FBB18 participates in preassembly of almost all axonemal dyneins independent of R2TP complex

Limei Wang; Xuecheng Li; Guang Liu; Junmin Pan

doi:10.1371/journal.pgen.1010374

. 2022 Aug 26;18(8):e1010374. doi: 10.1371/journal.pgen.1010374

FBB18 participates in preassembly of almost all axonemal dyneins independent of R2TP complex

Limei Wang ^1,^#, Xuecheng Li ^1,^#, Guang Liu ¹, Junmin Pan ^1,^2,^*

Editor: Susan K Dutcher³

PMCID: PMC9455862 PMID: 36026524

Abstract

Assembly of dynein arms requires cytoplasmic processes which are mediated by dynein preassembly factors (DNAAFs). CFAP298, which is conserved in organisms with motile cilia, is required for assembly of dynein arms but with obscure mechanisms. Here, we show that FBB18, a Chlamydomonas homologue of CFAP298, localizes to the cytoplasm and functions in folding/stabilization of almost all axonemal dyneins at the early steps of dynein preassembly. Mutation of FBB18 causes no or short cilia accompanied with partial loss of both outer and inner dynein arms. Comparative proteomics using ¹⁵N labeling suggests partial degradation of almost all axonemal dynein heavy chains (DHCs). A mutant mimicking a patient variant induces particular loss of DHCα. FBB18 associates with 9 DNAAFs and 14 out of 15 dynein HCs but not with IC1/IC2. FBB18 interacts with RuvBL1/2, components of the HSP90 co-chaperone R2TP complex but not the holo-R2TP complex. Further analysis suggests simultaneous formation of multiple DNAAF complexes involves dynein folding/stability and thus provides new insights into axonemal dynein preassembly.

Author summary

Motile cilia are important for human physiology and defects in cilia motility may cause human disorders such as male infertility and primary ciliary dyskinesia. The motility of cilia requires preassembly of axonemal dyneins. Using a combination of genetic and other approaches, we have studied the working mechanism of FBB18, a Chlamydomonas homologue of CFAP298, defects in which result in primary ciliary dyskinesia. We found that FBB18 participates in dynein folding/stability in the cytoplasm, which is distinct from its proposed function in ciliary targeting of dynein complexes or stabilization of dynein arms within cilia. In addition, we have provided evidence that multiple distinct complexes are simultaneously formed to participate in dynein folding, thus providing new insights into dynein preassembly. Last but not least, we showed that RuvBL1/2 of the HSP90 co-chaperone R2TP complex may function independently of the R2TP complex in dynein disassembly. This work has both scientific and medical significance and will be of general interest to the fields of ciliary biology and protein folding/stability.

Introduction

Motile cilia and flagella (exchangeable terms) are highly conserved throughout eukaryotes. The beating of a cilium provides forces for cell swimming in unicellular organisms. Its role is expanded in mammals. In addition to propelling sperm movement, it functions in circulation of cerebrospinal fluid in the brain, mucus clearance in airways, egg movement in fallopian tubes, sperm transport from testis to epididymis and nodal flow in embryonic node. Ciliary beating requires axonemal dyneins, which consist of heavy chains (HCs), intermediate chains (ICs), light intermediate chains (LICs) and light chains (LCs) while the exact composition of each dynein differs [1, 2]. The axonemal dyneins are organized into outer and inner dynein arms (ODAs and IDAs) as revealed by electron microscopy. ODAs from Chlamydomonas, a unicellular organism widely used for ciliary studies, are three-headed dyneins that are composed of three HCs (DHCα, β, γ), a pair of IC1/IC2 and several other subunits. IDAs include one two-headed dynein and 10 monomeric dyneins. A total of 15 axonemal dynein HCs including three outer arm dynein HCs and 12 inner arm dynein HCs are present in Chlamydomonas while mammals have 5 outer and 8 inner arm dynein HCs [3, 4]. Mutations in dynein subunits impair ciliary motility, leading to primary ciliary dyskinesia characterized by chronic respiratory infections, male infertility and laterality defects [5].

The dynein arm assembly requires preassembly of the dynein complexes in the cytoplasm before they are transported to cilia and docked on the ciliary axoneme (see review [6]). In the cytoplasm, dynein HCs as well as ODA ICs are properly folded and stabilized, assembled into a complex with other dynein subunits followed by maturation, packaging and targeting to cilia. More than a dozen factors have been shown to be dedicated to these processes and termed dynein, axonemal, assembly factors (DNAAFs) [2,6–8]. DNAAF12/LRRC56 and DNAAF11/LRRC6 are predicted to mediate later steps of dynein preassembly or transport because the dynein HCs and ODA IC1/IC2 showed normal abundance in the cytoplasm with loss of dyneins in cilia [9–11]. DNAAF9/Shulin packages outer arm dynein complexes [12] and DNAAF18/ODA16 serves as a cargo adapter of outer arm dynein complexes for ciliary targeting via the intraflagellar transport machinery [13]. The other factors appear to be critical for folding and stabilization of dynein HCs which is reflected by reduced abundance upon mutations in DNAAFs [6, 8, 14] while ZMYND10 is also involved in stability of IC1/IC2 of ODA [14]. How these factors interact and cooperate to mediate folding and stabilization of dyneins remain obscure.

Chaperone HSP90 functions in the folding, stability and assembly of a wide array of protein complexes [15]. Several DNAAFs have been found to associate with HSP90 by immunoprecipitation [8], suggesting that DNAAFs function in recruiting dynein clients to HSP90. DNAAFs that directly interact with HSP90 from experimental evidence or prediction include TPR domain containing proteins DNAAF4/DYX1C1, DNAAF13/SPAG1 and its paralog RPAP3 [16]. DYX1C1 interacts with DNAAF2/PF13 or DNAAF6/TWI1 [16, 17]. SPAG1/RPAP3 are homologues of yeast Tah1, a component of a R2TP complex (Rvb1, Rvb2, Tah1 and Pih1 in yeast and RuvBL1, RuvBL2, RPAP3 and PIH1D1 in mammal), which is a HSP90 co-chaperone [16, 18, 19]. Mutations in the components of R2TP or its like complex in different organisms lead to instability of axonemal dyneins [16, 20–27]. In addition, DNAAF10/WDR92 interacts with the R2TP complex in vivo and it thus has been proposed that DNAAFs also recruit the dynein clients to HSP90 via the R2TP complex for folding and assembly [23, 24]. RuvBL1/2 may function independent of R2TP in the assembly of other protein complexes and have been proposed to possess chaperone activities [28]. However, it is not clear whether RuvBL1/2 alone may function in dynein folding and assembly.

CFAP298 (aka C21orf59) is a conserved protein among organisms with motile cilia [29, 30]. Mutations in human patient and zebrafish and RNAi depletion in planaria cause loss of both ODAs and IDAs [31, 32]. It has been proposed that CFAP298 functions in cilia to enhance ODA stability in addition to possible roles in cytoplasmic preassembly and transport of dynein complexes. However, no experimental data has been shown for its working mechanism in the cytoplasm. In this work, by studying FBB18, a Chlamydomonas homologue of CFAP298, we found that FBB18 is involved in folding and stabilization of almost all outer and inner dynein HCs but not in ciliary targeting or stabilization ODAs within cilia. FBB18 forms an interacting network(s) with other nine DNAAFs. More importantly, this study shows that multiple DNAAF complexes are simultaneously formed during folding and stabilization of dynein HCs and RuvBL1/2 may function independently of a R2TP complex in dynein preassembly.

Results

An fbb18 mutant shows defects in ciliogenesis and ciliary motility

FBB18, a Chlamydomonas homologue of CFAP298, is a protein of 277 residues with two conserved domains: a coiled-coil domain and a domain of unknown function DUF2870, which are also present in its human homologue (Fig 1A). We identified an fbb18 mutant (fbb18-1) in which the 2068–2069 nt in the last exon is replaced by a foreign DNA insert (Fig 1B). In contrast to wild-type strain, which is unicellular with two cilia of about 12 μm in length, most of the cells in fbb18-1 formed multicellular aggregates known as palmelloids or sporangia when grown in exponential growth phase. Cells were hatched from sporangia from unknown reason when grown at stationary growth phase and had no or short cilia (Fig 1C and 1D). The cells with cilia were immotile as revealed under microscopic observation (see also Fig 2). Thus, the mutant is defective in both ciliogenesis and motility.

Fig 1 — (A) Diagrams showing the domain structures of FBB18 and its human counterpart CFAP298. The numbers indicate the positions of the residues. CC, coiled-coil domain. (B) Schematic illustration of the *FBB18* gene shows the insertion of a foreign DNA fragment containing paromomycin resistance gene *aphVIII*. The insertion was identified by PCR and DNA sequencing. Grey box, exon; line, intron; numbers, nucleotide positions. (C) Ciliary phenotypes of *fbb18-1* grown in exponential (E) and stationary (S) growth phases. (D) DIC cell images show the ciliary phenotype of wild-type (WT), *fbb18-1*, transformants expressing full-length (FL) or truncated (Δ252–277) gene. Bar, 5 μm. (E) Immunoblot analysis of wild-type (WT), *fbb18-1* and rescued cells expressing *FBB18*:*HA-TG* with the indicated primary antibodies.

Fig 2 — (A) TEMs of cilia cross-sections show a partial loss of axonemal outer (ODA, black arrowhead) and inner (IDA, white arrowhead) dynein arms in mutants. Bar, 100 nm. (B) Statistical analysis of ODA loss in the mutants. Numbers, TEM cross-sections examined. (C) Reduced abundance of axonemal dyneins in cilia of *fbb18* mutants. Representative axonemal dynein HCs were examined from isolated cilia of cells as indicated by immunoblotting. α-tubulin was used as a control. The abundances of dynein HCs were quantitated by densitometry. The ratios of dynein HC abundance (*wt/fbb18-1/Δ252–277*) with normalization to α-tubulin are the following: DHCα, 1.00:0.02:0.02; DHCβ, 1.00:0.73:1.00; dynein b, 1.00:0.00:0.75; dynein c, 1:0.38:0.90; dynein e, 1:0.44:1.01. (D-E) *FBB18*^Δ252–277 shows no defect in ciliary length (D) but with reduced beating frequency (D). Data were presented as mean ± SD. ns, not significant; *, p<0.05; ***, p<0.0001. (F-G) *FBB18*^Δ252–277 exhibits loss of DHCα as shown in TEM cross-section (F) and statistical analysis (G). White arrowhead shows loss of DHCα in the mutants. Bar in F, 10 nm; Numbers in G, ODAs examined.

FBB18 antibody recognized a band around the predicted molecular weight of FBB18 in the wild-type cells but not in the mutant, indicating that FBB18 expression was disrupted (Fig 1E). Expression of a full-length FBB18 tagged with HA (FBB18:HA-TG) in fbb18-1 rescued the ciliary defects of the mutant in motility and ciliary length (Fig 1D and 1E). 23% of the transformants from at least three independent transformations showed normal ciliary phenotype, indicating that the rescue was caused by incorporation of the transgene and not by random disruption of some other genes. Thus, we have identified a null mutant of FBB18 with defects in both ciliogenesis and ciliary motility.

Null mutation of FBB18 causes partial loss of ODAs and IDAs

CFAP298 mutation in human or RNAi depletion of its homologues in zebrafish and planaria respectively alters cilia motility with loss of ODAs [31, 32]. Consistent with this, transmission electron microscopy (TEM) showed that fbb18-1 cilia exhibit partial loss of both ODAs and IDAs (Fig 2A and 2B).

To further confirm the dynein arm defects in the cilia of fbb18-1 mutant, we examined two ODA heavy chains (ODAα and ODAβ) and three IDA heavy chains (dynein b, c and e) by immunoblotting. Isolated cilia from wild-type, rescue, oda1, oda5 and pf22 cells were used as controls. ODA1 encodes a component of ODA-docking complex (ODA-DC) and mutation of which induces loss of ODA [33]. ODA5 is involved in cytoplasmic maturation of ODAs for their binding to doublet microtubules [34]. PF22/DNAAF3 regulates the assembly of both ODAs and IDAs [7]. As expected, DHCα and DHCβ were not detected in oda1 and oda5 cilia while IDA components were not affected; and reductions in both ODA and IDA components were observed in pf22 cilia (Fig 2C). In cilia of fbb18-1, DHCα and dynein b were undetectable while the levels of DHCβ, dynein c and dynein e were reduced compared to the wild-type and rescued cells (Fig 2C). The reduced protein levels of the dynein heavy chains are consistent with the partial loss dynein arms (Fig 2A and 2B). Thus, FBB18 is required for proper assembly of both ODA and IDA in cilia.

FBB18^Δ252–277 that mimics truncated mutation in human patients leads to particularly loss of DHCα in cilia

FBB18 and its human homologue CFAP298 both have a coiled-coil and a DUF2870 domain (Fig 1A). To test the function of these domains, HA-tagged FBB18 with deletion of either domain was expressed respectively in fbb18-1 (S1A Fig). The ciliary defects of fbb18-1 in the transformants were not rescued. We found that the expression levels of the deletion mutants were much lower than that of the wild-type gene (S1B Fig), thus preventing us to establish the functions of the deleted domains. The lower expression suggests that the mutated proteins may not be stable or the corresponding mRNAs may be subjected to decay.

A PCD patient carrying truncation mutations (p.Arg98*, p.Tyr264*) exhibits less severe defects in dynein arms than patients with homozygous truncating mutations (p.Tyr245*) [31]. Examination of the pathogenicity of the mutation (p.Tyr264*) in zebrafish was not successful because the mutated protein was expressed at extremely low level [31]. p.Tyr264* mutation may lead to nonsense mediated mRNA decay, it might also result in a truncated protein with disruption of the DUF2870 domain (Fig 1A). To test the pathogenicity of a truncated protein in Chlamydomonas, a corresponding mutant FBB18^Δ252–277 tagged with HA was expressed in fbb18-1. The expression level of FBB18^Δ252–277 was comparable to that of wild-type FBB18 (S1B Fig). FBB18^Δ252–277 mutant had normal length of cilia while the ciliary beating frequency was reduced ~40% compared to the wild-type control (Figs 1D and 2D and 2E and S1–S3 Videos). TEM examination showed that it had partial loss of dynein arms but not as severe as the fbb18-1 null mutant (Fig 2A and 2B). Immunoblotting of isolated cilia showed that it specifically lost DHCα compared to the wild-type and the null mutant (Fig 2C). Chlamydomonas ODA forms a three heads architecture with DHCα on the outermost position [35]. Indeed, closer examination of the ODAs revealed that DHCα was deficient from the ODAs of fbb18^Δ252–277 cilia as well as in the fbb18-1 cilia (Fig 2F and 2G). These data suggest that the assembly of DHCα particularly depends on the function of the C-terminal DUF2870 domain. Whether FBB18^Δ252–277 is a good model for studying the pathogenicity of the human variant (p.Tyr264*) depends on future verification if this mutation leads to synthesis of a truncated protein.

FBB18 is involved in cytoplasmic preassembly of axonemal dyneins

The mRNA level of FBB18 is immediately up-regulated after deciliation and returned to pre-deciliation level at 60 min post-deciliation [36]. Immunoblot analysis showed that the protein level of FBB18 increased at 30 min after deciliation when about half length of cilia are assembled (Fig 3A), indicating cells utilize a preexisting pool of FBB18 during earlier ciliogenesis [37]. It was intriguing how FBB18 regulates the assembly of dynein arms. It was reported that FBB18 was present in cilia with predominant localization in the ciliary membrane/matrix. In addition, its protein level increased in the cell as well as in cilia of several motility mutants of Chlamydomonas [31]. It was thus proposed that FBB18 functions in the stability of dynein arms in cilia and/or ciliary targeting of dynein arms. We confirmed the distribution of FBB18 in cilia (Fig 3B). However, we failed to observe significantly increased level of FBB18 in the whole cell samples of several motility mutants. The changes in the level of FBB18 in cilia from these mutants were not consistently associated with the impaired motility phenotype, which are different from a previous report [31] (S2 Fig). One possible explanation for this discrepancy is that the loading control was not shown in that study.

Immunoblotting of FBB18 in the cell body versus cilia showed that it was predominantly localized in the cell body (Fig 3C), which is consistent with the prominent presence of CFAP298 in the cell body of rat trachea ciliated cells shown by immunostaining [31]. FBB18-HA was distributed in the cell body with punctate patterns (Fig 3D), which is in contrast to the reported enrichment at the basal bodies of its mammalian homolog [31]. Whether the FBB18-HA puncta represent the dynein axonemal particles (DynAPs) as being reported in Xenupus remains to be tested [38].

The predominant localization of FBB18 in the cell body and loss of dynein arms in the fbb18 mutants suggest that FBB18 is likely involved in dynein preassembly. We examined dynein heavy chains in fbb18 mutants by immunoblotting. Unlike oda1 and oda5 mutants, but similar to the pf22 mutant, both fbb18-1 and FBB18^Δ252–277 mutants showed decreased level of DHCα and dynein b compared to the wild-type control (Fig 3E). Thus, FBB18 likely functions to stabilize dynein HCs.

To demonstrate whether FBB18 is associated with dynein heavy chains, wild-type cell extract was subjected to immunoprecipitation with anti-FBB18 or rabbit IgG (control) followed by immunoblotting. FBB18 antibody but not IgG pulled down two axonemal dyneins that were examined (Fig 3F). Taken together, a predominantly localization of FBB18 in the cell body, its association with dynein HCs and reduced abundance of dynein HCs in the fbb18 mutants suggest that FBB18 is a bona fide DNAAF to serve as a co-chaperone to fold/stabilize axonemal dyneins.

Differential regulation of the stability of axonemal dyneins by FBB18

After confirmation that FBB18 is indeed a DNAAF, we next determined which axonemal dynein is regulated by FBB18 by comparative proteomics. The cell extracts from ¹⁵N labelled fbb18-1 and unlabeled rescued cells (¹⁴N) or vice versa were mixed in an equal amount followed by SDS-PAGE. The top gel region that contains the dynein HCs was sliced for mass spectrometry analysis. All dynein HCs except four IDA minor HCs were identified (Table 1) [3]. Loss of FBB18 induced reduction in the abundance of axonemal dyneins in different degrees. For the IDA heavy chains, it resulted in 10% to 70% reductions depending on the dynein species. For the ODA heavy chains, DHCγ had no changes, DHCβ showed about 10% reduction while DHCα was reduced ~90%, which is consistent with the immunoblotting data (Fig 3E). Please note, the extent of reduction in the abundance of dyneins shown by immunoblotting does not match very well with the proteomic data, which may reflect the limitations in protein quantifications by using immunoblotting. It is intriguing that FBB18 strongly affect the stability of DHCα relative to DHCβ and DHCγ. The three HCs of ODA show high similarity in the amino sequences in the motor domains, however, the N-terminus of DHCα possesses a unique kelch-type_b-propeller domain (residues 4–953). It raises a possibility that FBB18 may directly interact with this region, which could be tested in future studies. Interestingly, the abundance of IFT dynein was increased around 3 fold in the mutant (Table 1), suggesting that its folding/stability does not depend on FBB18. This is consistent with a previous report in which DNAAF is not involved in the folding of cytoplasmic dynein [39]. This increase in abundance may reflect a feedback control due to no or short cilia in the mutant. Taken together, FBB18 differentially regulates the stability of dynein HCs of both ODAs and IDAs.

Table 1. Changes in protein abundance of axonemal dyneins in fbb18-1 versus rescued cells.

	Protein/Gene	fbb18-1 labeling		control labeling		Average Ratio (fbb18/control)
	Protein/Gene	PSM Count	fbb18/ control	PSM Count	fbb18/ control	Average Ratio (fbb18/control)
IDA HCs	DHC 1α/DHC1	132	0.91	107	0.89	0.9
	DHC 1β/DHC10	70	0.31	33	0.46	0.38
	dynein a/DHC6	48	0.77	35	0.86	0.81
	dynein b/DHC5	33	0.5	15	0.42	0.46
	dynein c/DHC9	92	0.26	54	0.32	0.29
	dynein d/DHC2	52	0.52	39	0.70	0.61
	dynein e/DHC8	19	0.67	9	0.53	0.6
	dynein g/DHC7	73	0.37	52	0.43	0.4
ODA HCs	DHC α/DHC13	374	0.09	235	0.14	0.11
	DHC β/DHC14	291	0.88	240	0.94	0.91
	DHC γ/DHC15	130	0.99	131	1.06	1.02
IFT dynein	DHC1b/DHC16	404	3.75	450	2.22	2.98

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The cell extracts from ¹⁵N labeled fbb18-1 and unlabeled control cells with equal amount of proteins were mixed, or vice versa, and analyzed by SDS-PAGE and mass spectrometry.

FBB18 physically associates with almost all axonemal dyneins

Next, we determined which dynein heavy chain species might physically associate with FBB18. To this end, FBB18:YFP-TG was expressed in fbb18-1. The transgenic strains restored the wild-type ciliary phenotypes. Cell lysates of the rescued cells were subjected to immunoprecipitation with anti-GFP antibody, wild-type cells as a control. (S3 Fig). Mass spectrometry analysis of the immunoprecipitates showed that 14 out of a total of 15 axonemal dyneins were associated with FBB18 (Table 2).

Table 2. FBB18 associates with almost all axonemal dyneins.

Group	Gene/Protein	Gene ID	Control Spectral count (unique peptides)	FBB18:YFP-TG Spectral count (unique peptides)
ODA components	DHC13/DHCα	Cre03.g145127	22 (19)	63 (46)
	DHC14/DHCβ	Cre09.g403800	58 (37)	111 (66)
	DHC15/DHCγ	Cre11.g476050	15 (12)	52 (29)
IDA components	DHC1/DHC1α	Cre12.g484250	15 (14)	57 (43)
	DHC2/dynein d	Cre09.g392282	7 (6)	98 (57)
	DHC3 (minor type dynein)	Cre06.g265950	-	2 (2)
	DHC4 (minor type dynein)	Cre02.g107350	-	11 (1)
	DHC5/dynein b	Cre02.g107050	-	14 (4)
	DHC6/dynein a	Cre05.g244250	-	15 (13)
	DHC7/dynein g	Cre14.g627576	-	4 (1)
	DHC8/dynein e	Cre16. g685450	1 (1)	20 (13)
	DHC9/dynein c	Cre02.g141606	-	252 (106)
	DHC10/DHC1β	Cre14.g624950	3 (3)	34 (30)
	DHC11 (minor type dynein)	Cre12.g555950	4 (3)	11 (10)
	DHC12 (minor type dynein)	Cre06.g297850	-	-

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Immunoprecipitates with anti-GFP antibody from fbb18-1 cells expressing FBB18:YFP-TG or wild-type cells as control were analyzed by mass spectrometry. Please note, except for the minor dynein DHC12, all the other dyneins were enriched more than 2 fold in the immunoprepitate of FBB18:YFP-TG.

To learn whether FBB18 associates with dynein HCs during or after their translation, we examined the identified peptides of DHCα because its stability is severely affected by the loss of FBB18. We found that several identified peptides are located within the C-terminal region of DHCα (S4 Fig), suggesting that FBB18 may mediate folding of completely translated dynein HCs though we could not exclude the possibility that it also functions during dynein translation. We also examined the accessory components of the outer and inner arm dyneins in the immunoprecipitation–mass spectrometry data (S1 Table). For outer arm dyneins, none of the accessory components including IC1 and IC2 were enriched with FBB18-YFP. Interestingly, only three but none of the other subunits of IDA were enriched. IC140 and IC97 are components of double-headed dyneins while p28 is a component of dynein a, c, and d [1]. Taken together, these data suggest that FBB18 participates in the folding/maturation of most of the DHCs and likely some of the IDA accessory components before they are assembled into large dynein complexes for transporting into cilia though we do not exclude other possibilities.

FBB18 is in association with several DNAAFs, HSP90 and RuvBL1/2 of the R2TP complex

DNAAFs often form a complex with other DNAAFs, R2TP and chaperones [6, 23, 24]. To learn the protein factors that associate with FBB18 and function in dynein preassembly, we searched known DNAAFs, R2TP components and chaperones in the immunoprecipitates as described above. 16 out of 18 designated DNAAFs are conserved in Chlamydomonas [2]. 10 of 16 conserved DNAAFs including FBB18, chaperones HSP90 and RuvBL1/2 of the R2TP complex were identified (Table 3). These data suggest that FBB18 forms a network(s) with these factors to confer its function in axonemal dynein preassembly.

Table 3. Factors associated with FBB18 that are involved in dynein preassembly.

Proteins	Gene ID	Control Spectral count (unique peptides)	FBB18-YFP Spectral count (unique peptides)
DNAAFs that are associated with FBB18
DNAAF1/DAU1/ODA7	Cre01.g029150	-	11 (6)
DNAAF2/ PF13	Cre09.g411450	-	7 (5)
DNAAF3/ PF22	Cre01.g001657	-	28 (7)
DNAAF4/ DYX1C1/ PF23	Cre11.g467560	-	3 (3)
DNAAF5/ HEATR2	Cre09.g395500	44 (21)	94 (25)
DNAAF6/ TWI1/ PIH1D3	Cre07.g335800	-	9 (5)
DNAAF10/WDR92	Cre16.g672600	-	3 (3)
DNAAF11/MOT47/ LRRC6	Cre17.g739850	1(1)	5 (4)
DNAAF16/FBB18/CFAP298	Cre16.g688450	-	582 (31)
DNAAF17/FBB5/ C11orf70/CFAP300	Cre12.g556300	3 (3)	37 (14)
DNAAFs that are not associated with FBB18
DNAAF7/ZMYND10	Cre08.g358751	19(7)	12(6)
DNAAF9/Shulin/C20orf194	Cre11.g467556	1(1)	1(1)
DNAAF12/LRRC56/ODA8	Cre01.g043650	-	-
DNAAF13/SPAG1	Cre12.g556100	1(1)	(1)
DNAAF14/MOT48	Cre10.g467050	-	-
DNAAF18/DAW1/ODA16/WDR69	Cre04.g216902	-	-
RPAP3	Cre02.g084900	-	-
Chaperones and co-chaperones
RuvBL1	Cre09.g414050	9 (5)	220 (25)
RuvBL2	Cre10.g442700	7 (5)	188 (29)
HSP90A	Cre09.g386750	17 (9)	48 (17)
HSP90C	Cre12.g514850	-	8 (6)
HSP70E	Cre16.g677000	3 (2)	8 (3)

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Immunoprecipitates from cells expressing FBB18:YFP-TG or control cells with anti-GFP antibody were analyzed by mass spectrometry. Known dynein preassembly factors, R2TP components and chaperones were searched in the data set and their abundances are presented. Please note that the homologues of DNAAF8 and 15 are not present in the Chlamydomonas proteome. RPAP3 forms a R2TP complex with DNAAF14 and RuvBL1/2 and is thus being considered as a possible DNAAF (PMID: 30428028).

The other six DNAAFs that were not found to be associated with FBB18 include DNAAF7, 9, 12–14 and 18 (Table 3). DNAAF7/ZMYND10 is involved in stabilization of IC1/2 [14] though different results have been reported [40]. DNAAF9/Shulin functions in packaging outer arm dynein complexes for ciliary targeting while DNAAF12/LRRC56 and DNAAF18/ODA16 are involved in maturation and dynein transport, respectively [9, 12, 13]. Thus, these data suggest that FBB18 functions in the chaperoning steps of axonemal dynein HCs but not in IC1/2 stabilization and in maturation/transport of the dynein complexes. Interestingly, the other two DNAAFs that were not associated with FBB18 are DNAAF13/SPAG1 and DNAAF14/MOT48, a PIH domain containing protein. R2TP complexes consist of RuvBL1/2, SPAG1 or RPAP3 and one of the PIH domain containing proteins [16]. SPAG1 or RPAP3 interacts with the PIH containing proteins in the R2TP complex. RPAP3, the paralog of SPAG1, was also not identified (Table 3). Previously, we have shown that MOT48 forms a R2TP complex with RuvBL1/2 and RPAP3 [23]. Thus, these data suggest that FBB18 does not associate with a R2TP or its like complex.

Deciphering the interaction map of the FBB18-associated DNAAFs

Given the association of FBB18 with other DNAAFs revealed by affinity purification, it is intriguing how these factors are organized. Based on previously published data and the data obtained in the current study (see below), we propose a model for their interactions in Chlamydononas (Fig 4A). The solid lines indicate interactions derived from previous studies while dashed lines show the interactions revealed by the current study. FBB18 directly interacts with WDR92, ODA7 and LRRC6 as shown by yeast two-hybrid (Y2H) and/or in vitro pull-down assays [23, 32]. LRRC6 directly associates with RuvBL2 in Y2H assay [22]. DYX1C1 directly interacts with either of the two PIH proteins PF13 and TWI1 as shown by LUMIER interaction assay [16], and PF13 but not TWI1 interacts directly with FBB5 as shown by the Y2H and co-immunoprecipitation assays [41].

Fig 4 — (A) A model of molecular interaction map of FBB18. Dotted and solid lines represent interactions revealed by current and previous studies, respectively. (B) Y2H assays show direct interaction of FBB18 with LRRC6, DY1X1C1 but not the other proteins tested. Yeast cells that were transformed with each pair of constructs were grown under selection media lacking leucine, tryptophan, histidine, and adenine (-4) or leucine and tryptophan (-2). Empty AD or BD vectors were used as controls. (C) Reciprocal pull-down assays show interaction between FBB18 and DNAAF3/PF22. Bacterial expressed proteins were mixed as indicated and pulled down with glutathione beads followed by immunoblotting with GST and His antibodies, respectively. GST was used as a control. (D) Pull-down assays show interaction between FBB18 and RuvBL1/2. An assay similar to (C) was performed. GST and His antibodies were used for immunoblotting, respectively. (E) Sucrose gradient assay shows that FBB18 forms complexes with RuvBL1/2. Bacterial expressed proteins as indicated were subjected to sucrose gradient analysis followed by immunoblotting with His or Myc antibodies.

Previously published data could not place PF22, HEATR2, DYX1C1 in the above mentioned network associating with FBB18. We first considered possible direct interaction of FBB18 with PF22, DYX1C1, RuvBL1/2 or FBB5 due to their relative high abundance in the FBB18-YFP immunoprecipitates. HEATR2 was not considered because of high spectral counts in the control immunoprecipitation and only ca. 2 fold enrichment (Table 3). In Y2H assays, FBB18 interacted with LRRC6, confirming a previous study [22] and DYX1C1, but not with PF22, RuvBL1, RuvBL2 and FBB5 (Fig 4B). PF22 and RuvBL1/2 are among the most enriched proteins associating with FBB18 (Table 3). Given the limitations of the Y2H assays, we employed pull-down assays with bacterial expressed proteins. FBB18 and PF22 interacted in reciprocal pull-down assays (Fig 4C). FBB18 interacted with RuvBL1/2 complexes (Fig 4D). To further demonstrate the interaction between FBB18 and RuvBL1/2, bacterial expressed FBB18 and RuvBL1/2 were mixed and analyzed by sucrose gradient and immunoblot analysis (Fig 4E). A portion of FBB18 co-migrated with RuvBL1/2, confirming their interactions. Thus, we have provided an interacting network(s) of FBB18 with other DNAAFs. However, given a small protein like FBB18, how can it directly interact with several DNAAFs? One possibility is that these interactions may not occur simultaneously.

Discussion

Regulation of ciliary assembly and length by the assembly of axonemal dynein arms

The null mutant of FBB18 exhibits defects not only in dynein arm assembly but also in ciliogenesis for having short or no cilia. This phenomenon has been reported in Chlamydomonas mutants of several other DNAAFs including DNAAF10/WDR92 [23, 39], DNAAF14/MOT48 [20], DNAAF2/PF13 [42, 43], DNAAF4/DYX1C1/PF23 [43, 44] and DNAAF3/PF22 [7, 45]. Furthermore, a double mutant of pf13/mot48 that causes sever loss of outer and inner dynein arms exhibits much shorter cilia than either single mutant [27]. Thus, it suggests that axonemal dynein arms play a role in ciliary assembly and length control, which is supported by studies of mutants of dynein motors. Complete loss of ODAs in an IC1 mutant (oda9) or a DHCγ mutant (pf28) does not or slightly reduce ciliary length [45, 46]; the same is true for single loss of several inner arm dyneins [47]. However, double mutants with complete loss of outer dynein arms and individual inner arm species exhibit significantly shorter cilia [45, 47]. Thus, the occupancy of dynein arms in the ciliary axoneme facilitates the stabilization of axoneme and controls ciliary length. This view is supported by the data in which knockout of a tubulin polyglutamylase in a wdr92 mutant partially restores ciliary length [39]. In several DNAAF mutant patients, the sperm flagellar length is also significantly reduced [48]. However, the length of motile cilia in mammals may not be controlled by the presence of dynein arms. Knockout of DNAAF7/ZMYND10 or DNAAF11/LRRC6 in mouse does not change ciliary length of motile cilia [10, 40]. Thus it appears that the stability of the motile ciliary axoneme may be mainly controlled by other microtubule binding proteins and/or tubulin post-translational modifications [49, 50].

FBB18 is involved in chaperoning axonemal dynein HCs at the early steps of dynein preassembly

FBB18 was proposed to function in stabilization of dynein arms in cilia and/or in ciliary targeting of dynein complexes [31]. Our data demonstrate that it likely acts at the early steps of axonemal dynein preassembly in chaperoning dynein HCs before formation of dynein holo-complexes. First, FBB18 is predominantly localized in the cytoplasm, similar to what has been described in rat tracheal cells and KV of zebrafish embryos [31, 32]. Second, loss of FBB18 causes instability of multiple axonemal dynein HCs. Third, FBB18 is associated with 14 out of a total of 15 axonemal dynein HCs. None of the accessory components of the outer arm dyneins including IC1/IC2 were associated with FBB18. Except for the three accessory components (IC140, IC97 and p28), the other accessory subunits of the inner arm dyneins were not associated with FBB18. Fourth, DNAAFs involved in dynein maturation (DNAAF12/LRRC56) [9], packaging (DNAAF9/Shulin) [12] and transport (DNAAF18/ODA16) [13] are not associated with FBB18. Taken together, we propose that FBB18 with its associated DNAAFs is involved in the folding of dynein HCs and likely the three accessory components as well before a holo-complex of dyneins is formed.

The specificity of DNAAF in chaperoning axonemal dynein HCs

One feature for most of the DNAAFs is that its loss causes severe instabilities of axonemal dynein HCs. To learn the specificity of each DNAAF towards folding/stability of axonemal dyneins, it requires examination of all the dyneins present in particular ciliated cells or organisms. Traditional TEM microscopy, immunoblotting using a few dynein antibodies or even cryo-electron tomography without molecular resolution would not provide such a detailed information. Analysis of dyneins from whole cell sample by mass spectrometry would be an attractive method [40]. By using comparative proteomics with ¹⁵N labeling, we have detected 11 out of a total of 15 axonemal dynein species, the other four undetected species are minor type dyneins [3]. The abundance of all detected dyneins except for DHCγ was reduced with different degrees in fbb18-1 mutant. Given that FBB18 physically associates with all of these dyneins, it is proposed to participate in the preassembly of all these dyneins. In a comparative proteomics of mouse testes samples, 9 out of 13 mammalian axonemal dynein species were detected with reduced abundance in zmynd10 null mouse [40]. This study combined with our current study suggest that DNAAFs may have a plasticity nature and have broad functional specificity. Each DNAAF may contribute to the assembly of multiple dyneins with differential preference. Thus, mutation in each DNAAF would cause different outcomes in the instability of dyneins. However, other possibilities exist. The fact that FBB18 associates with almost all of the HCs but has differential regulation of their stability suggests that FBB18 may not directly interact with all of them. It may serve as a part of a network of DNAAFs to facilitate dynein folding. Disruption of the network may result in differential folding of the dyneins, leading to differential instability.

Formation of multiple DNAAF complexes during folding and stabilization of axonemal dyneins

In the immuoprecipiates of FBB18, a total of 10 DNAAFs have been found including FBB18. Using Y2H, pull-down and other biochemical assays combined with previous literatures, we have proposed an interacting network(s) of these DNAAFs. These DNAAFs may at least form two distinct complexes. FBB18 interacts with DNAAF4/DYX1C1, which interacts with either PF13 or TWI1, both are PIH domain containing proteins [16, 17, 20, 27]. A third PIH domain containing protein in Chlamydomonas is DNAAF14/MOT48 [20]. Previously, we found that MOT48 forms a R2TP like complex with RuvBL1/2 and RPAP3 [23]. SPAG1 is a paralog of RPAP3, which can also form a R2TP like complex [16]. Interestingly, MOT48, SPAG1 and RPAP3 all were not found to associate with FBB18. Thus, it suggests that a R2TP like complex exists independently of FBB18. DNAAF7/ZMYND10 involves stabilization of IC1/IC2 and axonemal dynein HCs in mammals [14, 40]. Though mutants of ZMYND10 in Chamydomonas have not been reported, its upregulation during ciliogenesis suggests that it may also function in axonemal dynein preassembly in Chlamydomonas [36]. We found that ZMYND10 was not associated with FBB18. ZMYND10 forms complexes with a few DNAAFs in mammalian cells [14]. Thus, it indicates that a ZMYND10 interacting module may also exist independently of FBB18 in Chlamydomonas. Taken together, in Chlamydomonas, at least 4 DNAAF complexes may be formed simultaneously to mediate folding of axonemal dyneins. It may be argued that most of the DNAAFs forms a loose network, which breaks apart during our immunoprecipitation. Though this is likely, three PIH domain containing proteins in Chlamydomonas and four in zebrafish are all involved in axonemal dynein stability [21, 27] and it is expected that each protein forms a discrete complex. These complexes may contribute to the folding of the same dynein species with different capacities, which can explain why mutations of different DNAAFs cause differential regulation of the same dynein species. For example, PIH1D1, KTU, and Twister all contribute to the assembly of inner arm dynein c [21]. Similar conclusions are also drawn in Chlamydomonas by systematic studies of PIH containing proteins [27]. Combined with discussion in the last section, we propose the rule governing the folding of dyneins by DNAAFs include two parts: each DNAAF may recruit multiple dyneins and each dynein may interact with more than one DNAAFs. This could explain the defects in one DNAAF lead to instability of multiple dyneins and the stability of one dynein is affected by more than one DNAAFs.

FBB18 associates with RuvBL1/2 but not a R2TP complex

R2TP complex is a HSP90 co-chaperone involved in folding and assembly of large protein complexes [18]. Recent evidences show that this complex is also involved in preassembly of axonemal dyneins [23, 24]. The conventional R2TP complex in mammal consists of RuvBL1/2, RPAP3 and PIH1D1. The TPR domains in RPAP3 interacts with HSP90 while PIH1D1 recruits chaperone clients [18]. Uncoventional R2TP complexes can be formed by substitution of RPAP3 with its paralog SPAG1 or by substitution of PIH1D1 with its paralogs PIH1D2 or DNAAF2/KTU [16]. In Chlamydomonas, we previously found that MOT48 forms a R2TP complex with RuvBL1/2 and RPAP3 [23]. Interestingly, our current data showed that FBB18 directly interacted with RuvBL1/2 while RPAP3, SPAG1 or MOT48 was not associated with FBB18, which suggests that RuvBL1/2 could function independently of formation of a R2TP complex. RuvBL1/2 belong to AAA+ superfamily of ATPases. RuvBL1/2 independently of R2TP complex formation have been shown to be involved in the assembly of many protein complexes including chromatin remodeling complexes, and have been proposed to be chaperones [28]. Thus, RuvBL1/2 are likely chaperones involved in axonemal dynein preassembly or serve as a molecular hub to organize DNAAFs.

Materials and methods

Strains and culture

Chlamydomonas reinhardtii wild-type strain 21gr (mt+; CC-1690) was obtained from the Chlamydomonas Resource Center (University of Minnesota, MN). Chlamydomonas mutants oda1, oda5, and pf22 were kindly provided by Dr. David Mitchell. Cells were routinely cultured in liquid M or TAP medium as indicated at 23°C with aeration under a 14:10h light/dark cycle.

Insertional mutagenesis, gene cloning and transformation

Insertional mutants were generated by electroporation of a DNA fragment containing AphVIII expressing cassette, which confers the transformants with paromomycin resistance [51, 52]. RESDA PCR combined with DNA sequencing was used to identify the genes with foreign DNA insertion [53]. FBB18 gene including an ~1.5-kb fragment upstream of the start codon was cloned by PCR. The gene was tagged with 3xHA or YFP followed by a rubisco terminator. The resulting constructs were cloned into a modified vector pHyg3 harbouring a hygromycin B resistance gene [54]. The constructs for the truncated mutants as indicated in the text were generated by PCR and cloning. The final constructs were linearized with KpnI for transformation.

Cell fractionation, ciliary regeneration, ciliary length measurement, and DIC cell imaging

Previous published methods were followed [55]. Briefly, cells were deciliated by pH shock followed by isolation of cell bodies and cilia or allowed for cilia regeneration. For cilia fractionation, isolated cilia were dissolved into HMDEK buffer containing 0.5% NP40 followed by centrifugation to separate the membrane/matrix and the axenemal fractions. For measurement of ciliary length or scoring ciliary phenotype, cells were fixed with 1% glutaraldehyde and imaged by DIC microscopy using Zeiss Axio Observer Z1 equipped with a CCD camera (QuantEM512SC, Photometrics, USA). Cell images were processed with Photoshop and Illustrator (Adobe, USA) and ciliary length was measured using ImageJ (NIH, USA). 50 cilia were measured to determine the mean length of cilia.

Ciliary beat analysis

Cells were imaged with a Nikon microscope (A1RSi) equipped with an ORCA-flash 4.0 camera (Hamamatsu, Japan). Phase-contrast optics and a 40x/0.65 NA objective were used. Images were captured at 400 fps and analyzed with HCImage Live software (Hamamatus, Japan). Beat frequency data were analyzed using Image J and graphed by GraphPad Prism 5 (GraphPad, USA). The average frequency was determined by analyzing 39 cells.

Primary antibody

Details for the primary antibodies for immunoblotting (IB) and immunofluorescence (IF) can be found in S2 Table. Please note, a rabbit polyclonal antibody of FBB18 was raised against a full-length polypeptide and affinity purified. DHCα and DHCβ antibodies were kindly provided by Dr. David Mitchell (SUNY Upstate Medical University), dynein b, c and e antibodies by Dr. Toshiki Yagi (Prefectural University of Hiroshima, Japan) and FMG1 antibody by Dr. Robert Bloodgood (University of Virginia).

SDS-PAGE, immunoblotting, immunoprecipitation and mass spectrometry

The methods for SDS-PAGE, immunoblotting and immunoprecipitation were essentially as described previously [56, 57]. For immunoprecipitation, 1x10⁹ of fbb18-1 rescue cells expressing FBB18-YFP were used with wild-type cells as a control. FBB18-YFP was pulled down with anti-GFP rabbit mAb conjugated agarose beads (Engibody, USA). The immunoprecipitates were subjected to SDS-PAGE and the gel slices were analyzed by mass spectrometry in the Core Facility of the Center for Biomedical Analysis (Tsinghua University). The mass spectrometry data presented for the immunoprecipitation are from one experiment.

For comparative proteomic analysis of axonemal dyneins, fbb18-1 mutant and rescued cells were labelled with ¹⁵N by culturing the cells in medium containing ¹⁵NH₄¹⁵NO₃, respectively. Equal amounts of proteins from ¹⁵N labelled fbb18-1 cells and unlabeled rescued cells were mixed, or vice versa. 200 μg proteins were subjected to SDS-PAGE (7.5%) followed by coomassie blue staining. The gel part above a 310 kD protein marker was sliced for mass spectrometry analysis. pFind algorithm was used for data analysis with default setting [58].

IF and thin-section electron microscopy

A previously published protocol for IF was followed [57]. The secondary antibodies used were Texas red-conjugated goat anti-mouse IgG (1:400) and Alexa Fluor 488-conjugated goat ani-rat IgG (1:400; Molecular Probes). The samples were viewed on a Zeiss LSM780 META Observer Z1 Confocal Laser Microscope. Images were acquired and processed by ZEN 2009 Light Edition (Zeiss) and Photoshop (Adobe). Analysis for electron microscopy followed published methods [52, 59]. The samples were imaged on an electron microscope (H-7650B; Hitachi Limited) equipped with a digital camera (ATM Company).

Pull-down assays

Bacterial expressed proteins were used for pull-down assays. cDNAs for Chlamydomonas genes were cloned into pGEX-6P-1, pET28a or pETDuet-1 vectors and expressed in E. coli Rosetta (DE3). Of note, His-RuvBL1 and GST-RuvBL2 or GST were co-expressed. The cell lysates were mixed before pull-down using GST glutathione beads (Smart LifeSci, China).

Sucrose gradient assay

A 2 ml of sucrose linear gradient (5–20%) was made [60]. 100–200 μl protein samples were loaded on top of the gradient followed by centrifugation at 200,000g for 4 h at 4°C (Beckman MAX-XP, rotor TLS-55). The fractions were subjected to immunoblotting. For co-migration assay of RuvBL1/2 and FBB18 from bacterial expressed proteins, co-expressed RuvBL1/2 were mixed with FBB18 before loading onto the sucrose gradient.

Yeast two-hybrid assays

Chlamydomonas cDNA of each gene was cloned into the prey vector pGADT7 (AD) and the bait vector pGBKT7 (BD), respectively. Each pair of constructs as indicated were co-transformed into the yeast strain AH109 following a previous method [61]. Transformants were grown at 30°C for 2–3 days on selection media (SD, -Leu, -Trp, -His, -Ade or SD, -Leu, -Trp) (Clontech, USA). Empty vectors were used as controls.

Statistical analysis

Data were presented as mean ± SD. Statistical significance was performed by using unpaired two-tailed Student’s t-test analysis. P < 0.01 was considered to be statistically significant. ***P < 0.0001.

Supporting information

S1 Table. The mass-spectrometry data of the accessary subunits of dynein from FBB18-YFP immunoprecipitation.

Immunoprecipitates with anti-GFP antibody from fbb18-1 cells expressing FBB18-YFP or wild-type cells as control were analyzed by mass spectrometry. Please note, only the subunits highlighted in red were identified both with more than 2 unique peptides and a more than two-fold increase in the spectral count (sample versus control).

(DOCX)

Click here for additional data file.^{(13.8KB, docx)}

S2 Table. Primary antibodies used in this study.

(DOCX)

Click here for additional data file.^{(18.1KB, docx)}

S1 Fig. Analysis of functional domains of FBB18.

(A) Domain organization of FBB18 and its truncated proteins. The coiled-coil domain and DUF2870 are deleted in FBB18^Δ25–53 and FBB18^Δ177–277, respectively. FBB18^Δ252–277 mimics a human mutation that results in a C-terminus truncated protein. Numbers denote the residue positions. (B) Immunoblot analysis of the expression of the deletion mutants. Deletion constructs with a wild-type construct as a control were expressed in fbb18-1 followed by immunoblotting. Wild-type (WT) and fbb18-1 cells were also used as controls. Arrow and arrowhead mark the truncated proteins FBB18^Δ25–53 and FBB18^Δ177–277, respectively. Band marked by asterisk indicates non-specific band.

(TIF)

Click here for additional data file.^{(751.5KB, tif)}

S2 Fig. Analysis of the abundance of FBB18 in whole cell and cilia samples of various motility mutants.

Whole cells (A) or isolated cilia (B) from cells as indicated were subjected to immunoblotting with the indicated antibodies. Please note, the ciliary abundance of FBB18 varies with the mutants. We failed to observe a correlation between the ciliary abundance of FBB18 with the phenotype of impaired cilia motility.

(TIF)

Click here for additional data file.^{(1.2MB, tif)}

S3 Fig. Immunoprecipitation of FBB18-YFP.

Cells expressing FBB18-YFP with wild type cells as a control were immunoprecipitated with anti-GFP antibody followed by immunoblotting with anti-GFP antibody (A) and SDS-PAGE (B). The gel slices were used for mass spectrometry analysis.

(TIF)

Click here for additional data file.^{(1.8MB, tif)}

S4 Fig. The amino acid sequence of DHCα and its identified unique peptides in FBB18-YFP immunoprecipitation.

Residues highlighted with red or blue are the peptides identified by mass spectrometry.

(PDF)

Click here for additional data file.^{(163.1KB, pdf)}

S1 Video. Time-lapse movies showing the motility of wild-type cells.

Cell images were captured at 400 fps and the movie is played at 10 fps.

(AVI)

Click here for additional data file.^{(1.6MB, avi)}

S2 Video. Time-lapse movies showing the motility of fbb18-1 cells expressing FBB18Δ252–277.

Cell images were captured at 400 fps and the movie is played at 10 fps.

(AVI)

Click here for additional data file.^{(2MB, avi)}

S3 Video. Time-lapse movies showing the motility of fbb18-1 cells expressing full-length FBB18 tagged with 3xHA.

Cell images were captured at 400 fps and the movie is played at 10 fps.

(AVI)

Click here for additional data file.^{(817.2KB, avi)}

Acknowledgments

We thank Drs. David Mitchell, Toshiki Yagi and Robert Bloodgood for providing antibodies and/or strains and the Core Facility of the Center for Biomedical Analysis (Tsinghua University) for assistance on cell imaging and mass spectrometry analysis.

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

This work was supported by the National Natural Science Foundation of China (31991191, 31972888) (https://www.nsfc.gov.cn/), by the Pilot National Laboratory for Marine Science and Technology (MS2019NO04) (http://www.qnlm.ac) and by the National Key R&D Program of China (2017YFA0503500) (https://www.most.gov.cn) to JP. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS Genet. doi: 10.1371/journal.pgen.1010374.r001

Decision Letter 0

Susan K Dutcher, Gregory P Copenhaver

20 May 2022

Dear Dr Pan,

Thank you very much for submitting your Research Article entitled 'CFAP298 participates in preassembly of almost all axonemal dyneins independent of R2TP complex' to PLOS Genetics.

The manuscript was fully evaluated at the editorial level and by independent peer reviewers. The reviewers appreciated the attention to an important problem, but raised some substantial concerns about the current manuscript. Based on the reviews, we will not be able to accept this version of the manuscript, but we would be willing to review a much-revised version. We cannot, of course, promise publication at that time. It is important to address the comments about the presence of FBB18/FAP298 in the ciia. This makes it very different from other DNAAFs and needs to be addressed.

The Chlamydomonas community has adopted the new nomenclature and I ask that you follow the new rules.

Should you decide to revise the manuscript for further consideration here, your revisions should address the specific points made by each reviewer. We will also require a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript.

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We are sorry that we cannot be more positive about your manuscript at this stage. Please do not hesitate to contact us if you have any concerns or questions.

Yours sincerely,

Susan K. Dutcher

Associate Editor

PLOS Genetics

Gregory P. Copenhaver

Editor-in-Chief

PLOS Genetics

Nomenclature

• You need to follow the nomenclature rules at the Chlamydomonas Resource Center. For example, a transgene should be FAP298:HA-TG. You need to indicate it is a transgene and not an edited gene. https://www.chlamycollection.org/resources/tools/gene-nomenclature/

• Genes should NEVER include Cr in the name.

• Do not use superscripts, but give them allele names. (see link to nomenclature)

• This gene has been named in Phytozome in 2005 and , it has been published as FBB19 (My choice for the Chlamy gene). It is also known as kurly, C21orf59 and finally as CFAP298. No matter what, it is not CFAP298.

• CFAP298 is the human protein name and not the Chlamydomonas name. You must use it, it is FBB18 or FAP298.

Introduction and Discussion points

• The manuscript states that DNAAF7 stabilizes IC1/IC2. The authors should look at the work from Mali (REF 47).

• Patients have laterality defects rather situs inversus as they are patients with heterotaxy. Laterality is a more general term.

• Wild-type should have a hyphen when used as an adjective (line 125 as one example).

• The American Society of Human Genetics recommends that changes in patients be referred as variants and not as mutations. (one example on line 171).

• Pleases avoid flagella and deflagellation. See line 190 on page 8.

Experimental Questions

• The FAP298:YFP-TG construct is never characterized but is used in several experiments. It must be characterized. The authors should at least look at cilia numbers and lengths. They should characterize at least three independent transformants.

• The ODA and IDA protein levels should be quantitated.

• The manuscript states that DNAAF7 stabilizes IC1/IC2. The authors should look at the work from Mali (REF 47).

• The authors state that cilia are isolated with 0.5% Non-Idet. I would guess that they have axonemes and not cilia.

• In the previous characterization of FBB18 in the cilia, it was stated that the level is low. Please compare your results to the published data.

• The proteomics and the blots do not agree very well. Please discuss

Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: Defects in CFAP298 (aka C21orf59 and FBB18) have previously been shown to affect dynein assembly/ciliary motility in zebrafish, humans and planaria; human patients exhibit primary ciliary dyskinesia phenotypes. Biochemical studies using Chlamydomonas found the protein both in the cytoplasm and the ciliary matrix. In this study, the authors identify and characterize a Chlamydomonas null mutant in CFAP298. They find it is present mainly in the cytoplasm but that it is also readily detectable in cilia. The mutant exhibits no or short cilia and proteomics/pull downs identify associations with dynein heavy chains (HCs) and several other factors known to be involved in the cytoplasmic preassembly of axonemal dyneins. They find that CFAP298 associates with the RuvBL1/2 ATPases but they find no evidence for association with other components (scaffolds and PIH proteins) of R2TP complexes and its variants.

Comment to the Journal

The figures provided as part of the manuscript pdf are very low quality. I can only assume that this reflects the automated manuscript processing system that generated the pdf as the supplemental data file figures provided directly by the authors are of high quality. Providing such low quality figures to reviewers does authors a great disservice.

Comments to the Authors

1. The abstract (and many other places) should indicate that the ODA/IDA loss is partial in the cfap298 mutant and that the amounts of some major HCs are barely affected. Also, the abstract refers to “new mechanisms of axonemal dynein preassembly” but no mechanistic detail is actually provided.

2. The authors show that the alpha HC is missing and the amount of beta HC in cfap298 cilia is reduced somewhat. Based on this and similar data for several inner arms they state that CFAP298 is “required for the assembly of both ODA and IDA in cilia” (lines 157-158) – considering many ODA/IDAs still assemble this seems a rather strong statement as the effect is clearly not “all or none” but is rather much more nuanced.

3. The observation that assembly of the alpha HC particularly depends on the CFAP298 DUF2870 domain is interesting as the alpha HC (which is closely related to the unaffected beta HC) is unusual in having a completely different N-terminal region compared to all other Chlamydomonas HCs. Although not a requirement, it might be interesting to test whether CFAP298 interacts specifically with this alpha HC region that consists of kelch domains and several Ig folds.

4. Although CFAP298 is said to associate with the beta and gamma ODA HCs, its loss appears to have little/no effect on their cytoplasmic assembly as they are present in the mutant at ratios of 0.9 and 1 compared to wildtype (it is quite possible that the small reduction in beta HC amount might reflect the total loss of its binding partner the alpha HC rather than a direct effect of CFAP298 loss). As these (beta and gamma) are 2 of the 3 most abundant axonemal HCs in Chlamydomonas, if CFAP298 is central to HC folding/stability why are they unaffected? From my understanding, the IP (table 2) was from cell extracts and so if CFAP298 binds alpha HC (which is greatly affected by cfap298 loss), they would also IP beta and gamma which are tightly bound to alpha even though CFAP298 may not be actually binding them directly at all. Similarly, if CFAP298 associates with one HC of the I1/f dynein one might expect to pull down the second HC as well even though it may not interact directly. Thus, this needs clarifying. Care should be taken not to imply that CFAP298 is a DNAAF for all HCs, which it clearly is not. Many sentences mentioning this concept are sweepingly broad (e.g. lines 333-334 and elsewhere) and it should be made clear they do not apply to all axonemal dynein HCs equally. Perhaps it might help if the authors could identify or discuss some common feature(s) of the HCs most dramatically affected compared to those that are not. What special feature of these HCs might CFAP298 affect?

5. In the IPs (table 2), where in the HCs do the identified unique peptides derive from? Do they include C-terminal peptides in which case one might predict that CFAP298 is associating with fully formed dyneins (i.e. post chaperone requiring) – how would this affect their interpretation of CFAP298 function? Also, the authors should detail what other dynein components (if any) were found in these IPs as this again directly impacts on the state of the dyneins with which CFAP298 is associating.

6. The authors observe that loss of cfap298 leads to an increase in the IFT dynein and suggest that its folding/stability does not depend on CFAP298. The observation that no DNAAFs (including CFAP298) have been reported to adversely affect cytoplasmic or IFT dynein assembly was made previously (see ref #39) and should be acknowledged.

7. The authors state that CFAP298 participates in dynein folding in cytoplasm based on interactions with several DNAAFs and contrast this with previous proposals that it might target dyneins to cilia or stabilize them within cilia as CFAP298 was found in cilia. As the authors also demonstrate and confirm the presence of CFAP298 in isolated cilia and the cilia membrane/matrix fraction what role do they predict it plays there? Presumably not an early step in preassembly? As many DNAAFs are totally absent from cilia this is very different and although the cilia signal in Fig 3C is quite low, a very robust signal is shown in Fig 3B. Perhaps including Coomassie stained gels would allow the gel loadings to be better appreciated. I think at several places in the manuscript there is an assumption that a role in cytoplasmic folding/stability precludes involvement in other activities or locations, but without providing any data to rule them out. This can be readily dealt with by rewriting several sections to be less assertive. Considering how long it takes a ribosome to actually synthesize a dynein HC, it is also unclear how “chaperoning” per se can be readily distinguished from many other steps in the pathway to functional dyneins as they are likely cotemporaneous and may occur prior to the completion of HC synthesis.

8. A previous study found more CFAP298 in flagella and cell bodies of several motility mutants compared to wildtype. The data provided here in Fig S2 using a different antibody show less increase and the authors say any differences are not significant (lines 200-202) but on what basis is unclear. Indeed, it does seem under some conditions that increases are actually observed. For example, in Fig S2A expt 1 there would appear to be clearly more CFAP298 in ida1 cells compared to wildtype and similarly in Fig. S2B expt 1 there is more in oda1 and oda5 cilia compared to wildtype (this may also be the case in expt 2 but the blot exposure is very dark). Also in Fig S2B, the amounts in pf20 cilia seem to be consistently much less than other strains. At the least, there does appear to be considerable variability in the signals obtained in the various mutants, which should be directly acknowledged even though the origin of the variation might be uncertain.

9. Fig 3C – why is there no tubulin signal in the whole cell/cell body samples while signal is readily visible in the cell extract blots shown in Fig 1E? Again, Coomassie stained gel/blots would aid interpretation here.

10. Also in Fig 1E, there are several non-specific bands detected by the CFAP298 antibody that are present in all cell extracts. However, in addition to CFAP298 itself, the CFAP298 antibody also detects a band at ~50 kDa in wildtype that disappears in the mutant and reappears in the rescued strain. Is this some other CFAP gene product or splice variant? Is it detected by the HA tag antibody (the WB strip shown is not large enough to tell)? Would it also be included in the IPs shown elsewhere in the paper, and how might this affect the interpretation of protein-protein interactions especially for proteins found in low abundance?

11. Immunostaining in Fig 3D indicates a punctate pattern throughout the entire cell including where I would anticipate the chloroplast, which fills much of the cell, is located. Is this a maximal projection image or ?????, the methods are very unclear on this. Please clarify.

12. The diagram in Fig 4A indicates that CFAP298 interacts directly with 6 different proteins. Given its rather small size, it seems unlikely this could all occur at the same time. Does binding of one or more of these components preclude binding of others? This information might significantly aid interpretation of CFAP298 function.

Reviewer #2: Summary. The authors use multiple complementary methods to analyze the function of CFAP298 in Chlamydomonas. The data from the insertional CFAP298-1 mutant suggests a role with the assembly of specific heavy chain outer dynein arm DHC alpha and inner dynein arm dynein b. For such function, CFAP298 interacts with many other dyneins. I have a few questions and requests for additional information that may improve the manuscript.

Questions

Line 135 and Figure 2C and Figure 3E. If only 23% of the transformants from the rescue had a normal phenotype, how does the level of DHC alpha and dynein b appear identical to the wild type levels?

Line 199 and Figure 3B, S1A. Please discuss the role of CFAP298 in the flagella/cilia. Such location is not consistent with other DNAAFs. Is it possible that there is an additional transport role.

Line 206 and Figure 3D. The figure 3D should be improved. The CFAP298-HA images be improved and especially brightened? It is difficult to see the puncta in green contrasted to black and observe if there is any signal in the flagella. Can the authors co-localize CFAP298 with axonemal dynein motor client cargo? Such experiments would strengthen the difference from prior published studies (ref 31) and support the IP experiments.

Lines 234-237 and Table 1. The explanation for finding more DHC alpha in the CFAP298-1 cells compared to control cells is not obvious. If CFAP298 if functioning to stabilize dynein (Line 214) would it not be likely that the target protein would be degraded? The western blot in of the mutants shows it is not detect in Fig 3E ?

Minor points

Table 1. Please explain and add to methods how were counts normalized for each mass spec sample and how the average ratio determined.

Line 131. Please include the site of the immunogen for the ab to CFAP298 in the main text. It is important for interpreting the data and I could not find that information in the methods or supplements.

Fig 4A. It would be useful to have a key for solid and dashed lines. Some interactions are previously reported interactions and identified in the current manuscript, so both types of lines would be used?

Reviewer #3: This study uses a CFAP298 null mutation in Chlamydomonas to add to biological understanding of axonemal dynein arm assembly, a complex chaperone-driven process. CFAP298 is not well understood after human disease and zebrafish studies and this report adds a useful critique of past papers and the current understanding. Defective CFAP298 is here linked to flagellar motility and length defects plus defects of both outer and inner arm dynein assembly. In constructs over-expressed on a CFAP298-null background, deletion of either the CFAP298 CC or DUF domain leads to protein instability. Over-expression of del252-277 was more useful, mimicking a reported human mutation causing PCD with dynein arm defects. This construct was less able to rescue flagella motility or ODA structures that FL. Mass spec interaction studies of CFAP298 to identify its protein network shed some fresh light on its protein partners and notably showed that CFAP298 is likely to interact with RuvBL1/2 without SPAG1 and RPAP3.

Generally speaking, this study adds information to the field, there are some additional controls that could have been done on protein experiments and some of the English grammar could benefit from review sub editing.

1. Have the authors looked at an alphafold model of CFAP298 and whether the model can tell something about functions of domains of the protein and mutations, in particular how it could fit into a R2TP type of complex.

2. Fig1B, it is unclear how exactly the gene insert affects the CFAP298 protein to create a null allele; e.g., a fusion protein? Early termination?

3. In the case of the human mutation p.Tyr245*, nonsense mutations are generally expected to lead not to an expressed truncated protein, but to nonsense mediated decay and degraded hence no protein and in this sense the Chlamy over expressed delta252-277 may not be a good model of the mutational effect. Is there any evidence for the human mutant protein and whether it is expressed or lost from patient cells?

4. Up to Fig3 the authors talked about delta252-277 but they don’t mention anything about this construct in Fig4. It would be interesting to know how this truncation affects protein-protein interactions shown in Fig4.

5. Fig 2C and 3E cilia versus cells, in the discussion what is the relevant comparison for these similar experiments. If possible, it would be useful to create a summary cartoon of the predicted effect of the loss of CFAP298 on the axonemal dyneins structures.

6. Table 2 and 3 IP data – these experiments pick up a lot of interactions and some are much weaker than others without any subsequent following up of these results for most. Was an empty-YFP construct included to these experiments as a control, can this data be shown in comparison? Are synchronised cultures of Chlamydomonas being compared. The text could do more to acknowledge some of the study limitations.

Can the text clarify whether Table 2 and 3 use the same mass spec data, and how many times was this experiment done, to understand variance in the results. With no follow up coIP studies shown for most of these interactions except the PF22 and RUVBL1/2, this would strengthen the evidence.

7. Discussion – refer to the possible reasons why the difference is seen in expression levels of CFAP298 in Chlamydomonas mutants in FigS1, compared to the past report [31].

8. Punctate staining of CFAP298 in the cytosol – have authors seen evidence that it is present in DyNAPs, if TEM was used. Ref to work of Wallingford group PMID: 33263282.

9. Discussion finishes abruptly – is it suggested from this data that CFAP298 could be in a different type of R2TP like complex, or in a differently structured complex? The notion of multiple R2TO type complexes as set out in reference 16, is the author’s speculation that this is still most likely for CFAP298.

Technical comments, typos:

- Fig1C graph, refers to cell numbers with sizes – how is this important what does it say about phenotype? This graph also needs to show the equivalent data for WT to understand what the normal spread of cell size is.

- What does it mean that cells were ‘hatched from sporangia for unknown reason’ with reference to Fig1, this is not further discussed, what is the relevance?

- Text on Fig1 suggests we should be seeing evidence of a 23% rescued phenotype – what data does this refer to?

- Page 6 of text only refers to Figure 1 for motility/length evidence, this needs to refer to Fig 2 data graphs.

- Fig2B why does the WT lack cross sections containing the expected 9 ODAs.

- Fig2D, these graphs really should show the equivalent cfap298-1 mutant data as well as the rescue data, for cilia length and beat frequency. Technical details seem incomplete – how many times was the experiment done, number of cells tested?

- The Fig2D data could benefit from showing movies of the rescued or non-rescued beating and length.

- Page 6 first referring to CFAP298-HA, this needs to explain this is a FL HA tagged version.

- Fig3A the use of a nonspecific band as a control is clearly unusual and seems unacceptable if that protein is unidentified. What is pdf, a control? Where can we see the wildtype levels of protein, to judge the level of successful rescue?

- Fig3E would benefit from a normalised graph of protein levels compared to CBB.

- Page 10 starts ‘After ascertaining that CFAP298 is a DNAAF etc’, but this is already established with human gene nomenclature DNAAF16 well established and so on. Need to indicate this is a confirmation therefore, or is it the first finding of such.

- Fig4B the criticised Y2H data missing key CFAP298 interactions, this seems better to be moved to supplementary data then.

- Table 1 data could benefit from a graph to show clearly the changed protein abundances.

- Table 3 what is the relevance in R2TP complexes of the two HSP90s A, C, and the HSP70E, which have been associated to DNAAFs before.

- In text and figures ensure DYX1C1 has the final 1 on the end, this is missing a couple of times.

- Bottom of page 12 add reference to Figure 4E here in the text.

- Page 14 typo DNNAFs, check for a few more minor typos. Such as line 351 page 14 attractive, line 384 page 15 axonemal, line 385 loose network.

**********

Have all data underlying the figures and results presented in the manuscript been provided?

Large-scale datasets should be made available via a public repository as described in the PLOS Genetics data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No: Mass spec raw data has not been provided

**********

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

PLoS Genet. 2022 Aug 26;18(8):e1010374. doi: 10.1371/journal.pgen.1010374.r002

Author response to Decision Letter 0

21 Jun 2022

Attachment

Submitted filename: Response to reviewers.docx

Click here for additional data file.^{(146.5KB, docx)}

PLoS Genet. doi: 10.1371/journal.pgen.1010374.r003

Decision Letter 1

Susan K Dutcher, Gregory P Copenhaver

12 Jul 2022

Dear Dr Pan,

Thank you very much for submitting your Research Article entitled 'FBB18 participates in preassembly of almost all axonemal dyneins independent of R2TP complex' to PLOS Genetics.

The manuscript was fully evaluated at the editorial level and by independent peer reviewers. The reviewers appreciated the attention to an important topic but identified some concerns that we ask you address the minor revisions in a revised manuscript. There are only a few places where the current Chlamydomonas nomenclature is not used. The gene is FBB18:YFP-TG or FBB:HA-TG. Please put the TG in the name of the gene on lines 257 and 779. Figure 3 does not have a control for the level of contamination for the cytoplasm in the cila prep. Is the FBB18 signal just cytoplasmic contramination;? Please use the NAB antibody or PFOR antibody to ask about cytoplasmic contamination. This is key since ciliary localization in not observed in C. elegans or human cells.

We therefore ask you to modify the manuscript according to the review recommendations. Your revisions should address the specific points made by each reviewer.

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Gregory P. Copenhaver

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Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: I think the authors have addressed most of my comments. However, concerning the variable amounts of FBB18 in cilia samples from various mutants, although they state they now acknowledge there is variability, the legend to the supplemental figure S2 still contains the statement “no significant changes in the abundance of FBB18………”. If one looks for example at the pf20 cilia, there is very obviously much less FBB18 than in say oda5 cilia; a similar statement can be made about wildtype vs. oda5. The lower cilia blot is very over exposed but even there the differences in samples are clear. Surely, this is significant and it is also consistent. If not, what difference would the authors consider significant? So I still think the authors are seeing consistent changed levels of FBB18 between at least some of the strains they examined.

I am also not sure I agree with the author’s point concerning the immunostaining which shows a punctate pattern throughout the cell even where the chloroplast should be. The argument is that on fixation the cytoplasm can flow into the damaged chloroplast and thus the antigen appears to be everywhere. If correct, doesn’t this negate the entire point of the experiment, which is to localize FBB18? Shouldn’t every other cytoplasmic antigen show the same pattern in this case? Perhaps a different fixation scheme using an aldehyde rather than methanol should be used to avoid this “flow”.

Reviewer #2: Summary. The authors have submitted a revised text with additional explanations of their data. I have a few suggestions to improve comprehension of the data.

1. The writing and grammar should be improved, particularly in the new (revised) text. The abstract should especially be edited for clarity. For example, Line 22: The sentence confuses the wild type and mutant alleles. I did not see evidence that the mutation of FBB18 localizes to the cytoplasm. I suggest moving cytoplasmic localization to line 20: Here, we show that FBB18, a Chlamydomonas homologue of CFAP298, localizes to the cytoplasm and functions in the folding/stabilization…

2. Results. Lines 240-252 are difficult to follow in the revised form since the reader has to move between the immunoblots in Figure 3 and Table 1 mass spec data. I suggest inserting another reference to (Table 1) at the end of the sentence referring to IFT dynein on line 249.

3. Line 131. Please include the immunogen used to generate the ab to FBB18 in the main text. It is important for interpreting the data and I could not find that information in the methods, figure legend or supplement. Another reviewer asked for that same information. It is not useful to only reference that information.

**********

Have all data underlying the figures and results presented in the manuscript been provided?

Reviewer #1: Yes

Reviewer #2: Yes

**********

PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #1: No

Reviewer #2: No

PLoS Genet. 2022 Aug 26;18(8):e1010374. doi: 10.1371/journal.pgen.1010374.r004

Author response to Decision Letter 1

25 Jul 2022

Attachment

Submitted filename: Response to reviewers.docx

Click here for additional data file.^{(22KB, docx)}

PLoS Genet. doi: 10.1371/journal.pgen.1010374.r005

Decision Letter 2

Susan K Dutcher, Gregory P Copenhaver

4 Aug 2022

Dear Dr Pan,

We are pleased to inform you that your manuscript entitled "FBB18 participates in preassembly of almost all axonemal dyneins independent of R2TP complex" has been editorially accepted for publication in PLOS Genetics. Congratulations!

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----------------------------------------------------

Comments from the reviewers (if applicable):

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PLoS Genet. doi: 10.1371/journal.pgen.1010374.r006

Acceptance letter

Susan K Dutcher, Gregory P Copenhaver

21 Aug 2022

PGENETICS-D-22-00477R2

FBB18 participates in preassembly of almost all axonemal dyneins independent of R2TP complex

Dear Dr Pan,

We are pleased to inform you that your manuscript entitled "FBB18 participates in preassembly of almost all axonemal dyneins independent of R2TP complex" has been formally accepted for publication in PLOS Genetics! Your manuscript is now with our production department and you will be notified of the publication date in due course.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Table. The mass-spectrometry data of the accessary subunits of dynein from FBB18-YFP immunoprecipitation.

(DOCX)

Click here for additional data file.^{(13.8KB, docx)}

S2 Table. Primary antibodies used in this study.

(DOCX)

Click here for additional data file.^{(18.1KB, docx)}

S1 Fig. Analysis of functional domains of FBB18.

(TIF)

Click here for additional data file.^{(751.5KB, tif)}

S2 Fig. Analysis of the abundance of FBB18 in whole cell and cilia samples of various motility mutants.

(TIF)

Click here for additional data file.^{(1.2MB, tif)}

S3 Fig. Immunoprecipitation of FBB18-YFP.

(TIF)

Click here for additional data file.^{(1.8MB, tif)}

S4 Fig. The amino acid sequence of DHCα and its identified unique peptides in FBB18-YFP immunoprecipitation.

Residues highlighted with red or blue are the peptides identified by mass spectrometry.

(PDF)

Click here for additional data file.^{(163.1KB, pdf)}

S1 Video. Time-lapse movies showing the motility of wild-type cells.

Cell images were captured at 400 fps and the movie is played at 10 fps.

(AVI)

Click here for additional data file.^{(1.6MB, avi)}

S2 Video. Time-lapse movies showing the motility of fbb18-1 cells expressing FBB18Δ252–277.

Cell images were captured at 400 fps and the movie is played at 10 fps.

(AVI)

Click here for additional data file.^{(2MB, avi)}

S3 Video. Time-lapse movies showing the motility of fbb18-1 cells expressing full-length FBB18 tagged with 3xHA.

Cell images were captured at 400 fps and the movie is played at 10 fps.

(AVI)

Click here for additional data file.^{(817.2KB, avi)}

Attachment

Submitted filename: Response to reviewers.docx

Click here for additional data file.^{(146.5KB, docx)}

Attachment

Submitted filename: Response to reviewers.docx

Click here for additional data file.^{(22KB, docx)}

Data Availability Statement

All relevant data are within the manuscript and its Supporting Information files.

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PERMALINK

FBB18 participates in preassembly of almost all axonemal dyneins independent of R2TP complex

Limei Wang

Xuecheng Li

Guang Liu

Junmin Pan

Roles

Abstract

Author summary

Introduction

Results

An fbb18 mutant shows defects in ciliogenesis and ciliary motility

Fig 1. Isolation and characterization of an fbb18 mutant.

Fig 2. Loss of dynein arms in fbb18 mutants.

Null mutation of FBB18 causes partial loss of ODAs and IDAs

FBB18Δ252–277 that mimics truncated mutation in human patients leads to particularly loss of DHCα in cilia

FBB18 is involved in cytoplasmic preassembly of axonemal dyneins

Fig 3. FBB18 localizes predominantly to cytoplasm and mediates stability of cytoplasmic axonemal dyneins.

Differential regulation of the stability of axonemal dyneins by FBB18

Table 1. Changes in protein abundance of axonemal dyneins in fbb18-1 versus rescued cells.

FBB18 physically associates with almost all axonemal dyneins

Table 2. FBB18 associates with almost all axonemal dyneins.

FBB18 is in association with several DNAAFs, HSP90 and RuvBL1/2 of the R2TP complex

Table 3. Factors associated with FBB18 that are involved in dynein preassembly.

Deciphering the interaction map of the FBB18-associated DNAAFs

Fig 4. An interacting network(s) of FBB18 with other DNAAFs.

Discussion

Regulation of ciliary assembly and length by the assembly of axonemal dynein arms

FBB18 is involved in chaperoning axonemal dynein HCs at the early steps of dynein preassembly

The specificity of DNAAF in chaperoning axonemal dynein HCs

Formation of multiple DNAAF complexes during folding and stabilization of axonemal dyneins

FBB18 associates with RuvBL1/2 but not a R2TP complex

Materials and methods

Strains and culture

Insertional mutagenesis, gene cloning and transformation

Cell fractionation, ciliary regeneration, ciliary length measurement, and DIC cell imaging

Ciliary beat analysis

Primary antibody

SDS-PAGE, immunoblotting, immunoprecipitation and mass spectrometry

IF and thin-section electron microscopy

Pull-down assays

Sucrose gradient assay

Yeast two-hybrid assays

Statistical analysis

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Susan K Dutcher

Gregory P Copenhaver

Roles

Author response to Decision Letter 0

Decision Letter 1

Susan K Dutcher

Gregory P Copenhaver

Roles

Author response to Decision Letter 1

Decision Letter 2

Susan K Dutcher

Gregory P Copenhaver

Roles

Acceptance letter

Susan K Dutcher

Gregory P Copenhaver

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

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FBB18^Δ252–277 that mimics truncated mutation in human patients leads to particularly loss of DHCα in cilia