Dataset Information

TTC30A and TTC30B redundancy protects IFT complex B integrity and its pivotal role in ciliogenesis

ABSTRACT: Intraflagellar transport (IFT) is a microtubule-based system that supports the assembly and maintenance of cilia, an essential organelle for development and maintenance of cellular integrity. Dysfunction of IFT was shown to lead to a wide spectrum of disease phenotypes, so called ciliopathies of variable severity. In previous studies two distinct complexes (IFT-A; IFT-B) were identified, containing multiple proteins that are part of the IFT machinery. Two of the IFT-B components are TTC30A and TTC30B and in contrast to all other IFT components, only these two are paralogues. To investigate whether these two proteins have a similar and essential ciliary or constitute redundant functions, CRIPSR/Cas9 was used to specifically knock-out TTC30A or B and to generate a double knock-out. Localization studies showed a redundancy of both proteins in ciliogenesis while polyglutamylation of cilia was affected by both single knockouts. Further, the localization of other IFT components was not affected by the depletion of a single paralogue. A loss of both proteins led to a severe ciliogenesis defect, resulting in no cilia formation which can be rescued by expression of TTC30A or B. The redundancy can be explained by highly similar interaction patterns of both paralogues, both equally interact with the IFT-B machinery. In summary our study demonstrates that a loss of one TTC30 paralogue can mostly compensate severe ciliary defects, due to redundancy. However, cells assemble shorter cilia which are potentially limited in their functions, especially because of impaired polyglutamylation. A complete loss of both proteins leads to a deficit in IFT complex B integrity followed by disrupted IFT and subsequently no cilia formation

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell, Cell Culture

SUBMITTER: Tina Beyer

LAB HEAD: Dr. Tina Beyer

PROVIDER: PXD034412 | Pride | 2022-08-12

REPOSITORIES: Pride

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Publications

TTC30A and TTC30B Redundancy Protects IFT Complex B Integrity and Its Pivotal Role in Ciliogenesis.

Hoffmann Felix F Bolz Sylvia S Junger Katrin K Klose Franziska F Schubert Timm T Woerz Franziska F Boldt Karsten K Ueffing Marius M Beyer Tina T

Genes 20220701 7

Intraflagellar transport (IFT) is a microtubule-based system that supports the assembly and maintenance of cilia. The dysfunction of IFT leads to ciliopathies of variable severity. Two of the IFT-B components are the paralogue proteins TTC30A and TTC30B. To investigate whether these proteins constitute redundant functions, CRISPR/Cas9 was used to generate single TTC30A or B and double-knockout hTERT-RPE1 cells. Ciliogenesis assays showed the redundancy of both proteins while the polyglutamylatio ...[more]

PMID: 35885974

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Project description:Cilia assembly, maintenance and the localization of signal-transduction proteins necessitate a functioning intraflagellar transport (IFT). The IFT machinery moves cargo along a microtubule scaffold from the proximal to the distal end of the cilium. This anterograde transport is facilitated by a large multiprotein complex IFT-B, which consists of 16 proteins in total and recruits motor protein Kinesin-2 for active movement. TTC30A and TTC30B are integral components of this complex and were shown before to have redundant function in context of IFT. One paralogue could compensate the loss of the other, preventing the disruption of IFT-B and thus a severe ciliogenesis defect with no formation of the cilium. Affinity-based protein complex analysis of endogenously tagged cells, generated via CRISPR/Cas9 system as described before, revealed paralogue specific protein interactors proposing the involvement of TTC30A or TTC30B in other ciliary functions, particularly Sonic hedgehog signaling (Shh). Defects in this ciliary signaling pathway are often correlated to synpolydactyly, which intriguingly is also linked to a rare TTC30 variant. In this study we focus on the so far unknown interaction of TTC30A with protein kinase A catalytic subunit α PRKACA, which is a negative regulator of Shh pathway. For an in-depth analysis of this unique interaction and the influence on Shh, we used previously, via the CRISPR/Cas9 system, generated TTC30A or B single- and double-knockout hTERT-RPE1 cells as well as rescue cells harboring the TTC30 mutation. Our systematic approach revealed the paralogue specific influence of TTC30A KO and mutated TTC30A on the activity of PRKACA as well as the uptake of Smoothened into the cilium resulting in a downregulation of Sonic hedgehog signaling. Affinity-based protein complex analysis of wildtype versus mutated TTC30A or TTC30B uncovered differences in interaction pattern. These interactome alterations combined with Shh downregulation suggest a possible mechanism of how mutant TTC30A and respectively TTC30A KO are linked to synpolydactyly.

Project description:Study Smoking and COPD are associated with decreased mucociliary clearance and healthy smokers have shorter cilia in the large airway than nonsmokers. Intraflagellar transport (IFT) is the process by which cilia are produced and maintained. We assessed expression of IFT-related genes in smokers and nonsmokers and evaluated cilia length in the large and small airway of nonsmokers, healthy smokers, and smokers with COPD. Methods Airway epithelium was obtained via bronchoscopic brushing. Affymetrix microarrays were used to evaluate IFT gene expression in 2 independent data sets from large and small airway. Cilia length was assessed by measuring 100 cilia (10 cilia on each of 10 cells) per subject. Results All 40 IFT genes were expressed in the human large and small airway epithelium. In the large airway, 10 IFT genes were down-regulated and 1 up-regulated in smokers. In the small airway, 11 genes were down-regulated and 3 up-regulated in smokers. A set of 8 IFT genes was down-regulated in both data sets. In the large and small airway epithelium, cilia were significantly shorter in healthy smokers than nonsmokers, and significantly shorter in COPD smokers than in both healthy smokers and nonsmokers. Answer to the Question These results support the concept that loss of cilia length contributes to defective mucociliary clearance in COPD, and that smoking-induced changes in expression of IFT genes may be one mechanism of abnormally short cilia in smokers. Strategies to normalize cilia length may be an important avenue for novel COPD therapies. Gene expression was assessed for 40 intraflagellar transport related genes in the LAE of nonsmokers (n=21) and healthy smokers (n=31) and the SAE of an independent group of nonsmokers (n=28) and healthy smokers (n=69). Cilia length was assessed in a total of 228 airway epithelium samples, including 120 LAE samples and 108 SAE samples; a subset of the 228 samples is represented among the 149 samples in this Series.

Dataset Information

TTC30A and TTC30B redundancy protects IFT complex B integrity and its pivotal role in ciliogenesis

Publications

TTC30A and TTC30B Redundancy Protects IFT Complex B Integrity and Its Pivotal Role in Ciliogenesis.

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