Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2011 Apr;22(7):976-87.
doi: 10.1091/mbc.E10-04-0331. Epub 2011 Feb 2.

Regulation of Flagellar Motility by the Conserved Flagellar Protein CG34110/Ccdc135/FAP50

Affiliations
Free PMC article

Regulation of Flagellar Motility by the Conserved Flagellar Protein CG34110/Ccdc135/FAP50

Yong Yang et al. Mol Biol Cell. .
Free PMC article

Abstract

Eukaryotic cilia and flagella are vital sensory and motile organelles. The calcium channel PKD2 mediates sensory perception on cilia and flagella, and defects in this can contribute to ciliopathic diseases. Signaling from Pkd2-dependent Ca²+ rise in the cilium to downstream effectors may require intermediary proteins that are largely unknown. To identify these proteins, we carried out genetic screens for mutations affecting Drosophila melanogaster sperm storage, a process mediated by Drosophila Pkd2. Here we show that a new mutation lost boys (lobo) encodes a conserved flagellar protein CG34110, which corresponds to vertebrate Ccdc135 (E = 6e-78) highly expressed in ciliated respiratory epithelia and sperm, and to FAP50 (E = 1e-28) in the Chlamydomonas reinhardtii flagellar proteome. CG34110 localizes along the fly sperm flagellum. FAP50 is tightly associated with the outer doublet microtubules of the axoneme and appears not to be a component of the central pair, radial spokes, dynein arms, or structures defined by the mbo waveform mutants. Phenotypic analyses indicate that both Pkd2 and lobo specifically affect sperm movement into the female storage receptacle. We hypothesize that the CG34110/Ccdc135/FAP50 family of conserved flagellar proteins functions within the axoneme to mediate Pkd2-dependent processes in the sperm flagellum and other motile cilia.

Figures

FIGURE 1:
FIGURE 1:
The sperm storage phenotype of lobo. (A) A schematic diagram of the female reproductive tract. (B, C) Confocal images of freshly dissected uteri from wild-type females at 30 min after mating with the wild-type (B) and CG34110lobo homozygous males (C). Both types of sperm were transferred into the uteri as indicated by the green fluorescence derived from the reporter transgene dj-GFP that labels the sperm flagellum. The mating plug is located at the posterior uterus, where it lacked the GFP fluorescence. The wild-type sperm entered the SR (arrowhead) and spermatheca (arrow), whereas very few of the mutant sperm entered the two storage organs. Low levels of auto-fluorescence were present in the fly tissue.
FIGURE 2:
FIGURE 2:
Western blot, male fertility, and CG34110 immunofluorescence staining. (A) The CG34110 protein was detected by either anti-CG34110861–874 or anti-CG34110828–842 as a band running at ∼167 kDa (arrowhead) on the Western blot. Protein samples were from adult wild-type females (lane 1); wild-type testes (lane 2); mutant testes from CG34110lobo (lane 3); and CG34110MB00722 (lane 4) and wild-type mature sperm from the male SV (lane 5). The band near 60 kDa was α-tubulin detected by mouse anti–α-tubulin (Sigma DM1A) as a loading control. Two other bands were likely nonspecific bands that were strong in the testis but undetectable in the mature sperm. (B) Male fertility was measured as the average number of F1 progeny (y axis) produced during the first 48 h by one wild-type female following mating with one male of different genotypes (x axis). The male genotypes are as follows: 1) wild type (F1 = 60, n = 10), 2) CG34110lobo (F1 = 9.6, n = 12), 3) CG34110lobo/Df(3R)Exel6201, (F1 = 6.5, n = 11), 4) CG34110Q46/Df(3R)Exel6201 (F1 = 6.1, n = 10), 5) CG34110MB00722/Df(3R)Exel6201 (F1 = 2.2, n = 24), and 6) CG34110MB00722/CG34110lobo (F1 = 8.7, n = 10). (C, D) Mature sperm were collected from the male SVs and stained with the anti-CG34110 antibody. The immunoreactivity (red) was observed along the entire sperm tail from wild type (C) but not in sperm from the mutant male (D; CG34110MB00722/CG34110lobo). The DNA of the sperm heads was stained blue by DAPI.
FIGURE 3:
FIGURE 3:
Association of C. reinhardtii FAP50 with the ODs. Western blots of C. reinhardtii isolated whole flagella (WF), whole cells (WC), the 1% NP-40-soluble flagellar membrane-plus-matrix fraction (M+M), the axonemal fraction remaining after extraction with 0.6 M KCl (EX-AX), and the 0.6 M KCl extract (KCL-EX). (A) Anti-FAP50 detected three bands in WF, one of which (arrowhead) is FAP50 based on the gel region that yielded FAP50 peptides in the flagellar proteomic analysis (Pazour et al., 2005). (B) The first two lanes were loaded at a ratio of 1 cell-to-2 flagella; lanes 2–5 were loaded with protein from equivalent numbers of flagella. Blots were probed with antibodies to FAP50 (1:4000 dilution), and to the outer dynein arm intermediate chain IC2 and the intraflagellar transport protein IFT46 to verify purity and correct loading of the fractions. Thin, white, vertical lines indicate where marker lanes were excised from the image. (C) Whole flagella from wild type (WT), the CP-less mutant pf18, and the radial spoke-less mutant pf14 were probed with antibodies to FAP50 and to IC2 as a loading control. (D) Isolated flagella or axonemes (obtained after 1% NP-40 treatment) from WT and the three mbo mutants were probed with anti-FAP50 and anti-IC2.
FIGURE 4:
FIGURE 4:
Movement of sperm from the SV into the ED in the male. The sperm heads and tails were marked with GFP, and the images were obtained by GFP imaging. (A, B) Video snap views from Supplemental Movie 1, which captured sequential events occurring in the reproductive tract of a wild-type male within 1–4 min following its copulation with a wild-type female. The sperm exited out of the bilateral SV through a converging tubule (CT, arrow) and entered the ED. Sperm heads were visible as GFP specks (arrowheads) at the leading edges of the moving front (MF). (C, D) Still images obtained from fast-freezing copulating wild-type (C) and lobo mutant males (D, CG34110MB00722/CG34110lobo). Scale bars are all 0.1 mm.
FIGURE 5:
FIGURE 5:
Sperm transitory movements in between different compartments of the female reproductive tract. These images were obtained by imaging sperm labeled with head (short specks, arrowheads) and tail GFP. Sperm are wild type in (A–D, K); CG34110MB00722/CG34110lobo in (E, F, G, J); Pkd2KO67/Pkd2KO67 in (H, I); and the Pkd2 lobo double mutant in (L). (A) A video snap view of moving sperm inside the uterus (Supplemental Movie 2). The flagella were propagating circular-shaped bends and the heads were dragged by their flagella during the movement. (B–J) Various segments of the PSR or DSR with the uterus (UT) positioned on the right and the DSR on the left. (B) A section of the PSR tubule is shown with its lumen filled with wild-type sperm flagella and the heads are visible along its length. (C) When the tubular wall was teased open at a central point of the PSR, it revealed a parallel bundle of sperm inside the lumen. Arrowheads point to two heads that were apparently in tail-leading orientation toward the DSR on the left. (D) The tail end of a wild-type sperm entered the PSR first. (E) In contrast, the heads of multiple lobo mutant sperm entered the PSR first, as did Pkd2 mutant sperm (not shown). (F) A lobo mutant sperm flagellum formed a hairpin-shaped fold (arrow) in the PSR lumen. (G) Two lobo mutant sperm became folded into large tangles, as did flagella of Pkd2 mutant sperm (not shown). Such tangles were still present in the PSR after the bulk sperm was extruded out of the UT, suggesting that they were stuck. (H) After entering the DSR, the wild-type, Pkd2, and lobo mutant sperm were similarly folded with heads distributed at random positions, as shown here for the Pkd2 mutant sperm. (I) In some images, the sperm heads in the DSR were found to reorient toward the UT, as if they were ready for DSR exit. (J) Many lobo sperm in the DSR had their heads oriented toward the UT, and two of the lobo sperm were returning to the UT with a head-leading orientation, just like the wild-type sperm in (K). (K) The reproductive tract of an egg-laying wild-type female following mating with a wild-type male. The egg is large enough to occupy the entire uterus and the sperm entry point on the egg shell, the micropyle (arrow), faces the openings of the storage organs. The arrowhead points to a group of head-leading sperm in the PSR that leads to the egg (see larger version of the image in Supplemental Figure 3). (L) The uterus from a female after mating with the double-mutant male (Pkd2ko67/MB06703; CG34110MB00722/lobo) contained a large number of sperm (only heads are visible with the head GFP marker). Scale bars are all 0.1 mm.
FIGURE 6:
FIGURE 6:
Comparison of sperm counts in the DSR with the progeny produced for the Pkd2 and lobo single mutants and the double mutant. The x axis indicates sperm counts; “In” for the number of sperm stored in the DSR per female per mating and “Out” for the number of viable F1 progeny produced per female per mating. Each data point was obtained from one wild-type female (collected as virgin) after mating with one mutant male. The four male mutant genotypes are (A) Pkd2Ko67, (B) Pkd2Ko67/MB06703, (C) CG34110lobo/MB00722, and (D) Pkd2Ko67/MB06703; CG34110lobo/MB00722 (also carrying one copy of Protamine-GFP). The y axis represents the percentages of the “In” or “Out” in the tested populations (n). All progeny were checked for the expected genotype to make sure that they were fathered by the experimental males. The wild-type male consistently stores an average of 330 sperm (range 275–404, Supplemental Table1).

Similar articles

See all similar articles

Cited by 22 articles

See all "Cited by" articles

References

    1. Baccetti B, Gibbons BH, Gibbons IR. Bidirectional swimming in spermatozoa of Tephritid flies. J Submicrosc Cytol Pathol. 1989;21:619–625. - PubMed
    1. Baker K, Beales PL. Making sense of cilia in disease: the human ciliopathies. Am J Med Genet C Semin Med Genet. 2009;151C:281–295. - PubMed
    1. Barr MM, Sternberg PW. A polycystic kidney-disease gene homologue required for male mating behaviour in C. elegans. Nature. 1999;401:386–389. - PubMed
    1. Bessen M, Fay RB, Witman GB. Calcium control of waveform in isolated flagellar axonemes of Chlamydomonas. J Cell Biol. 1980;86:446–455. - PMC - PubMed
    1. Bloch Qazi MC, Wolfner MF. Emergence of sperm from female storage sites has egg-influenced and egg-independent phases in Drosophila melanogaster. Biol Lett. 2006;2:128–130. - PMC - PubMed

Publication types

MeSH terms

Feedback