Outer dense fiber 2 is a widespread centrosome scaffold component preferentially associated with mother centrioles: its identification from isolated centrosomes

Abstract

Because centrosomes were enriched in the bile canaliculi fraction from the chicken liver through their association with apical membranes, we developed a procedure for isolation of centrosomes from this fraction. With the use of the centrosomes, we generated centrosome-specific monoclonal antibodies. Three of the monoclonal antibodies recognized an antigen of ~90 kDa. Cloning of its cDNA identified this antigen as a chicken homologue of outer dense fiber 2 protein (Odf2), which was initially identified as a sperm outer dense fiber-specific component. Exogenously expressed and endogenous Odf2 were shown to be concentrated at the centrosomes in a microtubule-independent manner in various types of cells at both light and electron microscopic levels. Odf2 exhibited a cell cycle-dependent pattern of localization and was preferentially associated with the mother centrioles in G0/G1-phase. Toward G1/S-phase before centrosome duplication, it became detectable in both mother and daughter centrioles. In the isolated bile canaliculi and centrosomes, Odf2, in contrast to other centrosomal components, was highly resistant to KI extraction. These findings indicate that Odf2 is a widespread KI-insoluble scaffold component of the centrosome matrix, which may be involved in the maturation event of daughter centrioles.

Figure 1

Centrosomes in the isolated bile canaliculi from chicken liver. (a) Ultrathin sections of the isolated bile canaliculi. The lumen of the bile canaliculus (asterisk) is delineated with apical membranes of several surrounding hepatocytes. Note several junctional complexes (arrowheads) and two centrosomes (arrows). (b) Fibrous structures (arrow) were occasionally detected between centrosomes and apical membranes (asterisk). (c and d) Double immunofluorescence staining of the isolated bile canaliculi on coverslips with anti-ZO-1 pAb (green)/anti-β-tubulin (tub) mAb (red) (c) or with anti-β-tubulin mAb (green)/anti-γ-tubulin pAb (red) (d). Many β-/γ-tubulin double-positive dots, i.e., centrosomes, were associated with individual isolated bile canaliculi, the tight junctions of which were visualized as parallel lines with anti-ZO-1 pAb. (e) Ultrathin sections of isolated centrosomes from the bile canaliculi fraction. In addition to centrosomes, contamination with fragmented membranous structures, fibrous structures such as collagen fibers, and amorphous structures can be seen. Bars: (a, b, and e) 0. 5 μm; (c and d) 10 μm.

Figure 2

Identification of Odf2 as a centrosomal component. (A) Immunoblotting analyses of the isolated bile canaliculi from the chicken liver with three independent anti-centrosome mAbs, mAb101, mAb184, and mAb1019. All of the mAbs detected a band of approximately 90 kDa (arrow). CBB, Coomassie brilliant blue staining. (B) The deduced aa sequence of the antigen for mAb101. This antigen showed striking similarity to mouse Odf2/1(M. Odf2/1) (an isotype of Odf2), although its C-terminal fragment sequence is different from that of mouse Odf2/1. Conserved aa are boxed. This antigen was designated as chicken Odf2 (C. Odf2). (C) Reactivities of mAb101, mAb184, and mAb1019 with chicken Odf2 and mouse Odf2/1. GST-fusion proteins with full-length (c-F), parts of C-terminal half (aa 297–659 [c-C1], aa 297–630 [c-C2], and aa 297–588 [c-C3]) of chicken Odf2 and full-length of mouse Odf2/1 (m-F) were produced in E. coli, and then the crude lysate of E. coli was immunoblotted with three mAbs. D, Localization of exogenously expressed Odf2. HA-tagged chicken Odf2 (a) or mouse Odf2/1 (b) was expressed in human HeLa cells, followed by double immunofluorescence staining with anti-HA mAb (HA-tag; green)/anti-γ-tubulin pAb (γ-tub; red). In cells transiently expressing a large amount of chicken Odf2 (a), Odf2 formed huge fibrous aggregates throughout the cytoplasm in which centrosomes were detected by γ-tubulin staining. In stable transfectants expressing lower levels of Odf2 (b), mouse Odf2/1 showed centrosomal localization. Bars, 10 μm.

Figure 3

Localization of Odf2 in the isolated bile canaliculi. (A) Double immunofluorescence staining of the isolated bile canaliculi on coverslips with an anti-Odf2 mAb 101 (Odf2; green)/anti-γ-tubulin mAb (γ-tub; red). Each centrosome was resolved into two dots, both of which were stained equally for γ-tubulin at low (a) and high (b and c) magnifications. In sharp contrast, one dot of the pair was stained more intensely for Odf2 than the other dot. Bars: top, 10 μm; bottom, 1 μm. (B) Immunoelectron microscopic staining of the isolated bile canaliculi with anti-Odf2 mAb (mAb101) and then with anti-rat IgG pAb conjugated with 15-nm (a, b, and d) or 10-nm (c and e) gold particles. In the longitudinal (a and b) and transverse (c–e) sectional images of centrioles, the high levels of gold labeling of Odf2 were associated with the mother centrioles, especially with the distal appendages (arrows), whereas the lower levels were associated with daughter centrioles (arrowheads). Bar, 200 nm.

Figure 4

Subcellular localization of endogenous Odf2 in the liver and cultured cells. (a and b) Localization of Odf2 in frozen sections of the chicken liver and cultured human HeLa cells at low magnification. The samples were double stained with anti-Odf2 mAb101 (Odf2; green) and anti-γ-tubulin mAb (γ-tub; red). In the liver (a), Odf2 appeared to be preferentially associated with one of the paired γ-tubulin-positive centrioles. In HeLa cells (b), the ratio of staining intensity with anti-Odf2 mAb between paired centrioles appeared to vary among cells. In the majority of cells, both of the paired centrioles were stained equally. However, in some cells, one of the paired centrioles was stained more intensely than the other (arrows). (c–e) Cell cycle-dependent localization of Odf2 in the centrosomes of human HeLa cells at higher magnification. The cells were double stained with anti-Odf2 mAb101 (Odf2; green) and anti-γ-tubulin mAb (γ-tub; red). In G1-phase (c), Odf2 (Odf2; green) was preferentially associated with the mother centriole, whereas γ-tubulin (γ-tub; red) was equally associated with both centrioles. Toward G1/S-phase before duplication of centrioles, Odf2 (Odf2; green) was associated almost equally with both centrioles (γ-tub; red; d and e). The distributions of Odf2 and γ-tubulin were not precisely identical (e). (f–h) Cell cycle-dependent localization of Odf2 and ninein in the centrosomes of mouse L cells at higher magnification. The cells were triple stained with anti-Odf2 mAb101 (Odf2; green), anti-γ-tubulin mAb (γ-tub; red), and anti-ninein pAb(ninein; blue). In G1-phase (f), Odf2 (Odf2; green) was preferentially associated with the mother centriole, which was identified as the ninein staining. The Cy5 staining of ninein was pseudocolored in blue. Toward G1/S-phase before duplication (g and h), Odf2 and ninein were associated equally with both of the paired centrioles. The distributions of Odf2, γ-tubulin and ninein were not precisely identical (e and h). Bars: (a and b) 5 μm; (c–h) 2 μm.

Figure 5

KI insolubility of Odf2 in centrosomes. (A) immunofluorescence analysis of the untreated bile canaliculi (a and c; −KI) or 2 M KI-treated bile canaliculi (b and d; +2 M KI) on coverslips were doubly stained with anti-Odf2 mAb101 (Odf2; green)/anti-ZO-1 pAb (ZO-1; red; a and b) or with mAb101(Odf2; green)/anti-γ-tubulin mAb (γ-tub; red; c and d). Incubation (20 min) with 2 M KI removed ZO-1 and γ-tubulin from bile canaliculi, whereas Odf2 was highly resistant to KI extraction. Bar, 10 μm. (B) Western blot analysis of the untreated (−) or 2 M KI-treated (KI) samples of bile canaliculi (BC) or isolated centrosomes (Centro). The untreated or KI-extracted samples, prepared from the bile canaliculi (30 μg) and isolated centrosome fractions (3 μg), were applied to the SDS-PAGE and blotted with anti-Odf2 mAb1019 and anti-γ-tubulin mAbs (a). Because >90% of the total proteins of the bile canaliculi and isolated centrosomes were extracted by the KI treatment, the total protein applied on the gel is reduced to <10% in KI-treated samples as compared with the untreated samples. However, the signals for Odf2 were almost the same in their intensity between the KI-insoluble and the untreated samples in the bile canaliculi as well as the isolated centrosomes, indicating high resistance of Odf2 to the extraction by KI (ns; nonspecific signal; see Figure 2A). In contrast, the signals for γ-tubulin were extremely attenuated by the KI treatment, indicating high extractability of γ-tubulin by KI. Next, to estimate the degree of enrichment of Odf2 into the KI-insoluble fractions, the gels loaded with equal protein (3 μg) from the untreated and KI-treated samples of the bile canaliculi or isolated centrosomes were blotted with anti-Odf2 mAb1019 and anti-γ-tubulin mAb, with the result that Odf2 is highly enriched in the KI-treated samples versus the untreated samples (b).