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. 2015 Apr 15;26(8):1463-75.
doi: 10.1091/mbc.E14-11-1545. Epub 2015 Feb 18.

The CSC Proteins FAP61 and FAP251 Build the Basal Substructures of Radial Spoke 3 in Cilia

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Free PMC article

The CSC Proteins FAP61 and FAP251 Build the Basal Substructures of Radial Spoke 3 in Cilia

Paulina Urbanska et al. Mol Biol Cell. .
Free PMC article

Abstract

Dynein motors and regulatory complexes repeat every 96 nm along the length of motile cilia. Each repeat contains three radial spokes, RS1, RS2, and RS3, which transduct signals between the central microtubules and dynein arms. Each radial spoke has a distinct structure, but little is known about the mechanisms of assembly and function of the individual radial spokes. In Chlamydomonas, calmodulin and spoke-associated complex (CSC) is composed of FAP61, FAP91, and FAP251 and has been linked to the base of RS2 and RS3. We show that in Tetrahymena, loss of either FAP61 or FAP251 reduces cell swimming and affects the ciliary waveform and that RS3 is either missing or incomplete, whereas RS1 and RS2 are unaffected. Specifically, FAP251-null cilia lack an arch-like density at the RS3 base, whereas FAP61-null cilia lack an adjacent portion of the RS3 stem region. This suggests that the CSC proteins are crucial for stable and functional assembly of RS3 and that RS3 and the CSC are important for ciliary motility.

Figures

FIGURE 1:
FIGURE 1:
The CSC components FAP61, FAP91, and FA251 localize to Tetrahymena cilia but do not affect the length of cilia. (A–G) Localization of CSC proteins. FAP61 (A, D), FAP91 (B, E) and FAP251 (C, F) were expressed in FAP61-KO–rescued cells tagged with GFP (A) or tagged with 3HA in the native loci in wild-type background (B, C) or overexpressed as GFP-tagged (D, F) or 3HA-tagged (E) proteins. Note that all proteins localize to cilia; as expected, in the case of overexpression, the proteins also accumulated in the cell body. (G) Western blot of cilia (isolated from GFP-FAP61–rescued cells) and total cell extract (isolated from FAP91-3HA– and FAP251-3HA–expressing cells) probed with anti-GFP or anti-HA antibodies, respectively. Predicted molecular weight: GFP-FAP61, 223 kDa; FAP91-3HA, 79 kDa; and FAP251-3HA, 119 kDa. (H–J) Ciliary phenotype in knockout strains. FAP61-KO (I) and FAP251-KO (J) cells assemble cilia of normal length compared with wild-type cells (H) based on immunofluorescence with anti–α-tubulin antibodies. Bar, 10 μm. (K) Graph showing the average length of cilia. At least 50 cilia were measured for each genotype. Bars represent standard errors.
FIGURE 2:
FIGURE 2:
Deletions of FAP61 or FAP251 strongly affect ciliary motility. (A–C) Video captures of paths of swimming wild-type (A), FAP61-KO (B), and FAP251-KO (C) cells recorded for 2.5 s. (D) Average swimming distances per 2.5 s normalized to the wild-type value (n = 100 for each strain); knockout cells swam 37 ± 5.5% (FAP61-KO) and 25 ± 1.5% (FAP251-KO) of the wild-type distance. (E) Paths of swimming cells untreated or treated with 0.5 mM IBMX. (F, F′) Average distance of swimming paths normalized to either untreated wild-type cells (F) or untreated cells with the same genetic background (F′); note that IBMX treatment partially rescues the swimming defect in the mutants. (G) Growth curves of wild-type and knockout strains grown at 30°C in SPP medium with or without shaking. (H) Evaluation of the efficiency of phagocytosis based on the number of India ink–labeled food vacuoles per cell formed during 10 min (averages of three experiments). Wild type, 6.2 food vacuoles, n = 322 cells; FAP61-KO, 2.8, n = 477; and FAP251-KO, 2.1, n = 452.
FIGURE 3:
FIGURE 3:
Comparison of the 3D structures of the 96-nm repeats from Tetrahymena WT, FAP61-KO, and FAP251-KO axonemes. Tomographic slices (A, D, G) and isosurface renderings (B, C, E, F, H, I) show the averaged 96-nm repeats in longitudinal views in WT (A–C) and the two CSC-knockout mutants, FAP61-KO (D–F) and FAP251-KO (G–I). In the WT strain, the central stem region of RS3 (green) connects to the arch-like basal region (red), the main distal part of RS3 (orange), and the tails of the two dynein IDAs g and d (pink; B, C). Both CSC-knockout mutants show structural defects in the basal half of RS3: in FAP61-KO (D, E), the central stem region (green arrowhead in A), and in FAP251-KO (G, H), the base (red arrowhead in A) are barely visible. Note that the main distal part of RS3 (orange) is present in all strains, but in FAP61-KO, its electron microscopic density is significantly weaker (D). The other two radial spokes, RS1 (dark gray) and RS2 (blue/yellow), appear structurally normal in the mutants compared with WT; note that also the back prong of RS2 (yellow), which was reduced in the artificial microRNAi mutants of FAP61 and FAP91 in Chlamydomonas (Heuser et al., 2012), seems unaffected in the Tetrahymena FAP61-KO and FAP251-KO mutants studied here. Bar, 20 nm.
FIGURE 4:
FIGURE 4:
Classification analysis of RS3 in cilia from FAP61-KO and FAP251-KO. Longitudinal slices of the averaged 96-nm repeats show the presence (class 1: +RS3) or absence (class 2: –RS3) of the main distal part of RS3 in WT (A, B) and the CSC-knockout mutants FAP61-KO (C–E) and FAP251-KO (FH). Whereas RS3 is present in all WT 96-nm repeats (100%, B), it is missing from 36% and 16% of the 96-nm repeats from FAP61-KO (E) and FAP251-KO (H), respectively. The loss of RS3 on some of the 96-nm repeats resulted in reduced RS3 density in the mutant averages, including all repeats (C, F), especially in FAP61-KO (C). Even in the class average in which RS3 is present (D, G), the mutant RS3 lacks parts of the stem region in FAP61-KO (D) or the arch-like RS base in FAP251-KO (G), respectively. Note that although the distal part of RS3 was present in 64% of FAP61-KO repeats (D), the class 1 (+RS3) average still shows a slightly reduced distal RS3 density; this could be because a large part of the RS3 stem is missing in the FAP61-KO, so that the stem connection between the distal and basal parts of RS3 is greatly reduced and likely less stable, leading to a somewhat flexible position of the distal part of RS3 in individual repeats (positional heterogeneity) and blurring of the structure in averages as compared with WT. Bar, 20 nm.
FIGURE 5:
FIGURE 5:
Classification analysis of IDAs g and d in axonemes from FAP61-KO and FAP251-KO. Longitudinal slices of averaged 96-nm repeats show the presence (g/d in pink) and absence (g/d in white) of IDAs g and d in WT (A, D, G, J, M), FAP61-KO (B, E, H, K, N), and FAP251-KO (C, F, I, L, O). The densities of IDAs g and d, in the average of all 96-nm repeats from FAP61-KO (B), are slightly weaker than those of WT (A), whereas FAP251-KO (C) shows a density similar to that of WT. Classification analysis of WT 96-nm repeats revealed that they lack IDA g (G) and IDA d (M) in a small subset (10%) of repeats. A similar fraction (13%) of 96-nm repeats in FAP251-KO lacks IDA g (I) and IDA d (O), whereas in FAP61-KO mutants, two to three times more repeats lacked IDA g (25%; H) and IDA d (38%; N) compared with WT. Bar, 20 nm.

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