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doi: 10.1242/10.1242/jcs.00088

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The mitotic-spindle-associated protein astrin is essential for progression through mitosis

Jens Gruber^1,*, Jens Harborth^1,*, Jörg Schnabel¹, Klaus Weber¹ and Mechthild Hatzfeld^2,

¹ Max Planck Institute for Biophysical Chemistry, Department of Biochemistry, Am Fassberg 11 37070 Göttingen, Germany
² Department of Biochemistry and Pathobiochemistry, Medical Faculty of the University of Halle, 06097 Halle/Saale, Germany

View larger version (19K): [in a new window]   Fig. 1. (A) Secondary structure prediction and domain organization of astrin. (B) cDNA constructs used in this study. cc denotes the coiled-coil regions, L the linker region.

View larger version (29K): [in a new window]   Fig. 2. (A) Purification of recombinant astrin. A Coomassie-stained gel showing bacterial extracts before (-IPTG) and 4 hours post-induction (+IPTG). The protein aggregated into inclusion bodies (IB) and was purified under denaturating conditions by anion exchange chromatography (Mono Q). Western blot (WB) analysis using a monoclonal T7-antibody against the tag-sequence and the two astrin antibodies (DC and DN) confirmed the identity of purified astrin. (B) CD spectroscopy of purified astrin in phosphate buffer containing 0.5 mM DTT.

View larger version (62K): [in a new window]   Fig. 3. (A) Electron microscope analysis of recombinant astrin-T7 in low salt phosphate buffer containing 2.5 mM DTT. The gallery of dimers shows that the flexible linker in the rod domain can be bent to varying degrees. (B) Electron microscope analysis of recombinant astrin-T7 in PBS containing 2.5 mM DTT. The overview shows formation of higher order oligomers. Examples of oligomers containing two to five dimers are shown in the gallery. Oligomerization is exclusively mediated by the globular head domains. Bar, 100 nm.

View larger version (44K): [in a new window]   Fig. 4. siRNA-induced knockdown of astrin in HeLa cells. Cells were transfected with the luciferase control siRNA (pGL2 siRNA) or with the astrin-specific siRNA (A). After 44 hours, cells were stained for -tubulin, astrin and DNA (Hoechst). Note the loss of astrin from the abnormal mitotic figures induced after astrin depletion. (B) After 44 hours, cells were stained for tubulin, -tubulin and DNA (Hoechst). Astrin depletion affects chromosome congression and proper spindle formation. No normal metaphase cells were found. Bars in (A) and (B), 15 µm. (C) Silencing of astrin was confirmed by the immunoblotting. The lower parts show comparable reactions of the blots of control and astrin-siRNA-treated cells with vimentin antibody.

View larger version (80K): [in a new window]   Fig. 5. Astrin silencing induces apoptosis. The TUNEL assay shows that many of the cells transfected with the astrin siRNA undergo apoptosis (green colour) 60 hours post-transfection (A-C). Cells transfected with the luciferase siRNA (pGL2) continue to grow (D-F). A single apoptotic cell was detected in this field. The phase image and the fluorescent image arising from the incorporation of FITC-labeled deoxynucleotides are superimposed (for details see Materials and Methods).