scholarly journals Scaffold subunits support associated subunit assembly in the Chlamydomonas ciliary nexin-dynein regulatory complex

2019 ◽  
Author(s):  
Long Gui ◽  
Kangkang Song ◽  
Douglas Tristchler ◽  
Raqual Bower ◽  
Yan Si ◽  
...  
Keyword(s):  
Electron Tomography ◽  
Core Complex ◽  
Ciliary Motility ◽  
C Terminus ◽  
Large N ◽  
Cryo Electron Tomography ◽  
N Terminus ◽  
Regulatory Complex ◽  
Cilia And Flagella ◽  
Linker Domain

ABSTRACTThe nexin-dynein regulatory complex (N-DRC) in motile cilia and flagella functions as a linker between neighboring doublet microtubules, acts to stabilize the axonemal core structure, and serves as a central hub for the regulation of ciliary motility. Although the N-DRC has been studied extensively using genetic, biochemical, and structural approaches, the precise arrangement of the eleven (or more) N-DRC subunits remains unknown. Here, using cryo-electron tomography, we have compared the structure of Chlamydomonas wild-type flagella to that of strains with specific DRC subunit deletions or rescued strains with tagged DRC subunits. Our results show that DRC7 is a central linker subunit that helps connect the N-DRC to the outer dynein arms. DRC11 is required for the assembly of DRC8, and DRC8/11 form a sub-complex in the proximal lobe of the linker domain that is required to form stable contacts to the neighboring B-tubule. Gold labeling of tagged subunits determines the precise locations of the previously ambiguous N-terminus of DRC4 which is now shown to contribute to the core scaffold of the N-DRC and C-terminus of DRC5. Our results reveal the overall architecture of N-DRC, with the three subunits, DRC1/2/4 forming a core complex that serves as the scaffold for the assembly of the “functional subunits” associate, namely DRC3/5-8/11. These findings shed light on N-DRC assembly and its role in regulating flagellar beating.Significance StatementCilia and flagella are small hair-like appendages in eukaryotic cells that play essential roles in cell sensing, signaling, and motility. The highly conserved nexin-dynein regulatory complex (N-DRC) is one of the key regulators for ciliary motility. At least 11 proteins (DRC1–11) have been assigned to the N-DRC, but their precise arrangement within the large N-DRC structure is not yet known. Here, using cryo-electron tomography combined with genetic approaches, we have localized DRC7, the sub-complex DRC8/DRC11, the N-terminus of DRC4, and the C-terminus of DRC5. Our results provide insights into the N-DRC structure, its function in the regulation of dynein activity, and the mechanism by which n-drc mutations can lead to defects in ciliary motility that cause disease.

scholarly journals Scaffold subunits support associated subunit assembly in the Chlamydomonas ciliary nexin–dynein regulatory complex

2019 ◽  
Vol 116 (46) ◽  
pp. 23152-23162 ◽  
Author(s):  
Long Gui ◽  
Kangkang Song ◽  
Douglas Tritschler ◽  
Raqual Bower ◽  
Si Yan ◽  
...  
Keyword(s):  
Electron Tomography ◽  
Wild Type ◽  
Core Complex ◽  
The Core ◽  
C Terminus ◽  
Cryo Electron Tomography ◽  
Regulatory Complex ◽  
Cilia And Flagella ◽  
Linker Domain ◽  
Shed Light

The nexin–dynein regulatory complex (N-DRC) in motile cilia and flagella functions as a linker between neighboring doublet microtubules, acts to stabilize the axonemal core structure, and serves as a central hub for the regulation of ciliary motility. Although the N-DRC has been studied extensively using genetic, biochemical, and structural approaches, the precise arrangement of the 11 (or more) N-DRC subunits remains unknown. Here, using cryo-electron tomography, we have compared the structure of Chlamydomonas wild-type flagella to that of strains with specific DRC subunit deletions or rescued strains with tagged DRC subunits. Our results show that DRC7 is a central linker subunit that helps connect the N-DRC to the outer dynein arms. DRC11 is required for the assembly of DRC8, and DRC8/11 form a subcomplex in the proximal lobe of the linker domain that is required to form stable contacts to the neighboring B-tubule. Gold labeling of tagged subunits determines the precise locations of the previously ambiguous N terminus of DRC4 and C terminus of DRC5. DRC4 is now shown to contribute to the core scaffold of the N-DRC. Our results reveal the overall architecture of N-DRC, with the 3 subunits DRC1/2/4 forming a core complex that serves as the scaffold for the assembly of the “functional subunits,” namely DRC3/5–8/11. These findings shed light on N-DRC assembly and its role in regulating flagellar beating.

scholarly journals The CSC connects three major axonemal complexes involved in dynein regulation

2012 ◽  
Vol 23 (16) ◽  
pp. 3143-3155 ◽  
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.

scholarly journals DRC3 connects the N-DRC to dynein g to regulate flagellar waveform

2015 ◽  
Vol 26 (15) ◽  
pp. 2788-2800 ◽  
Author(s):  
Junya Awata ◽  
Kangkang Song ◽  
Jianfeng Lin ◽  
Stephen M. King ◽  
Michael J. Sanderson ◽  
...  
Keyword(s):  
Electron Tomography ◽  
Motor Domain ◽  
Flagellar Motility ◽  
Wild Type ◽  
Cryo Electron Tomography ◽  
Radial Spokes ◽  
Regulatory Complex ◽  
And Function ◽  
Linker Domain ◽  
Distal Lobe

The nexin-dynein regulatory complex (N-DRC), which is a major hub for the control of flagellar motility, contains at least 11 different subunits. A major challenge is to determine the location and function of each of these subunits within the N-DRC. We characterized a Chlamydomonas mutant defective in the N-DRC subunit DRC3. Of the known N-DRC subunits, the drc3 mutant is missing only DRC3. Like other N-DRC mutants, the drc3 mutant has a defect in flagellar motility. However, in contrast to other mutations affecting the N-DRC, drc3 does not suppress flagellar paralysis caused by loss of radial spokes. Cryo–electron tomography revealed that the drc3 mutant lacks a portion of the N-DRC linker domain, including the L1 protrusion, part of the distal lobe, and the connection between these two structures, thus localizing DRC3 to this part of the N-DRC. This and additional considerations enable us to assign DRC3 to the L1 protrusion. Because the L1 protrusion is the only non-dynein structure in contact with the dynein g motor domain in wild-type axonemes and this is the only N-DRC–dynein connection missing in the drc3 mutant, we conclude that DRC3 interacts with dynein g to regulate flagellar waveform.

scholarly journals CFAP70 Is a Novel Axoneme-Binding Protein That Localizes at the Base of the Outer Dynein Arm and Regulates Ciliary Motility

Cells ◽  
2018 ◽  
Vol 7 (9) ◽  
pp. 124 ◽  
Author(s):  
Noritoshi Shamoto ◽  
Keishi Narita ◽  
Tomohiro Kubo ◽  
Toshiyuki Oda ◽  
Sen Takeda
Keyword(s):  
Electron Tomography ◽  
Intraflagellar Transport ◽  
Tetratricopeptide Repeat ◽  
Flagellar Motility ◽  
C Terminus ◽  
Mouse Tissues ◽  
N Terminus ◽  
Motile Cilia ◽  
Cilia And Flagella ◽  
Ciliary Localization

In the present study, we characterized CFAP70, a candidate of cilia-related protein in mice. As this protein has a cluster of tetratricopeptide repeat (TPR) domains like many components of the intraflagellar transport (IFT) complex, we investigated the domain functions of particular interest in ciliary targeting and/or localization. RT-PCR and immunohistochemistry of various mouse tissues demonstrated the association of CFAP70 with motile cilia and flagella. A stepwise extraction of proteins from swine tracheal cilia showed that CFAP70 bound tightly to the ciliary axoneme. Fluorescence microscopy of the cultured ependyma expressing fragments of CFAP70 demonstrated that the N-terminus rather than the C-terminus with the TPR domains was more important for the ciliary localization. When CFAP70 was knocked down in cultured mouse ependyma, reductions in cilia beating frequency were observed. Consistent with these observations, a Chlamydomonas mutant lacking the CFAP70 homolog, FAP70, showed defects in outer dynein arm (ODA) activity and a reduction in flagellar motility. Cryo-electron tomography revealed that the N-terminus of FAP70 resided stably at the base of the ODA. These results demonstrated that CFAP70 is a novel regulatory component of the ODA in motile cilia and flagella, and that the N-terminus is important for its ciliary localization.

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.

scholarly journals Targeted dynein inhibition generates flagellar beating

2017 ◽  
Author(s):  
Jianfeng Lin ◽  
Daniela Nicastro
Keyword(s):  
Molecular Mechanism ◽  
Structural Changes ◽  
Direct Evidence ◽  
Electron Tomography ◽  
Asymmetric Distribution ◽  
Conformational Switching ◽  
Cryo Electron Tomography ◽  
Cilia And Flagella ◽  
Biochemical Analyses ◽  
Sea Urchin Sperm

Motile cilia and flagella are highly conserved organelles that are essential for the normal development and health of many eukaryotes including humans. To reveal the molecular mechanism of motility, we used cryo-electron tomography of active sea urchin sperm flagella to directly visualize the macromolecular complexes and their structural changes during flagellar beating. We resolved distinct conformations of dynein motors and regulators, and showed that many of them are distributed in bend-direction-dependent fashion in active flagella. Our results provide direct evidence for the conformational switching predicted by the ‘switch-point-hypothesis’. However, they also reveal a fundamentally different mechanism of generating motility by inhibiting dyneins, rather than activating them, causing an asymmetric distribution of force and thus bending. Our high-resolution structural and biochemical analyses provide a new understanding of the distinct roles played by various dyneins and regulators in ciliary motility and suggest a molecular mechanism for robust beating in an all-or-none manner.

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 Detailed structural and biochemical characterization of the nexin-dynein regulatory complex

2015 ◽  
Vol 26 (2) ◽  
pp. 294-304 ◽  
Author(s):  
Toshiyuki Oda ◽  
Haruaki Yanagisawa ◽  
Masahide Kikkawa
Keyword(s):  
Biochemical Characterization ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Dynein Motor ◽  
Cryo Electron Tomography ◽  
Regulatory Complex ◽  
And Function

The nexin-dynein regulatory complex (N-DRC) forms a cross-bridge between the outer doublet microtubules of the axoneme and regulates dynein motor activity in cilia/flagella. Although the molecular composition and the three-dimensional structure of N-DRC have been studied using mutant strains lacking N-DRC subunits, more accurate approaches are necessary to characterize the structure and function of N-DRC. In this study, we precisely localized DRC1, DRC2, and DRC4 using cryo–electron tomography and structural labeling. All three N-DRC subunits had elongated conformations and spanned the length of N-DRC. Furthermore, we purified N-DRC and characterized its microtubule-binding properties. Purified N-DRC bound to the microtubule and partially inhibited microtubule sliding driven by the outer dynein arms (ODAs). Of interest, microtubule sliding was observed even in the presence of fourfold molar excess of N-DRC relative to ODA. These results provide insights into the role of N-DRC in generating the beating motions of cilia/flagella.

scholarly journals Analysis of the Intact Surface Layer of Caulobacter crescentus by Cryo-Electron Tomography

2010 ◽  
Vol 192 (22) ◽  
pp. 5855-5865 ◽  
Author(s):  
Fernando Amat ◽  
Luis R. Comolli ◽  
John F. Nomellini ◽  
Farshid Moussavi ◽  
Kenneth H. Downing ◽  
...  
Keyword(s):  
Caulobacter Crescentus ◽  
Electron Tomography ◽  
Range Order ◽  
Double Layers ◽  
Negative Stain ◽  
C Terminus ◽  
Opposite Vertex ◽  
Cryo Electron Tomography ◽  
Subtomogram Averaging ◽  
Intact Surface

ABSTRACT The surface layers (S layers) of those bacteria and archaea that elaborate these crystalline structures have been studied for 40 years. However, most structural analysis has been based on electron microscopy of negatively stained S-layer fragments separated from cells, which can introduce staining artifacts and allow rearrangement of structures prone to self-assemble. We present a quantitative analysis of the structure and organization of the S layer on intact growing cells of the Gram-negative bacterium Caulobacter crescentus using cryo-electron tomography (CET) and statistical image processing. Instead of the expected long-range order, we observed different regions with hexagonally organized subunits exhibiting short-range order and a broad distribution of periodicities. Also, areas of stacked double layers were found, and these increased in extent when the S-layer protein (RsaA) expression level was elevated by addition of multiple rsaA copies. Finally, we combined high-resolution amino acid residue-specific Nanogold labeling and subtomogram averaging of CET volumes to improve our understanding of the correlation between the linear protein sequence and the structure at the 2-nm level of resolution that is presently available. The results support the view that the U-shaped RsaA monomer predicted from negative-stain tomography proceeds from the N terminus at one vertex, corresponding to the axis of 3-fold symmetry, to the C terminus at the opposite vertex, which forms the prominent 6-fold symmetry axis. Such information will help future efforts to analyze subunit interactions and guide selection of internal sites for display of heterologous protein segments.

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