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A WDR35-dependent coatomer transports ciliary membrane proteins from the Golgi to the cilia

ABSTRACT: Intraflagellar transport (IFT) is a highly conserved mechanism for motor-driven transport of cargo inside cilia. However, the mechanism of selective transport of cargo to cilia and entry across the diffusion barrier is not well understood. WDR35/IFT121 is a component of the IFT-A complex, best known for ciliary retrograde transport. Small Wdr35 mutant cilia are formed but fail to enrich in diverse classes of ciliary membrane proteins. Whilst the assembly of the IFT-B complex is unaffected, these exhibit retrograde trafficking defects in Wdr35 mutants. In contrast, the IFT-A peripheral components are degraded and core components remain stuck at the cilia base. The deep sequence homology and structural similarity of WDR35 and other IFT-As to the coatomer COPI proteins α and ß’, and accumulation of electron-light vesicles around Wdr35 mutant cilia, suggest that WDR35 is required to form coated vesicles delivering cargo from the Golgi necessary for cilia elongation. This is the first insitu proof towards a novel coatomer function for WDR35 and likely other IFT-A proteins in transporting ciliary membrane proteins from the Golgi.

INSTRUMENT(S): Q Exactive Plus

ORGANISM(S): Mus Musculus (mouse)

SUBMITTER: Jimi Wills

LAB HEAD: Pleasantine Mill

PROVIDER: PXD022652 | Pride | 2021-05-03

REPOSITORIES: Pride

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Source:

			Action	DRS
	2016-12-14_02_GFP-8-2.raw	Raw
	2016-12-14_03_IFT-8-2.raw	Raw
	2016-12-14_04_GFP-mut-8-3.raw	Raw
	2016-12-14_05_IFT-mut-8-3.raw	Raw
	2016-12-14_06_GFP-mut-9-1.raw	Raw

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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.

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.

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A WDR35-dependent coatomer transports ciliary membrane proteins from the Golgi to the cilia

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