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doi: 10.1242/10.1242/jcs.00057

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Anaphase onset does not require the microtubule-dependent depletion of kinetochore and centromere-binding proteins

Julie C. Canman^1,, Nitin Sharma^1,*, Aaron Straight^2,*, Katie B. Shannon¹, Guowei Fang³ and E. D. Salmon¹

¹ University of North Carolina, Department of Biology, 607 Fordham Hall, CB#3280, Chapel Hill, NC 27599, USA
² Harvard Medical School, Department of Cell Biology, Seely Mudd 529, 240 Longwood Avenue, Boston, MA 021 15, USA
³ Stanford University, Department of Biological Sciences, Gilbert Building, Room 345, Stanford, CA 94305-5020, USA

View larger version (29K): [in a new window]   Fig. 1. Mad1 constructs. (A) Full length Homo sapiens Mad1 protein showing Mad1-Mad1 oligomerization domain, Mad2-binding domains, and RLK sequence essential for Bub1 and Bub3 interactions. (B) GST-Mad1F10, which lacks the first 320 amino acids of full length Mad1, but contains the Mad1-Mad1 oligomerization domain and the Mad2-binding domain. Notably, GST-Mad1F10 is also missing the last 162 amino acids, which includes the RLK Bub1 and Bub3-interaction sequence.

View larger version (115K): [in a new window]   Fig. 2. Mad1F10 can activate the APC/C in vitro. SDS-PAGE of Xenopus mitotic extracts that were incubated with buffer alone (panel I) or with 20 µM Mad2 (panel II), in the presence of increasing amounts of Mad1F10 (a-e at 0, 10, 20, 40 and 60 µM). Asterisks indicate APC/C activation by Mad1F10.

View larger version (128K): [in a new window]   Fig. 3. GST-Mad1F10 can induce premature anaphase onset in prometaphase mammalian tissue cells. Prometaphase PtK1 cells were microinjected with either GST alone (top row) or GST-Mad1F10 (middle row). Microinjection occurred at the 0:00 time point for all rows. The injection of GST-Mad1F10 triggered premature anaphase onset prior to alignment of all chromosomes at the metaphase plate (middle row). Arrows mark unaligned chromosomes. GST-Mad1F10 also induced anaphase onset in cells pre-treated with 10 µM nocodazole to depolymerize all microtubules (bottom row). Asterisks indicates the injection needle. Bar, 5 µM.

View larger version (118K): [in a new window]   Fig. 4. Microtubules are required for depletion of Mad2, cytoplasmic dynein, BubR1, and the phosphoepitope 3F3/2 at kinetochores in anaphase in PtK1 cells. (A-D) For all images the top row shows a prometaphase cell, the second row shows a cell induced to enter precocious anaphase by Mad1F10 injection, the third row shows a cell treated with 10 µM nocodazole, and the bottom row shows a cell treated with 10 µM nocodazole and induced to enter anaphase by Mad1F10 injection. Bar, 5 µM.

View larger version (29K): [in a new window]   Fig. 6. Kinetochore (Mad2, dynein, 3F3/2, BubR1 and CENP-E) and centromere (INCENP) fluorescence intensity measurements in prometaphase and anaphase with and without microtubules. The figure provides a graphical representation of the data shown in Table 1 after translating the raw measurements to a percentage of unattached prometaphase kinetochore intensities for each experimental condition respectively.

View larger version (97K): [in a new window]   Fig. 5. Microtubules are required for depletion of INCENP at centromeres in anaphase in PtK1 cells. A prometaphase cell fixed and stained for INCENP (top row). A cell induced to enter precocious anaphase by Mad1F10 injection (second row). A cell treated with 10 µM nocodazole (third row). A cell treated with 10 µM nocodazole and induced to enter precocious anaphase by Mad1F10 injection (bottom row). Bar, 5 µM.

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