Essential role of Cenexin1, but not Odf2, in ciliogenesis

Abstract

Primary cilia are microtubule-based solitary sensing structures on the cell surface that play crucial roles in cell signaling and development. Abnormal ciliary function leads to various human genetic disorders, collectively known as ciliopathies. Outer dense fiber protein 2 (Odf2) was initially isolated as a major component of sperm-tail fibers. Subsequent studies have demonstrated the existence of many splicing variants of Odf2, including Cenexin1 (Odf2 isoform 9), which bears an unusual C-terminal extension. Strikingly, Odf2 localizes along the axoneme of primary cilia, whereas Cenexin1 localizes to basal bodies in cultured mammalian cells. Whether Odf2 and Cenexin1 contribute to primary cilia assembly by carrying out either concerted or distinct functions is unknown. By taking advantage of odf2-/- cells lacking endogenous Odf2 and Cenexin1, but exogenously expressing one or both of these proteins, we showed that Cenexin1, but not Odf2, was necessary and sufficient to induce ciliogenesis. Furthermore, the Cenexin1-dependent primary cilia assembly pathway appeared to function independently of Odf2. Consistently, Cenexin1, but not Odf2, interacted with GTP-loaded Rab8a, localized to the distal/subdistal appendages of basal bodies, and facilitated the recruitment of Chibby, a centriolar component that is important for proper ciliogenesis. Taken together, our results suggest that Cenexin1 plays a critical role in ciliogenesis through its C-terminal extension that confers a unique ability to mediate primary cilia assembly. The presence of multiple splicing variants hints that the function of Odf2 is diversified in such a way that each variant has a distinct role in the complex cellular and developmental processes.

Keywords: Cenexin1; Chibby; Odf2; Rab8a; ciliogenesis; primary cilia.

Figure 1. hCenexin1, but not hOdf2, binds to Rab8a in a GTP-dependent manner. (A) Schematic diagram of primary amino acid sequences showing hCenexin1 (also called hOdf2 isoform 9; accession number DQ444714 or NP_002531.3) and hOdf2 (also called hOdf2 isoform 6; accession number AF012549 or NP_702915.1). Dark blue, hCenexin1-specific C-terminal extension (residues 614–805); dark red, hOdf2-specific 20 residues (residues 619–638 in hOdf2); N-terminal light blue and light red regions, N-terminal 37 or 41 residue-long variable regions in hCenexin1 or hOdf2, respectively. (B) Asynchronously growing HEK293T cells were co-transfected with Rab8a and hOdf2 variant (var.) (hOdf2 isoform 4 containing a 19-residue insertion in the N-terminus; accession number NP_702914.1), hCenexin1, or hOdf2. Twenty-four hours after transfection, cells were serum-starved for 30 h, and the resulting cells were lysed with TBSN buffer. Total cellular lysates were incubated with 200 μM of GTPγS, GTP, or GDP for 1 h and then subjected to immunoprecipitation with an anti-HA antibody. Immunoprecipitates were subjected to immunoblotting analyses.

Figure 2. hCenexin1, but not hOdf2, is necessary and sufficient to induce primary cilia assembly. (A) F9 ODF2^+/+ or odf2^−/− cells expressing the indicated control vector or hCenexin1/hOdf2 constructs were generated as described in “Materials and Methods.” Total cell lysates were prepared and subjected to immunoblotting analyses to comparatively examine the levels of exogenously expressed proteins with that of endogenous mCenexin1. The arrows indicate exogenous hCenexin1/hOdf2 proteins, while the arrowhead denotes endogenous mCenexin1. (B and C) The same cells in (A) were subjected to immunostaining analyses with anti-Cenexin1/Odf2 (red) and anti-acetylated tubulin (green) antibodies (B). Insets show enlarged images of the respective panels. Magnified images are from the dotted squares in the Merge panels. The number of cells with primary cilia in (C) was quantified from more than 200 cells per each of three independent experiments. Error bars indicate the mean ± standard error of the mean (SEM). (D and E) F9 odf2^−/− cells expressing hCenexin1 were superinfected with lentiviruses expressing the indicated constructs, and the lysates prepared from these cells were subjected to immunoblotting analysis with an anti-Cenexin1/Odf2 antibody (D). Arrows indicate exogenously expressed hCenexin1 proteins co-migrating with endogenous mCenexin1. The number of cells with the primary cilia was quantified from more than 200 cells obtained from each of three independent experiments (E). Error bars represent the mean ± SEM.

Figure 3. Cenexin1 localizes to the distal/subdistal appendages of basal bodies and induces apparently normal primary cilia. (A) F9 ODF2^+/+ control cells and odf2^−/− cells expressing hCenexin1 were subjected to IEM using an anti-Cenexin1/Odf2 antibody. Arrows indicate endogenous mCenexin1 (ODF2^+/+ panels) and exogenously expressed hCenexin1 (odf2^−/− panels) localized to the distal/subdistal appendage. A total of 800–1,000 thin-sectioned cells were examined for each sample. Among them, approximately 5–10% contained centrosomes. Dotted triangles, distal/subdistal appendages; asterisks, daughter centrioles. (B) F9 odf2^−/− cells expressing vector, hOdf2, or hCenexin1 were subjected to transmission electron microscopy (TEM). The odf2^−/− cells expressing either the control vector or hOdf2 exhibited basal bodies without any detectable ciliary structure (top two panels), whereas the cells expressing hCenexin1 displayed a basal body with a morphologically normal-looking primary cilium (bottom panel). Representative images for each group are shown.

Figure 4. hCenexin1, but not hOdf2, is required to recruit Chibby to basal bodies under serum-starved conditions. (A and B) F9 ODF2^+/+ control cells and odf2^−/− cells expressing control vector, hCenexin1 or hOdf2 were serum starved for 18 h and co-stained with anti-Cenexin1/Odf2 and anti-Chibby antibodies (A) or with anti-Chibby and anti-γ-tubulin antibodies (,B). The resulting samples were quantified from more than 200 cells obtained from each of three independent experiments (B). Statistics: **p < 0.01 (unpaired two-tailed t-test). Note that, regardless of the presence or absence of hCenexin1, γ-tubulin is normally recruited to the odf2^−/− basal bodies.