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. 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 polyglutamylation of cilia was affected in single knockouts. 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 was rescued by TTC30A or B. The redundancy can be explained by the highly similar interaction patterns of the paralogues; both equally interact with the IFT-B machinery. Our study demonstrates that a loss of one TTC30 paralogue can mostly be compensated by the other, thus preventing severe ciliary defects. However, cells assemble shorter cilia, which are potentially limited in their function, 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.

Paralog-specific TTC30 regulation of Sonic hedgehog signaling

“…However, cilia length is decreased, and tubulin polyglutamylation is reduced upon the expression of one paralog only. Concomitant loss of TTC30A and B results in the absence of ciliogenesis, which emphasizes the relevance of TTC30 in IFT-B-mediated ciliary assembly (Fan et al, 2010;Takei et al, 2018;Hoffmann et al, 2022).…”

“…IFT is a bidirectional transport process that is facilitated by large protein complexes and the support of motor proteins. The movement in an anterograde manner is driven by kinesin-2, and the retrograde transport is driven by dynein-2 (Vashishtha et al, 1996;Pazour et al, 1999;Lechtreck, 2015;Hoffmann et al, 2022). The IFT particles are large multiprotein complexes that can be classified into two subcomplexes, IFT-A and IFT-B (Lechtreck, 2015).…”

“…There are two paralogs present in humans, TTC30A and B, which are essential for IFT-B core complex stability. TTC30A and B have an almost identical nucleotide sequence and, as a result, also have a highly similar protein structure, which is conserved across species (Du et al, 2019;Hoffmann et al, 2022). Each human paralog individually interacts with the IFT-B complex and is able to maintain cilia assembly.…”

Defective airway intraflagellar transport underlies a combined motile and primary ciliopathy syndrome caused byIFT74mutations

“…HEK293 transfections were followed by affinity purification of Strep-FLAG fusion proteins then mass spectrometry and LFQ of the eluates, using methods previously described ( 40 ). For identification and quantification of proteins, the raw spectra were searched against the human SwissProt databases (03/2018) using MaxQuant software (version 1.5.0.3) with a minimum ratio count of 2.…”

TTC6-Mediated Stabilization of the Flagellum Annulus Ensures the Rapid and Directed Motion of Sperm

“…The tetratricopeptide repeat protein family, including TTC21a, TTC26, TTC29, TTC30a, and TTC30b, plays a significant role in forming and operating the ciliar and flagellar structures that are pivotal for cell movement and signal transduction [ 24 , 25 , 26 , 27 , 28 ]. To date, only mutations in TTC21a and TTC29 have been associated with multiple morphological abnormalities of the flagella, a condition that precipitates morphological abnormalities in sperm flagella and can result in male infertility [ 25 , 26 ].…”