head Protein 4 homolog A ( RSPH4A) can lead to primary ciliary dyskinesia (PCD), a life-shortening disease characterized by chronic respiratory tract infections, abnormal organ positioning, and infertility. Despite its importance for human health, the location of RSPH4A in human cilia has not been resolved, and the structural basis of RSPH4A-/- PCD remains elusive. Here, we present the native, three-dimensional structure of RSPH4A-/- human respiratory cilia using samples collected non-invasively from a PCD patient. Using cryo-electron tomography and subtomogram averaging, we compared the structures of control and RSPH4A-/- cilia, revealing primary defects in two of the three radial spokes (RSs) within the axonemal repeat and secondary (heterogeneous) defects in the central pair complex. Similar to RSPH1-/- cilia, the radial spoke heads of RS1 and RS2, but not RS3, were missing in RSPH4A-/- cilia. However, RSPH4A-/- cilia also exhibited defects within the arch domains adjacent to the RS1 and RS2 heads, which were not observed with RSPH1 loss. Our results provide insight into the underlying structural basis for RSPH4A-/- PCD and highlight the benefits of applying cryo-ET directly to patient samples for molecular structure determination. [Media: see text]

Download Full-text

Faculty Opinions recommendation of Three-dimensional structure of herpes simplex virus from cryo-electron tomography.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1016499.200752 ◽

2003 ◽

Author(s):

Lindsey Hutt-Fletcher

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Electron Tomography ◽

Three Dimensional ◽

Dimensional Structure ◽

Three Dimensional Structure ◽

Cryo Electron Tomography ◽

Simplex Virus

Download Full-text

Three-Dimensional Structure of Herpes Simplex Virus from Cryo-Electron Tomography

Science ◽

10.1126/science.1090284 ◽

2003 ◽

Vol 302 (5649) ◽

pp. 1396-1398 ◽

Cited By ~ 393

Author(s):

Kay Grünewald ◽

Prashant Desai ◽

Dennis C. Winkler ◽

J. Bernard Heymann ◽

David M. Belnap ◽

...

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Electron Tomography ◽

Three Dimensional ◽

Dimensional Structure ◽

Three Dimensional Structure ◽

Cryo Electron Tomography ◽

Simplex Virus

Download Full-text

Three-dimensional Structure of the Tiny Bacterium Pelagibacter ubique Studied by Cryo-electron Tomography

Microscopy and Microanalysis ◽

10.1017/s1431927606067456 ◽

2006 ◽

Vol 12 (S02) ◽

pp. 180-181 ◽

Cited By ~ 6

Author(s):

D Nicastro ◽

C Schwartz ◽

J Pierson ◽

J-C Cho ◽

SJ Giovannoni ◽

...

Keyword(s):

Electron Tomography ◽

Three Dimensional ◽

Dimensional Structure ◽

Three Dimensional Structure ◽

Cryo Electron Tomography

Extended abstract of a paper presented at Microscopy and Microanalysis 2006 in Chicago, Illinois, USA, July 30 – August 3, 2005

Download Full-text

Faculty Opinions recommendation of Three-dimensional structure of herpes simplex virus from cryo-electron tomography.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1016499.201225 ◽

2003 ◽

Author(s):

Greg Smith

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Electron Tomography ◽

Three Dimensional ◽

Dimensional Structure ◽

Three Dimensional Structure ◽

Cryo Electron Tomography ◽

Simplex Virus

Download Full-text

Cryo-Electron Tomography Reveals the Comparative Three-Dimensional Architecture of Prochlorococcus, a Globally Important Marine Cyanobacterium

Journal of Bacteriology ◽

10.1128/jb.01948-06 ◽

2007 ◽

Vol 189 (12) ◽

pp. 4485-4493 ◽

Cited By ~ 61

Author(s):

Claire S. Ting ◽

Chyongere Hsieh ◽

Sesh Sundararaman ◽

Carmen Mannella ◽

Michael Marko

Keyword(s):

Cell Wall ◽

Electron Tomography ◽

Three Dimensional ◽

Membrane System ◽

Niche Differentiation ◽

Dimensional Structure ◽

Comparative Genomic ◽

Photosynthetic Membrane ◽

Peptidoglycan Biosynthesis ◽

Cryo Electron Tomography

ABSTRACT In an age of comparative microbial genomics, knowledge of the near-native architecture of microorganisms is essential for achieving an integrative understanding of physiology and function. We characterized and compared the three-dimensional architecture of the ecologically important cyanobacterium Prochlorococcus in a near-native state using cryo-electron tomography and found that closely related strains have diverged substantially in cellular organization and structure. By visualizing native, hydrated structures within cells, we discovered that the MED4 strain, which possesses one of the smallest genomes (1.66 Mbp) of any known photosynthetic organism, has evolved a comparatively streamlined cellular architecture. This strain possesses a smaller cell volume, an attenuated cell wall, and less extensive intracytoplasmic (photosynthetic) membrane system compared to the more deeply branched MIT9313 strain. Comparative genomic analyses indicate that differences have evolved in key structural genes, including those encoding enzymes involved in cell wall peptidoglycan biosynthesis. Although both strains possess carboxysomes that are polygonal and cluster in the central cytoplasm, the carboxysomes of MED4 are smaller. A streamlined cellular structure could be advantageous to microorganisms thriving in the low-nutrient conditions characteristic of large regions of the open ocean and thus have consequences for ecological niche differentiation. Through cryo-electron tomography we visualized, for the first time, the three-dimensional structure of the extensive network of photosynthetic lamellae within Prochlorococcus and the potential pathways for intracellular and intermembrane movement of molecules. Comparative information on the near-native structure of microorganisms is an important and necessary component of exploring microbial diversity and understanding its consequences for function and ecology.

Download Full-text

The CSC connects three major axonemal complexes involved in dynein regulation

Molecular Biology of the Cell ◽

10.1091/mbc.e12-05-0357 ◽

2012 ◽

Vol 23 (16) ◽

pp. 3143-3155 ◽

Cited By ~ 51

Author(s):

Thomas Heuser ◽

Erin E. Dymek ◽

Jianfeng Lin ◽

Elizabeth F. Smith ◽

Daniela Nicastro

Keyword(s):

Electron Tomography ◽

Structural Heterogeneity ◽

Flagellar Motility ◽

Wild Type ◽

Localization Model ◽

Cryo Electron Tomography ◽

Radial Spokes ◽

Regulatory Complex ◽

Health And Development ◽

Cilia And Flagella

Motile cilia and flagella are highly conserved organelles that play important roles in human health and development. We recently discovered a calmodulin- and spoke-associated complex (CSC) that is required for wild-type motility and for the stable assembly of a subset of radial spokes. Using cryo–electron tomography, we present the first structure-based localization model of the CSC. Chlamydomonas flagella have two full-length radial spokes, RS1 and RS2, and a shorter RS3 homologue, the RS3 stand-in (RS3S). Using newly developed techniques for analyzing samples with structural heterogeneity, we demonstrate that the CSC connects three major axonemal complexes involved in dynein regulation: RS2, the nexin–dynein regulatory complex (N-DRC), and RS3S. These results provide insights into how signals from the radial spokes may be transmitted to the N-DRC and ultimately to the dynein motors. Our results also indicate that although structurally very similar, RS1 and RS2 likely serve different functions in regulating flagellar motility.

Download Full-text

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

The Journal of Cell Biology ◽

10.1083/jcb.200808050 ◽

2008 ◽

Vol 183 (5) ◽

pp. 923-932 ◽

Cited By ~ 121

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.

Download Full-text