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A family of microtubule (MT)-binding proteins, Orbit/multiple asters/cytoplasmic linker protein-associated protein, has emerged as an important player during mitosis, but their functional mechanisms are poorly understood. In this study, we used meiotic egg extracts to gain insight into the role of the Xenopus laevis homologue Xorbit in spindle assembly and function. Xorbit immunodepletion or its inhibition by a dominant-negative fragment resulted in chromosome alignment defects and aberrant MT structures, including monopolar and small spindles. Xorbit-depleted extracts failed to nucleate MTs around chromatin-coated beads, indicating its essential requirement for spindle assembly in the absence of centrosomes and kinetochores. Xorbit's MT stabilizing effect was most apparent during anaphase, when spindle MTs depolymerized rapidly upon Xorbit inhibition. Biochemical interaction between a COOH-terminal Xorbit fragment and the kinetochore-associated kinesin centromeric protein E may contribute to Xorbit's role in chromosome congression. We propose that Xorbit tethers dynamic MT plus ends to kinetochores and chromatin, providing a stabilizing activity that is crucial for spindle assembly and chromosome segregation.
Figure 1. Xorbit inhibition results in aberrant spindle structures and chromosome alignment defects. (A) Western blot of CSF Xenopus extract and extracts depleted using IgG (Îmock) or Xorbit antibodies (ÎXorbit) probed with α-Xorbit antibody. (B) Metaphase spindles assembled in mock- and Xorbit-depleted extracts. All three ÎXorbit images display chromosome alignment defects, and the two bottom spindles are also smaller in size. (C) GST or GST-CT were added at the start of mitosis, and metaphase spindles were examined. (D) Immunofluorescence with α-GST, shown in green in merged images. Arrow indicates aggregates of added protein. MTs are red and DNA is blue. Bars, 10 μm.
Figure 2. Xorbit is required for MT stabilization by mitotic chromatin. (A) Spindles assembled around chromatin-coated beads in mock- and Xorbit-depleted extracts. (B) GST or GST-CT were added at the start of mitosis, and metaphase chromatin bead spindles were examined. MTs are red and DNA is blue. (C) Centrosome asters were assembled in mock- and Xorbit-depleted extracts and were fixed after 10 min. Bars, 10 μm. (D) MTs polymerized after a 10-min incubation in the presence or absence of DMSO in mock- and Xorbit-depleted extracts were pelleted and analyzed by immunoblotting with α-tubulin antibody.
Figure 3. Xorbit is essential for MT stabilization during anaphase. Chromosome segregation in mock- and Xorbit-depleted extracts. (A) Once metaphase spindles assembled (top), anaphase was induced, and samples were fixed after 5, 10, and 15 min. MTs are red and DNA is blue. (B) Time-lapse fluorescence microscopy of X-rhodamineâlabeled MTs in mock- and Xorbit-depleted spindles during anaphase. Time is in seconds after anaphase induction. Bars, 10 μm.
Figure 4. Xorbit is required to maintain kinetochore and nonkinetochore MTs at anaphase onset. (A) Wild-type metaphase spindles were assembled, and GST or GST-CT was added at the same time anaphase was induced. Spindles were fixed 5, 10, and 15 min later. (B) Wild-type spindles were assembled around chromatin-coated beads, and GST or GST-CT was added during metaphase arrest (bottom left) or at the same time anaphase was induced (right), and spindles were fixed 10 min later. MTs are red and DNA is blue. Bars, 10 μm.
Figure 5. Xorbit interacts with kinetochore kinesin 7 (CENP-E). (A) Silver-stained gel of GST and GST-CT pull-downs performed in Xenopus tropicalis extracts. GST-CTâinteracting proteins analyzed by mass spectrometry are indicated on the right. The band at â¼160 kD could not be identified. (B) Western blot of CSF extract, GST, and GST-CT pull-downs probed with αâCENP-E antibody. (C) Metaphase spindles in mock- and Xorbit-depleted extracts stained for CENP-E. (D and E) Duplicated metaphase chromosomes formed in the presence of GST and GST-CT stained for CENP-E (D and E) and GST (D) or BubR1 (E). In merged images, CENP-E is shown in green, DNA in blue, and either MTs (C), GST-CT (D), or BubR1 (E) in red. Arrows indicate aggregates of GST-CT and CENP-E. Bars, 10 μm.
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