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First published online 31 August 2004
doi: 10.1242/jcs.01303

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Outer dense fibre protein 2 (ODF2) is a self-interacting centrosomal protein with affinity for microtubules

¹ Göttinger Zentrum für Molekulare Biowissenschaften (GZMB), Entwicklungsbiologie, Georg-August-Universität Göttingen, Justus-von-Liebig-Weg 11, 37077 Göttingen, Germany
² Göttinger Zentrum für Molekulare Biowissenschaften (GZMB), Entwicklungsbiochemie, Georg-August-Universität Göttingen, Justus-von-Liebig-Weg 11, 37077 Göttingen, Germany

View larger version (41K): [in a new window]   Fig. 6. Copurification of ODF2 with tubulin from Xenopus oocyte extracts. Purified ODF2NC-GST or ODF2NC2-GST proteins were incubated in Xenopus oocyte extracts supplemented with tubulin and under conditions that promote tubulin polymerization. GST fusion proteins and associated proteins were isolated by affinity chromatography, separated using denaturing SDS-PAGE, transferred to Hybond-C and probed with anti-ODF2 antibodies (A), and anti--tubulin antibodies (B).

View larger version (34K): [in a new window]   Fig. 1. Structural organization of the ODF2 protein and subcellular localization of ODF2-GFP fusion proteins. (A) Scheme of ODF2 fusion constructs. As outlined, different parts of ODF2 were fused to GFP at its C-terminal end, resulting in a C-terminal GFP tag, or to GST at its N-terminal end. The leucine-zipper regions are shown as black boxes in the C-terminal region of ODF2. (B) Cytoplasmic versus nuclear distribution of ODF2-GFP fusion proteins expressed in cos-7 cells. Full length protein, ODF2NC, was slightly higher in the nucleus. The C-terminally truncated ODF2 proteins fused to GFP (ODF2NC1 and ODF2NC2) were mostly located outside of the nucleus, whereas the N-terminally truncated protein, ODF2N2C, fused to GFP was predominantly located inside the nucleus.

View larger version (72K): [in a new window]   Fig. 2. Full length and truncated ODF2 proteins form fibrillar structures in vivo partially associated with microtubules. Blue, DAPI counterstain; green, ODF2-GFP fluorescence; red, immunolocalization of -tubulin. C,F,J,M,P,S are merged images of ODF2-GFP, -tubulin and DAPI stainings. (A-G) Localisation of ODF2NC in cos-7 cells. ODF2NC fused to GFP was expressed in cos-7 cells. The protein is present in the nucleus and in the cytoplasm and shows a fibrillar structure (A,G). In G the fluorescence of the nucleus is too bright to discern the centrosome. Detection of microtubules with antibodies against -tubulin (B,C,E,F) revealed partial colocalisation to the microtubule network. Association with microtubules is also demonstrated by colocalisation of ODF2 to the mitotic spindle (D-F, arrows). Arrows in A and G indicate fibrous structures; arrowheads indicate MTOC. Scale bars: 10 µm. (H-S) Localisation of truncated ODF2-GFP fusion constructs transfected in cos-7 cells. Odf2NC1-GFP (H-J), Odf2NC2-GFP (K-M) and Odf2N2C-GFP (N-S) were transfected into cos-7 cells and the microtubule network highlighted by anti--tubulin staining. ODF2NC1 and ODF2NC2 showed cytoplasmic localisation whereas ODF2N2C was preferentially located inside the nucleus. All constructs are found to be concentrated in the centrosome (arrowheads) and have a fibrillar appearance (arrows). Colocalisation to the mitotic spindle is demonstrated for ODF2N2C (Q-S). Scale bars: 10 µm (H,K), 20 µm (N). (T-V) Cytoplasmic organisation of ODF2 is mostly MT independent. Odf2NC-GFP transfected cos-7 cells were treated with Nocodazol (2 µg/ml for 1 hour) for disruption of microtubules. Microtubules were detected by anti--tubulin staining (U,V). Whereas Nocodazol treatment disrupted MT organisation (U), the cytoplasmic ODF2 organisation is not affected (T).

View larger version (12K): [in a new window]   Fig. 3. ODF2 is a predicted overall coiled-coil protein. Secondary structure predictions for rat ODF2 were performed with the program `Coils' (Lupas et al., 1991) with a scanning window of 28 residues. The coiled-coil forming probability demonstrates that ODF2 is a predicted overall coiled-coil protein with the exception of the N-terminal 50 amino acids.

View larger version (24K): [in a new window]   Fig. 4. Self-interaction of ODF2 does not depend on the presence of the leucine zipper regions. ODF2-GST fusion proteins were incubated with ODF2-GFP fusion proteins, and GST proteins as well as associated proteins were isolated by affinity chromatography on glutathione-Sepharose beads. Proteins were separated using denaturing SDS-PAGE and probed with antibodies against GFP. The ODF2 constructs used for each assay are shown above with the first construct designating the GFP fusion detected by anti-GFP antibodies, and the second construct designating the GST fusion.

View larger version (42K): [in a new window]   Fig. 5. Cosedimentation of ODF2 with microtubules. (A,B) Microtubules were isolated from cos-7 cells expressing one out of four ODF2-GFP fusion proteins under conditions that promote microtubule polymerization. Proteins were separated using denaturing SDS-PAGE and transferred to Hybond-C. Probing with antibodies against GFP (A) revealed cosedimentation of the full-length ODF2NC as well as the truncated ODF2 proteins ODF2NC1, ODF2NC2 and ODF2N2C with microtubules. (B) Detection of tubulin by anti--tubulin antibodies in the same fractions as in A. (C) Control performed with cos-7 cells expressing ODF2NC-GFP demonstrating the dependence of ODF2 sedimentation on microtubules. No ODF2 proteins were found in the final pellet if the experiment was performed under microtubule destabilising conditions (2), whereas the cellular debris after the first centrifugation step do contain ODF2-GFP (1). Incubation with antibodies against GFP.

View larger version (38K): [in a new window]   Fig. 7. ODF2 does not bind directly to tubulin. Purified ODF2-GST fusion proteins were incubated with purified tubulin under conditions that promote microtubule formation. GST fusion proteins were isolated by affinity chromatography, separated using denaturing SDS-PAGE, transferred to Hybond-C, and probed with anti-ODF2 antibodies (A), and anti--tubulin antibodies (B). Whereas ODF2 fusion proteins were successfully isolated, -tubulin was not coprecipitated.