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. 2010 May 3;189(3):601-12.
doi: 10.1083/jcb.200912009. Epub 2010 Apr 26.

Pcdp1 Is a Central Apparatus Protein That Binds Ca(2+)-calmodulin and Regulates Ciliary Motility

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Free PMC article

Pcdp1 Is a Central Apparatus Protein That Binds Ca(2+)-calmodulin and Regulates Ciliary Motility

Christen G DiPetrillo et al. J Cell Biol. .
Free PMC article

Abstract

For all motile eukaryotic cilia and flagella, beating is regulated by changes in intraciliary calcium concentration. Although the mechanism for calcium regulation is not understood, numerous studies have shown that calmodulin (CaM) is a key axonemal calcium sensor. Using anti-CaM antibodies and Chlamydomonas reinhardtii axonemal extracts, we precipitated a complex that includes four polypeptides and that specifically interacts with CaM in high [Ca(2+)]. One of the complex members, FAP221, is an orthologue of mammalian Pcdp1 (primary ciliary dyskinesia protein 1). Both FAP221 and mammalian Pcdp1 specifically bind CaM in high [Ca(2+)]. Reduced expression of Pcdp1 complex members in C. reinhardtii results in failure of the C1d central pair projection to assemble and significant impairment of motility including uncoordinated bends, severely reduced beat frequency, and altered waveforms. These combined results reveal that the central pair Pcdp1 (FAP221) complex is essential for control of ciliary motility.

Figures

Figure 1.
Figure 1.
Anti-CaM antibodies precipitate four polypeptides, one of which exhibits calcium-sensitive CaM binding. (A) Diagram of the central apparatus and a single doublet microtubule with associated structures. Central pair projections are labeled. Inserted table lists WT and mutant strains used in this study along with the associated structural defects. (B) CaM gel overlay of WT, pf14, and pf18 axonemes in high and low calcium conditions. CaM binds to a polypeptide of ∼110 kD specifically in the presence of calcium (red arrowhead); this protein is missing from pf18 axonemes. (C) Silver-stained gels of anti-CaM immunoprecipitation experiments (IPs) performed in low and high calcium buffers from axonemal extracts isolated from WT and mutant axonemes. Four polypeptides are precipitated that are highly enriched in high calcium IPs (HC1–4). These four polypeptides are missing or reduced from pf18 and pf16 anti-CaM IPs (blue asterisks) and are present at WT levels in pf6, cpc1, and pf14 anti-CaM IPs (red asterisks). These results tentatively localize HC1–4 to the C1c or C1d projections of the central apparatus. (D) Corresponding CaM gel overlays of high calcium anti-CaM IPs from WT and mutant axonemal extracts. Only one of the precipitated proteins, HC4, exhibits calcium-sensitive CaM binding.
Figure 2.
Figure 2.
Pcdp1 is the mammalian orthologue of FAP221. (A) Diagram comparing the FAP221 and Pcdp1 protein coding sequences that share two major regions of identity. In the second region (shown in blue), both proteins have a predicted CaM-binding site (hatch marked region labeled “CaM”). Pcdp1 also has a predicted IQ motif at its C terminus (IQ). Regions that were expressed and used in the CaM gel overlay assay are marked with a black line; A and B for FAP221 and 1 and 2 for Pcdp1. (B) CaM gel overlay assay of FAP221 and Pcdp1 predicted CaM-binding regions. Overlays were performed with CaM from mouse (mCaM). The predicted CaM-binding regions shown in hatch marks only bind to CaM in the presence of high calcium. The predicted IQ motif at the C terminus of Pcdp1 does not bind to CaM under high or low calcium conditions. (C) Site-directed mutagenesis was used to alter three amino acids in the FAP221 CaM-binding site. Gel overlay analysis demonstrates the altered CaM-binding site no longer binds to C. reinhardtii CaM (CrCaM) in the presence of calcium.
Figure 3.
Figure 3.
FAP54, FAP46, FAP74, and FAP221 form a single complex. (A) Western blots of isolated axonemes. FAP74 and FAP221 antibodies recognize proteins of the correct molecular weight and that are missing or reduced from pf18 and pf16, but are present in WT, pf6, and cpc1 axonemes. (B) These proteins are also present in anti-CaM IPs using WT axonemal extracts but not pf18 and pf16 extracts. (C) Silver-stained gel of high calcium anti-FAP74 IPs from WT and pf18 NaCl axonemal extracts. All four proteins coprecipitate from WT extracts. The identities of FAP74 and FAP221 were confirmed by Western blot (not depicted). The identities of FAP54 and FAP46 were confirmed by mass spectrometry. (D) Western blots of WT axonemal extracts fractionated on a 5–20% sucrose gradient. FAP74 and FAP221 cosediment at ∼15S. (E) Silver-stained gel of high calcium anti-FAP74 IPs from pooled sucrose gradient fractions (see F). All four members of the complex coprecipitate from pool B (fractions 7–11), and not from pools A, C, or D. (F) Silver-stained gel of WT axonemal extracts fractionated on a 5–20% sucrose gradient. Odd fractions were pooled together in designated groups A–D and used in high calcium anti-FAP74 IPs shown in E. These data support the conclusion that all four proteins form a single complex.
Figure 4.
Figure 4.
Mutants with reduced expression of FAP74 lack the C1d central pair projection. (A) Diagram of FAP74 artificial miRNA construct. (B) Western blot of WT and mutant flagella showing that FAP74 amiRNA transformants 1G11, 2D4, and 7A4 have reduced levels of FAP74. FAP221 levels are not reduced in these mutants. IC138 is a dynein intermediate chain used as a loading control. (C) Top panel is a silver-stained gel of high calcium anti-CaM IPs from axonemal extracts. Reduced amounts of FAP54 and FAP46 are immunoprecipitated from 1G11, 2D4, and 7A4 axonemal extracts compared with WT. Bottom panels are anti-FAP74 and anti-FAP221 Western blots of high calcium anti-CaM IPs showing that FAP74 and FAP221 are not precipitated from FAP74ami transformants. (D) Diagram shows the identities of the central pair projections. Electron micrographs of transverse sections of WT, 1G11, pf14, and 1G11,pf14 mutant axonemes. (E) Enlarged view of the central apparatus from pf14 and 1G11,pf14 double-mutant axonemes. All micrographs are oriented with the axoneme viewed proximal to distal with the bar representing 25 nm; the C1 central microtubule in all images is to the left. The 1G11 and 1G11,pf14 axonemes lack the C1d density and the sheath connecting C1d to C1b (marked by red arrows and asterisks).
Figure 5.
Figure 5.
FAP74ami transformants have slow and uncoordinated flagella with abnormal waveforms. Montage of sequential frames from high speed recordings. Elapsed time in seconds is denoted on each frame. (A) WT cell displaying normal waveforms of two coordinated flagella (see Video 1). (B) 1G11 cell displaying uncoordinated flagellar movements. The right flagellum beats first, followed by the left flagellum. The corresponding waveform traces show an incomplete effective stroke and the lack of a normal recovery stroke (see Video 2). (C) 1G11 cell displaying uncoordinated flagella. The left flagellum completes two beats in the same time the right flagellum completes one effective stroke. The waveform traces also show the failure of the left flagellum to complete an effective stroke before beginning the recovery stroke. Examples of FAP74ami transformants 2D4 and 7A4 swimming can be seen in Videos 3 and 4, respectively.

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