Identification and mapping of central pair proteins by proteomic analysis

Abstract

16Cilia or flagella of eukaryotes are small micro-hair like structures that are indispensable to 17 single-cell motility and play an important role in mammalian biological processes. Cilia or flagella 18 are composed of nine doublet microtubules surrounding a pair of singlet microtubules called the 19 central pair (CP). Together, this arrangement forms the canonical and highly conserved 9+2 20 axonemal structure. The CP, which is a unique structure exclusive to motile cilia, is a pair of 21 structurally dimorphic singlet microtubules decorated with numerous associated proteins. 22Mutations of CP-associated proteins cause several different physical symptoms termed ciliopathies. 23Thus, it is crucial to understand the architecture of the CP. However, the protein composition of 24 the CP was poorly understood. This was because identification of CP proteins was mostly limited 25 by available Chlamydomonas mutants of CP proteins. In this study, we conducted a comprehensive 26 CP proteome analysis using several CP mutants and identified 37 novel CP protein candidates. By 27 using Chlamydomonas strains lacking specific CP sub-structures, we also present a more complete 28 model of localization of known and newly identified CP proteins. This work has established a new 29 foundation for CP protein analysis for future studies. 30 31 33 34 35 the central pair (CP) [8]. This specific arrangement defines the "9+2" structure of the axoneme 60 ( Fig. 1A). There exists axonemal dyneins (outer dynein arm, ODA; and inner dynein arm, IDA) 61 attached to DMTs which are responsible for the beating of cilia. Radial spoke (RS) complexes are 62 extending from DMTs toward the CP. Intraflagellar transport (IFT) driven by IFT dyneins and IFT 63 kinesins takes place on the DMTs [9]. This arrangement of the axoneme structure is highly 64 conserved in all eukaryotes with motile cilia, suggesting that there exists a similar set of proteins 65 and processes required for similar functional output. Thus, we can study the axoneme composition 66 using model organisms like a green alga Chlamydomonas reinhardtii. 67The presence of the CP distinguishes motile cilia from its immotile counterpart, primary 68 cilia. The CP works in a diverse array of function including the regulation of local Ca 2+ 69 concentration, ATP/ADP concentration and axonemal dynein activities through mechanical 70 interactions with RS[10-13]. The CP is a huge protein complex composed of a pair of structurally 71 and functionally dimorphic singlet microtubules named C1 and C2 and many other associated 72proteins [14]. The CP has a variety of sub-structures such as C1a, C1b, C1c and C1d on the C1 73 singlet, or C2a, C2b and C2c on the C2 singlet as characterized by traditional cross-sectional 74 electron microscopy (EM) (Fig. 1B). C1 and C2 microtubules are connected by a structure called 75 the "bridge" and "diagonal link". With recent higher resolution cryo-electron tomography (cryo-76 ET) structures, more details of these sub-structures have been characterized allowing the C1a ...

Remodeling and activation mechanisms of outer arm dyneins revealed by cryo‐EM

“…Using BLAST on UniProt database, Q22T00 was identified as a homolog for CCDC151. To see the MS profile of Q22T00, MS analyses were performed for untreated and salt-treated doublet samples as described in Dai et al (2020). The MS profiles of Q22T00 were similar to DC2 and DC3 (Appendix Table S1).…”

The inner junction complex of the cilia is an interaction hub that involves tubulin post-translational modifications

“…Sample preparation and mass spectrometry of FAP52 mutant and relative quantification compared to wild type Chlamydomonas was done according to Dai et al, 2019. (57).…”

Remodeling and activation mechanisms of outer arm dyneins revealed by cryo-EM

“…When we blasted CCDC151, we found Q22T00 as a homolog. To see the MS profile of Q22T00, MS analyses were performed for untreated and salt-treated doublet samples as described in Dai, D, et al, (33). The MS profiles of Q22T00 were similar to DC2 and DC3 (Table S1).…”