scholarly journals Systematic studies of all PIH proteins in zebrafish reveal their distinct roles in axonemal dynein assembly

2018 ◽  
Author(s):  
Hiroshi Yamaguchi ◽  
Toshiyuki Oda ◽  
Masahide Kikkawa ◽  
Hiroyuki Takeda
Keyword(s):  
Sperm Motility ◽  
Electron Tomography ◽  
Protein Family ◽  
Abnormal Sperm ◽  
Kupffer’S Vesicle ◽  
Kupffer's Vesicle ◽  
Cryo Electron Tomography ◽  
Organ Specific ◽  
Axonemal Dynein ◽  
Motile Cilia

AbstractConstruction of motile cilia/flagella requires cytoplasmic preassembly of axonemal dyneins before transport into cilia. Axonemal dyneins have various subtypes, but the roles of each dynein subtype and their assembly processes remain elusive in vertebrates. The PIH protein family, consisting of four members, has been implicated in the assembly of different dynein subtypes, although evidence for this idea is sparse. Here, we established zebrafish mutants of all four PIH-protein genes: pih1d1, pih1d2, ktu, and twister, and analyzed the structures of axonemal dyneins in mutant spermatozoa by cryo-electron tomography. Mutations caused the loss of specific dynein subtypes, which was correlated with abnormal sperm motility. We also found organ-specific compositions of dynein subtypes, which could explain the severe motility defects of mutant Kupffer’s vesicle cilia. Our data demonstrate that all vertebrate PIH proteins are differently required for cilia/flagella motions and the assembly of axonemal dyneins, assigning specific dynein subtypes to each PIH protein.

scholarly journals Systematic studies of all PIH proteins in zebrafish reveal their distinct roles in axonemal dynein assembly

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Hiroshi Yamaguchi ◽  
Toshiyuki Oda ◽  
Masahide Kikkawa ◽  
Hiroyuki Takeda
Keyword(s):  
Sperm Motility ◽  
Electron Tomography ◽  
Protein Family ◽  
Abnormal Sperm ◽  
Kupffer’S Vesicle ◽  
Kupffer's Vesicle ◽  
Cryo Electron Tomography ◽  
Organ Specific ◽  
Axonemal Dynein ◽  
Motile Cilia

Construction of motile cilia/flagella requires cytoplasmic preassembly of axonemal dyneins before transport into cilia. Axonemal dyneins have various subtypes, but the roles of each dynein subtype and their assembly processes remain elusive in vertebrates. The PIH protein family, consisting of four members, has been implicated in the assembly of different dynein subtypes, although evidence for this idea is sparse. Here, we established zebrafish mutants of all four PIH-protein genes: pih1d1, pih1d2, ktu, and twister, and analyzed the structures of axonemal dyneins in mutant spermatozoa by cryo-electron tomography. Mutations caused the loss of specific dynein subtypes, which was correlated with abnormal sperm motility. We also found organ-specific compositions of dynein subtypes, which could explain the severe motility defects of mutant Kupffer’s vesicle cilia. Our data demonstrate that all vertebrate PIH proteins are differently required for cilia/flagella motions and the assembly of axonemal dyneins, assigning specific dynein subtypes to each PIH protein.

scholarly journals Tubulin glycylation controls axonemal dynein activity, flagellar beat, and male fertility

Science ◽  
2021 ◽  
Vol 371 (6525) ◽  
pp. eabd4914
Author(s):  
Sudarshan Gadadhar ◽  
Gonzalo Alvarez Viar ◽  
Jan Niklas Hansen ◽  
An Gong ◽  
Aleksandr Kostarev ◽  
...  
Keyword(s):  
Posttranslational Modifications ◽  
Male Fertility ◽  
Electron Tomography ◽  
Structural Basis ◽  
Cellular Functions ◽  
Flagellar Beat ◽  
Cryo Electron Tomography ◽  
Axonemal Dynein ◽  
Dynein Arms ◽  
Cilia And Flagella

Posttranslational modifications of the microtubule cytoskeleton have emerged as key regulators of cellular functions, and their perturbations have been linked to a growing number of human pathologies. Tubulin glycylation modifies microtubules specifically in cilia and flagella, but its functional and mechanistic roles remain unclear. In this study, we generated a mouse model entirely lacking tubulin glycylation. Male mice were subfertile owing to aberrant beat patterns of their sperm flagella, which impeded the straight swimming of sperm cells. Using cryo–electron tomography, we showed that lack of glycylation caused abnormal conformations of the dynein arms within sperm axonemes, providing the structural basis for the observed dysfunction. Our findings reveal the importance of microtubule glycylation for controlled flagellar beating, directional sperm swimming, and male fertility.

scholarly journals PACRG and FAP20 form the inner junction of axonemal doublet microtubules and regulate ciliary motility

2019 ◽  
Vol 30 (15) ◽  
pp. 1805-1816 ◽  
Author(s):  
Erin E. Dymek ◽  
Jianfeng Lin ◽  
Gang Fu ◽  
Mary E. Porter ◽  
Daniela Nicastro ◽  
...  
Keyword(s):  
Cell Motility ◽  
Electron Tomography ◽  
Structural Studies ◽  
Ciliary Beating ◽  
Ciliary Motility ◽  
Functional Studies ◽  
Cryo Electron Tomography ◽  
Motile Cilia

We previously demonstrated that PACRG plays a role in regulating dynein-driven microtubule sliding in motile cilia. To expand our understanding of the role of PACRG in ciliary assembly and motility, we used a combination of functional and structural studies, including newly identified Chlamydomonas pacrg mutants. Using cryo-electron tomography we show that PACRG and FAP20 form the inner junction between the A- and B-tubule along the length of all nine ciliary doublet microtubules. The lack of PACRG and FAP20 also results in reduced assembly of inner-arm dynein IDA b and the beak-MIP structures. In addition, our functional studies reveal that loss of PACRG and/or FAP20 causes severe cell motility defects and reduced in vitro microtubule sliding velocities. Interestingly, the addition of exogenous PACRG and/or FAP20 protein to isolated mutant axonemes restores microtubule sliding velocities, but not ciliary beating. Taken together, these studies show that PACRG and FAP20 comprise the inner junction bridge that serves as a hub for both directly modulating dynein-driven microtubule sliding, as well as for the assembly of additional ciliary components that play essential roles in generating coordinated ciliary beating.

Proteomic analysis of microtubule inner proteins (MIPs) in Rib72 null Tetrahymena cells reveals functional MIPs

2020 ◽  
Author(s):  
Amy S. Fabritius ◽  
Brian A. Bayless ◽  
Sam Li ◽  
Daniel Stoddard ◽  
Westley Heydeck ◽  
...  
Keyword(s):  
Proteomic Analysis ◽  
Tetrahymena Thermophila ◽  
Electron Tomography ◽  
Functional Characterization ◽  
Ciliary Beating ◽  
Ciliary Function ◽  
Cryo Electron Tomography ◽  
Motile Cilia ◽  
Cilia And Flagella ◽  
Stable Populations

AbstractMotile cilia and flagella are built from stable populations of doublet microtubules that comprise their axonemes. Their unique stability is brought about, at least in part, by a network of Microtubule Inner Proteins (MIPs) found in the lumen of their doublet microtubules. Rib72A and Rib72B were identified as microtubule inner proteins (MIPs) in the motile cilia of Tetrahymena thermophila. Loss of these proteins leads to ciliary defects and loss of multiple MIPs. We performed mass spectrometry coupled with proteomic analysis and bioinformatics to identify the MIPs lost in RIB72A/B knockout (KO) Tetrahymena cells. From this analysis we identified a number of candidate MIPs and pursued one, Fap115, for functional characterization. We find that loss of Fap115 results in disrupted cell swimming and aberrant ciliary beating. Cryo-electron tomography reveals that Fap115 localizes to MIP6a in the A-tubule of the doublet microtubules. Overall, our results highlight the complex relationship between MIPs, ciliary structure, and ciliary function.

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

2012 ◽  
Vol 23 (1) ◽  
pp. 111-120 ◽  
Author(s):  
Cynthia F. Barber ◽  
Thomas Heuser ◽  
Blanca I. Carbajal-González ◽  
Vladimir V. Botchkarev ◽  
Daniela Nicastro
Keyword(s):  
Electron Tomography ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Flagellar Motility ◽  
Three Dimensional Structure ◽  
Radial Spoke ◽  
Cryo Electron Tomography ◽  
Radial Spokes ◽  
Axonemal Dynein ◽  
Subtomogram Averaging

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.

scholarly journals The molecular structure of primary cilia revealed by cryo-electron tomography

2020 ◽  
Author(s):  
Petra Kiesel ◽  
Gonzalo Alvarez Viar ◽  
Nikolai Tsoy ◽  
Riccardo Maraspini ◽  
Alf Honigmann ◽  
...  
Keyword(s):  
Molecular Structure ◽  
Primary Cilium ◽  
Actin Filaments ◽  
Primary Cilia ◽  
Electron Tomography ◽  
3D Structure ◽  
Molecular Composition ◽  
Structural Investigations ◽  
Cryo Electron Tomography ◽  
Motile Cilia

AbstractPrimary cilia are microtubule-based organelles involved in key signaling and sensing processes in eukaryotic cells. Unlike motile cilia, which have been thoroughly studied, the structure and the composition of primary cilia remain largely unexplored despite their fundamental role in development and homeostasis. They have for long been falsely regarded as simplified versions of motile cilia because they lack distinctive elements such as dynein arms, radial spokes, and central pair complex. However, revealing the detailed molecular composition and 3D structure of primary cilia is necessary in order to understand the mechanisms that govern their functions. Such structural investigations are so far being precluded by the challenging preparation of primary cilia for cryo-electron microscopy. Here, we developed an enabling method for investigating the structure of primary cilia at molecular resolution by cryo-electron tomography. We show that the well-known “9+0” arrangement of microtubule doublets is present only at the base of the primary cilium. A few microns away from the base the ciliary architecture changes into an unstructured bundle of EB1-decorated microtubule singlets and some actin filaments. Our results suggest the existence of a previously unobserved crosstalk between actin filaments and microtubules in the primary cilium. Our work provides unprecedented insights into the molecular structure of primary cilia and a general framework for uncovering their molecular composition and function in health and disease. This opens up new possibilities to study aspects of this important organelle that have so far been out of reach.

scholarly journals Inner lumen proteins stabilize doublet microtubules in cilia/flagella

2018 ◽  
Author(s):  
Mikito Owa ◽  
Takayuki Uchihashi ◽  
Haruaki Yanagisawa ◽  
Takashi Yamano ◽  
Hiro Iguchi ◽  
...  
Keyword(s):  
High Speed ◽  
Electron Tomography ◽  
Intraflagellar Transport ◽  
Force Microscopy ◽  
Microtubule Associated Proteins ◽  
New Class ◽  
Atomic Force ◽  
Associated Proteins ◽  
Axonemal Dynein ◽  
Motile Cilia

AbstractMotile cilia are microtubule-based organelles that play important roles in most eukaryotes. Although it is known that microtubules in cilia are sufficiently stable to withstand their beating motion, it remains unknown how they are stabilized while serving as tracks for axonemal dynein and intraflagellar transport. To address this question, we identified a new class of microtubule-associated proteins, named FAP45 and FAP52, in Chlamydomonas. These proteins are conserved among eukaryotes with motile cilia. Using cryo-electron tomography (cryo-ET) and high-speed atomic force microscopy (HS-AFM), we established that lack of these proteins leads to a loss of inner protrusions in B-tubules and less stable microtubules. These inner protrusions are located near the inner junctions of doublet microtubules and lack of FAP45, FAP52, and FAP20 results in detachment of the B-tubule from the A-tubule, as well as flagellar shortening. These results demonstrated that FAP45 and FAP52 bind to the inside of microtubules and stabilize ciliary axonemes.

scholarly journals Ciliary Proteins: Filling the Gaps. Recent Advances in Deciphering the Protein Composition of Motile Ciliary Complexes

Cells ◽  
2019 ◽  
Vol 8 (7) ◽  
pp. 730 ◽  
Author(s):  
Anna Osinka ◽  
Martyna Poprzeczko ◽  
Magdalena M. Zielinska ◽  
Hanna Fabczak ◽  
Ewa Joachimiak ◽  
...  
Keyword(s):  
Primary Cilia ◽  
Higher Plants ◽  
Electron Tomography ◽  
Protein Complexes ◽  
Protein Composition ◽  
Model Organisms ◽  
Cryo Electron Tomography ◽  
Motile Cilia ◽  
Biochemical Analyses ◽  
Eukaryotic Organisms

Cilia are highly evolutionarily conserved, microtubule-based cell protrusions present in eukaryotic organisms from protists to humans, with the exception of fungi and higher plants. Cilia can be broadly divided into non-motile sensory cilia, called primary cilia, and motile cilia, which are locomotory organelles. The skeleton (axoneme) of primary cilia is formed by nine outer doublet microtubules distributed on the cilium circumference. In contrast, the skeleton of motile cilia is more complex: in addition to outer doublets, it is composed of two central microtubules and several diverse multi-protein complexes that are distributed periodically along both types of microtubules. For many years, researchers have endeavored to fully characterize the protein composition of ciliary macro-complexes and the molecular basis of signal transduction between these complexes. Genetic and biochemical analyses have suggested that several hundreds of proteins could be involved in the assembly and function of motile cilia. Within the last several years, the combined efforts of researchers using cryo-electron tomography, genetic and biochemical approaches, and diverse model organisms have significantly advanced our knowledge of the ciliary structure and protein composition. Here, we summarize the recent progress in the identification of the subunits of ciliary complexes, their precise intraciliary localization determined by cryo-electron tomography data, and the role of newly identified proteins in cilia.

scholarly journals Proteomic analysis of microtubule inner proteins (MIPs) in Rib72 null Tetrahymena cells reveals functional MIPs

2021 ◽  
pp. mbc.E20-12-0786
Author(s):  
Amy S. Fabritius ◽  
Brian A. Bayless ◽  
Sam Li ◽  
Daniel Stoddard ◽  
Westley Heydeck ◽  
...  
Keyword(s):  
Proteomic Analysis ◽  
Tetrahymena Thermophila ◽  
Electron Tomography ◽  
Functional Characterization ◽  
Ciliary Beating ◽  
Ciliary Function ◽  
Luminal Side ◽  
Cryo Electron Tomography ◽  
Motile Cilia ◽  
Cilia And Flagella

The core structure of motile cilia and flagella, the axoneme, is built from a stable population of doublet microtubules. This unique stability is brought about, at least in part, by a network of Microtubule Inner Proteins (MIPs) that are bound to the luminal side of the microtubule walls. Rib72A and Rib72B were identified as MIPs in the motile cilia of the protist Tetrahymena thermophila. Loss of these proteins leads to ciliary defects and loss of additional MIPs. We performed mass spectrometry coupled with proteomic analysis and bioinformatics to identify the MIPs lost in RIB72A/B knockout Tetrahymena axonemes. We identified a number of candidate MIPs and pursued one, Fap115, for functional characterization. We find that loss of Fap115 results in disrupted cell swimming and aberrant ciliary beating. Cryo-electron tomography reveals that Fap115 localizes to MIP6a in the A-tubule of the doublet microtubules. Overall, our results highlight the complex relationship between MIPs, ciliary structure, and ciliary function.

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