Cfap91-Dependent Stability of the RS2 and RS3 Base Proteins and Adjacent Inner Dynein Arms in Tetrahymena Cilia

Abstract

Motile cilia and eukaryotic flagella are specific cell protrusions that are conserved from protists to humans. They are supported by a skeleton composed of uniquely organized microtubules—nine peripheral doublets and two central singlets (9 × 2 + 2). Microtubules also serve as docking sites for periodically distributed multiprotein ciliary complexes. Radial spokes, the T-shaped ciliary complexes, repeat along the outer doublets as triplets and transduce the regulatory signals from the cilium center to the outer doublet-docked dynein arms. Using the genetic, proteomic, and microscopic approaches, we have shown that lack of Tetrahymena Cfap91 protein affects stable docking/positioning of the radial spoke RS3 and the base of RS2, and adjacent inner dynein arms, possibly due to the ability of Cfap91 to interact with a molecular ruler protein, Ccdc39. The localization studies confirmed that the level of RS3-specific proteins, Cfap61 and Cfap251, as well as RS2-associated Cfap206, are significantly diminished in Tetrahymena CFAP91-KO cells. Cilia of Tetrahymena cells with knocked-out CFAP91 beat in an uncoordinated manner and their beating frequency is dramatically reduced. Consequently, CFAP91-KO cells swam about a hundred times slower than wild-type cells. We concluded that Tetrahymena Cfap91 localizes at the base of radial spokes RS2 and RS3 and likely plays a role in the radial spoke(s) positioning and stability.

An amino acid-resolution interactome for motile cilia illuminates the structure and function of ciliopathy protein complexes

“…A recent EM study showed that this elongated protein forms the base of the RS2 radial spoke, extends parallel to the CCDC39/40 ruler and through the N-DRC, and contacts RS3 70,82 . Moreover, defective CFAP91 function in both humans and Tetrahymena is associated with disruption specifically of the RS3 radial spoke 79,81 . Little else is known of CFAP91 function, so it is significant that multiple XLs in our dataset also linked CFAP91 directly to CCDC96 (Figure 6B , dark blue ), which connects RS3 to the NDR-C 83 .…”

“…These interactions in Tetrahymena prompted us to explore the effect of CFAP91 loss in vertebrate multiciliated cells. First, we showed that knockdown (KD) of CFAP91 in Xenopus multiciliated cells recapitulated the known effect of genetic loss in humans and Tetrahymena 79,81 . For example, we observed specific loss of the RS3 subunit CFAP251 (Figure 6G), while the RS1/RS2 subunit Rsph1 was unaffected (Figure 6H).…”

“…We also identified multiple XLs connecting CCDC39/40 and N-DRC subunits to CFAP91/MAATS1(Figure 6B, light blue), which is essential for cilia beating and is associated with human motile ciliopathy but is otherwise poorly understood [79][80][81] . A recent EM study showed that this elongated protein forms the base of the RS2 radial spoke, extends parallel to the CCDC39/40 ruler and through the N-DRC, and contacts RS3 70,82 .…”

CCDC113 stabilizes sperm axoneme and head-tail coupling apparatus to ensure male fertility

“…Recent advances in artificial intelligence, biochemical techniques, and cryo-electron microscopy (cryo-EM) facilitated the analysis of the axonemal structures, and numerous components were detected among RS, N-DRC and DMTs, which may serve as hubs to stabilize the axoneme (Bazan et al, 2021; Leung et al, 2023; Walton et al, 2023; Zhou et al, 2023). Such as, CFAP91 is identified to extend from the base of RS2 through the N-DRC base plate to the RS3, and stabilizes RS2 and RS3 on the DMTs (Bicka et al, 2022; Dymek et al, 2011; Gui et al, 2021). CFAP57 also extends through the N-DRC and interacts with RS3 via its C-terminal region (Ghanaeian et al, 2023).…”

A Standardized Protocol for Assuring the Validity of Proteomics Results from Liquid Chromatography–High-Resolution Mass Spectrometry

“…Modern proteomics methods have also recently become useful in neurodegenerative diseases in developing methods for the analysis [ 8 ] and identification of new biomarkers for early diagnosis and disease progression [ 9 , 10 , 11 , 12 ]. Many proteomics studies are also being conducted to identify novel proteins and biological functions in plants [ 13 , 14 ] and on model organisms such as Tetrahymena thermophila to elucidate the involvement of many key proteins in the basis of biological processes [ 15 ]. There are several reports in the literature on the use of proteomic methods in antidoping tests [ 16 ], early medical diagnosis [ 17 , 18 , 19 , 20 ], and candidates for vaccine production [ 21 ].…”