In Situ Localization of N and C Termini of Subunits of the Flagellar Nexin-Dynein Regulatory Complex (N-DRC) Using SNAP Tag and Cryo-electron Tomography

Song, Kangkang; Awata, Junya; Tritschler, Douglas; Bower, Raqual; Witman, George B.; Porter, Mary E.; Nicastro, Daniela

doi:10.1074/jbc.m114.626556

Cited by 55 publications

(65 citation statements)

References 45 publications

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“…However, cell models of the sup-pf4 and drc3 mutants , which retain most of the N-DRC linker (Fig. 1; (Awata et al 2015; Heuser et al 2009; Song et al 2015), could be reactivated like WT cells (91.5 ± 10.1% and 91.2 ± 4.8% of the time, respectively). The drc mutants pf3 , ida6 , sup-pf5 , sup- pf3 , and pf2 lack the linker domain (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Cryo-ET has defined several structural domains within the N-DRC (Awata et al 2015; Heuser et al 2009; Oda et al 2013; Oda et al 2015; Song et al 2015). In this study, we focus on the two major domains, the base plate that attaches to the underside of the A-tubule (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…drc3 lacks the most distal portion of the distal lobe and a strut that connects that domain to the base of the linker (Fig. 1, D–F; (Awata et al 2015; Song et al 2015). sup-pf4 lacks the small region that connects the proximal and distal lobes of the linker (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Analyses of drc mutants by biochemical and structural approaches have provided models for the localization of subunits within the substructures of the N-DRC (Fig. 1; Table S1; (Awata et al 2015; Bower et al 2013; Heuser et al 2009; Lin et al 2011; Oda et al 2015; Song et al 2015). The N-DRC is considered one of the major regulatory hubs in the axoneme (Awata et al 2015; Dymek et al 2011; Heuser et al 2012b; Heuser et al 2009; Oda et al 2013; Song et al 2015), and may serve several functions, including physical alignment of outer doublet microtubules (Bower et al 2013; Lin et al 2015).…”

Section: Introductionmentioning

confidence: 99%

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The nexin link and B‐tubule glutamylation maintain the alignment of outer doublets in the ciliary axoneme

Alford

Stoddard

et al. 2016

Cytoskeleton

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We developed quantitative assays to test the hypothesis that the N-DRC is required for integrity of the ciliary axoneme. We examined reactivated motility of demembranated drc cells, commonly termed “reactivated cell models.” ATP-induced reactivation of wild-type cells resulted in the forward swimming of ~90% of cell models. ATP-induced reactivation failed in a subset of drc cell models, despite forward motility in live drc cells. Dark-field light microscopic observations of drc cell models revealed various degrees of axonemal splaying. In contrast, >98% of axonemes from wild-type reactivated cell models remained intact. The sup-pf4 and drc3 mutants, unlike other drc mutants, retain most of the N-DRC linker that interconnects outer doublet microtubules. Reactivated sup-pf4 and drc3 cell models displayed nearly wild-type levels of forward motility. Thus, the N-DRC linker is required for axonemal integrity. We also examined reactivated motility and axoneme integrity in mutants defective in tubulin polyglutamylation. ATP-induced reactivation resulted in forward swimming of >75% of tpg cell models. Analysis of double mutants defective in tubulin polyglutamylation and different regions of the N-DRC indicate B-tubule polyglutamylation and the distal lobe of the linker region are both important for axonemal integrity and normal N-DRC function.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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The nexin link and B‐tubule glutamylation maintain the alignment of outer doublets in the ciliary axoneme

Alford

Stoddard

et al. 2016

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“…However, under the electron microscope, the rather homogeneous electron density, along with the antigen disruption caused by sectioning and antibody penetration issues, can have a negative effect on labeling and identification of specific proteins by immuno-EM. These limitations can be overcome in some instances by using smaller antibody probes for immuno-EM (Kilmartin, 2014) or by using protein tagging strategies in Cryo-EM (Choy, Kollman, Zelter, Davis, & Agard, 2009;Song et al, 2015). When compared to EM methods, subdiffraction fluorescence imaging has the overall advantage of a better labeling specificity and sensitivity, and it is relatively easy to implement, but it has the disadvantage of a lower resolution power.…”

Section: Introductionmentioning

confidence: 97%

Subdiffraction resolution microscopy methods for analyzing centrosomes organization

Mennella

Hanna

Kim

2015

Centrosome &Amp; Centriole

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Algal Ciliary Motility

Hunter

Penny

Dutcher

2021

Encyclopedia of Life Sciences

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The microtubule‐based cilium (also known as a flagellum) extends from the surface of most eukaryotic cells. This long, slender organelle is biochemically complex and is conserved in many eukaryotic organisms. The unicellular alga, Chlamydomonas reinhardtii, serves as a tractable organism for studying the assembly and function of cilia. Cilia provide locomotion and environmental sensing. To produce motility, specialised molecular motors called outer and inner dynein arms generate force to produce waveform. These motors are regulated by other macromolecular complexes in the cilium. The ease of genetic and biochemical approaches combined with electron microscopy has provided the ability to perform in‐depth studies of the regulation of ciliary assembly and function. Results from Chlamydomonas researchers have broadened our knowledge of motile cilia diseases in humans. There are still many outstanding questions to be addressed. Key Concepts Chlamydomonas reinhardtii is a haploid, motile, single‐celled alga that is useful for the study of cilia. The Chlamydomonas cilium is a complex structure that generates asymmetric and symmetric waveforms to allow forward and backward swimming, respectively, and orientation to environmental signals. During ciliary motility, dynein arms pull microtubule doublets past each other to generate sliding, which is converted into axonemal bending, although the mechanism for how this occurs is still controversial. Outer dynein arms control the ciliary beat frequency by changing the velocity of doublet sliding. Inner dynein arms in conjunction with the radial spokes and the central pair apparatus, control the bend amplitude and the shape of the waveform. The axoneme is divided into 96 nm repeating segments that contain regulatory, enzymatic and structural components essential for motility; these include the inner and outer dynein arms, N‐DRC, MIA complex and radial spokes as well as two central microtubules. IFT (intraflagellar transport) is essential for building the cilium by transporting cargo into (anterograde) and out of (retrograde) the cilium. Cell biological research about ciliary components, including the central pair apparatus, radial spokes and N‐DRC, provides key information about how ciliary motility is regulated. Genetic, biochemical and microscopic methods used in Chlamydomonas have allowed molecular resolution of interactions between regulatory complexes. Studies of Chlamydomonas cilia have provided additional insights to the structure, function and regulation of cilia, and have helped with the understanding of human cilia‐related diseases called ciliopathies.

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In Situ Localization of N and C Termini of Subunits of the Flagellar Nexin-Dynein Regulatory Complex (N-DRC) Using SNAP Tag and Cryo-electron Tomography

Cited by 55 publications

References 45 publications

The nexin link and B‐tubule glutamylation maintain the alignment of outer doublets in the ciliary axoneme

The nexin link and B‐tubule glutamylation maintain the alignment of outer doublets in the ciliary axoneme

Subdiffraction resolution microscopy methods for analyzing centrosomes organization

Algal Ciliary Motility

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