Radial Spokes—A Snapshot of the Motility Regulation, Assembly, and Evolution of Cilia and Flagella

Zhu, Xiaoyan; Liu, Yi; Yang, Pinfen

doi:10.1101/cshperspect.a028126

Cited by 48 publications

(47 citation statements)

References 94 publications

(118 reference statements)

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“…Additionally, this group contains a major wave of expression of genes involved in regulating ciliary motility including spag6, several radial spoke genes, and components of the dynein regulatory complex. None of these genes is required for the assembly of cilia in other model systems, but instead are involved in coordinating the activity of axonemal dyneins to generate motility [62]. In Chlamydomonas, radial spoke protein synthesis reaches its maximum rate 30-60 minutes after the flagella have begun assembling [52], which roughly matches the delay seen in our data between genes involved in ciliary assembly and genes encoding radial spokes during Stentor regeneration.…”

Section: Groupsupporting

confidence: 81%

The Transcriptional Program of Regeneration in the Giant Single Cell,Stentor coeruleus

Sood

McGillivary

Marshall

2017

Preprint

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The giant ciliate Stentor coeruleus is a classical model system for studying regeneration and morphogenesis at the level of a single cell. Stentor are polarized cells with a complex subcellular architecture. The anterior of the cell is marked by a complex polarized array of cilia, known as the oral apparatus. This feeding organelle can be induced to shed and regenerate in a series of reproducible morphological steps, previously shown to require transcription. We used RNAseq to assay the dynamic changes in Stentor's transcriptome during regeneration with high temporal resolution, allowing us to identify five distinct waves of gene expression. We show that the oral apparatus is a model for organelle regeneration, as well many conserved genes involved in centriole assembly and ciliogenesis are induced. Additionally, we find genes involved in signaling, cell cycle regulation, transcription, and RNA binding to be expressed at distinct stages of organelle regeneration, suggesting that the morphological steps of regeneration are driven by a complex regulatory system. Materials and Methods Inducing Regeneration and Staging StentorCells were obtained from Carolina Biological Supply and cultured as previously described [20]. Briefly, cells were maintained in Pasteurized Spring Water (Carolina Biological Supply) and fed with Chlamydomonas and wheat seeds. Cells were collected from the same culture for each RNAseq experimental replicate. To induce regeneration, cells were shocked with a 15% sucrose solution for 2 minutes [11], and then washed in Carolina Spring Water thoroughly. Samples of ~20 cells were collected before shock, then at 30 minutes post shock, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours and 8 hours. At each time point, a sample of cells was lysed into RNA-stabilizing buffers specified by the extraction kit, and then stored on ice until the end of the experiment when the RNA purification was performed in parallel on all samples (see below). 4 replicates were analyzed for each time-point. Total RNA extractionRNA was extracted at each time point using the Nucleospin RNA XS kit from Clontech (cat. num. 740902.250). RNA quality was assessed using a NanoDrop and then Bioanalyzer was used to quantify RNA amount. ERCC spike ins (ThermoFisher cat. num. 4456739) were added to each sample in a dilution ranging from 1:1000 to 1:10000 depending on the initial amount of RNA extracted. RNA-Seq library preparation and sequencingRNA-seq libraries were prepared with Ovation RNA-seq system v2 kit (NuGEN). In this method, the total RNA (50 ng or less) is reverse transcribed to synthesize the first-strand cDNA using a combination of random hexamers and a poly-T chimeric primer. The RNA template is then partially degraded by heating and the second strand cDNA is synthesized using DNA polymerase. The double-stranded DNA is then amplified using single primer isothermal amplification (SPIA). SPIA is a linear cDNA amplification process in which RNase H degrades RNA in DNA/RNA heteroduplex at the 5′-end of the double-st...

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Section: Groupsupporting

confidence: 81%

The Transcriptional Program of Regeneration in the Giant Single Cell,Stentor coeruleus

Sood

McGillivary

Marshall

2017

Preprint

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“…We observed that the ciliary localizations of multiple CP proteins, including CP MTs-associated Spef1, C2 MT-associated Hydin, C1 MT-associated Spag6, and C1-C2 bridge-associated Spag16 (Fig. 2a, S3) 22,35,44 , were abolished or markedly reduced in Wdr47 -/-mEPCs, whereas Rsph4a, a radial spoke component 45,46 , was not affected ( Fig. 2b; Fig.…”

Section: Wdr47 Is Critical For Both Ciliary Localization Of Cp Proteimentioning

confidence: 92%

Wdr47, Camsaps, and Katanin cooperate to generate ciliary central microtubules

Líu

Zheng

Zhu

et al. 2020

Preprint

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The axonemal central pair (CP) are non-centrosomal microtubules critical for planar ciliary beat. How they form, however, is poorly understood. Here, we show that mammalian CP formation requires cooperative activities of Katanin, Camsaps, and Wdr47. Katanin severs peripheral microtubules to produce central microtubule seeds in nascent cilia. Camsaps stabilize minus ends of the seeds to facilitate MT outgrowth, whereas Wdr47 concentrates Camsaps into the axonemal central lumen to properly position the central microtubules. Wdr47 deficiency in mouse multicilia results in complete loss of CP, rotatory beat, and primary ciliary dyskinesia. Overexpression of Camsaps induces central microtubules in Wdr47-/- ependymal cells but at the expense of low efficiency, abnormal numbers, and wrong location, whereas overexpression of a dominant inhibitor of Katanin impairs the CP formation. We propose that Wdr47, Camsaps, and Katanin constitute a general cooperative work team for the generation of non-centrosomal MT arrays in polarized subcellular compartments.

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“…The radial spokes extend in the direction of the central apparatus. It was proposed that the transient contacts between the radial spokes and projections of the central apparatus facilitate the transmission of the signals that originate at the central apparatus to the outer doubles and dynein arms, thus regulating cilia beating [20].…”

Section: Ciliary Ultrastructurementioning

confidence: 99%

Rare Human Diseases: Model Organisms in Deciphering the Molecular Basis of Primary Ciliary Dyskinesia

Poprzeczko

Bicka

Farahat

et al. 2019

Cells

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Primary ciliary dyskinesia (PCD) is a recessive heterogeneous disorder of motile cilia, affecting one per 15,000-30,000 individuals; however, the frequency of this disorder is likely underestimated. Even though more than 40 genes are currently associated with PCD, in the case of approximately 30% of patients, the genetic cause of the manifested PCD symptoms remains unknown. Because motile cilia are highly evolutionarily conserved organelles at both the proteomic and ultrastructural levels, analyses in the unicellular and multicellular model organisms can help not only to identify new proteins essential for cilia motility (and thus identify new putative PCD-causative genes), but also to elucidate the function of the proteins encoded by known PCD-causative genes. Consequently, studies involving model organisms can help us to understand the molecular mechanism(s) behind the phenotypic changes observed in the motile cilia of PCD affected patients. Here, we summarize the current state of the art in the genetics and biology of PCD and emphasize the impact of the studies conducted using model organisms on existing knowledge.

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Radial Spokes—A Snapshot of the Motility Regulation, Assembly, and Evolution of Cilia and Flagella

Cited by 48 publications

References 94 publications

The Transcriptional Program of Regeneration in the Giant Single Cell,Stentor coeruleus

The Transcriptional Program of Regeneration in the Giant Single Cell,Stentor coeruleus

Wdr47, Camsaps, and Katanin cooperate to generate ciliary central microtubules

Rare Human Diseases: Model Organisms in Deciphering the Molecular Basis of Primary Ciliary Dyskinesia

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