Mao Y , Desai A , Cleveland DW .

R01 GM 29513 NIGMS NIH HHS , R01 GM029513 NIGMS NIH HHS

	Figure 1. Motorless CENP-Etail activates BubR1 kinase activity and mitotic checkpoint signaling in Xenopus egg extracts. (A) Xenopus CENP-E protein structure showing the relative position of the NH2-terminal microtubule motor domain, the rod domain, and the COOH-terminal kinetochore binding tail domain. (B) Purification of recombinant Xenopus CENP-Etail. Initial Escherichia coli lysates encoding 6-His-CENP-Etail (lane 1) and the recombinant protein (arrowhead) after purification over Ni-NTA agarose beads (QIAGEN; lane 2). (C) CENP-Etail activates BubR1 kinase activity in Xenopus egg extracts. After immunodepletion of endogenous BubR1, recombinant GST–BubR1 was added to a molar level comparable to the initial level of endogenous BubR1 in CSF-arrested egg extracts. Sperm nuclei and nocodazole were then added to egg extracts containing either endogenous CENP-E (mock depleted) or CENP-Etail (CENP-E–depleted extracts supplemented with CENP-E tail fragment) as indicated. After 30 min, GST–BubR1 was immunoprecipitated using specific anti-GST antibody and immunoblotted with anti-BubR1 antibody (bottom row) or kinase activity assayed after addition of histone H1 and [γ-32P]ATP (top row). (D) CENP-Etail is sufficient for activating mitotic checkpoint signaling. Xenopus egg extracts were (a and b) mock depleted, or (c) CENP-E depleted, and then supplemented with either (d and e) recombinant CENP-E or (f and g) CENP-Etail. After incubation of sperm nuclei with or without nocodazole, as indicated, CSF activity was inactivated by addition of calcium. Aliquots were taken from each extract at indicated times and then assayed by autoradiography for Cdk1 kinase activity (left) using added histone H1 as a substrate and (right) maintenance of chromatin condensation. (E) CENP-Etail–induced mitotic arrest is dependent on the mitotic checkpoint. CSF-arrested egg extracts were depleted with anti-IgG (1 and 2) or anti-CENP-E antibodies and supplemented with CENP-Etail (3–5) and incubated with sperm nuclei and nocodazole as indicated for 30 min. IgG (1–4) or Mad2 antibodies (5) were then added to reactions as indicated. Subsequently, Cdk1 kinase activity was measured (on histone H1, top) at 0, 30, and 60 min (as indicated) after calcium addition, and chromatin was visualized with DAPI (bottom).
	Figure 2. Microtubule capture by CENP-E is required for preventing Mad2 recruitment to sister kinetochores under tension in Xenopus egg extracts. (A–C) CENP-E–depleted egg extracts containing sperm nuclei were cycled through interphase and arrested with CSF activity at the following metaphase. (A) Recombinant CENP-E or (B and C) CENP-Etail were added. (B) Nocodazole or (A and C) no drug was added for 30 min, as indicated, and kinetochore recruitment of Mad2 (green) and spindle microtubules (red) were visualized by indirect immunofluorescence, and chromatin was visualized with DAPI (blue). Paired kinetochores (labeled by [A] Mad2 and BubR1, or [B and C] Mad2 are boxed and shown magnified in insets [right]). (D and E) Quantification of (D) the spacing between paired kinetochores (blue: 0–0.4 μm, red: >0.4 μm); and (E) the normalized integrated intensity of Mad2 signals at kinetochores in D. At least 10 kinetochores from more than three spindles were quantified for each bar. P < 0.05. Error bars represent standard errors.
	Figure 3. Continued recruitment of mammalian Mad2 to bioriented, sister kinetochores under tension in the presence of CENP-Etail. (A–L) Transient expression of CENP-Etail in cultured human T98B cells. (A, D, G, and J) Flag:hCENP-Etail and (B, C, E, F, H, I, K, and L; red) Mad2 were observed by immunofluorescence with mouse anti-flag, rabbit anti-hMad2, and (C, F, I, and L; green) rat anti-tubulin antibodies, respectively. (C, F, I, and L; blue) Chromatin was visualized with DAPI. (M) The mitotic index and (N) the proportions of mitotic cells exhibiting prometaphase, metaphase, and anaphase chromosome alignments in cells transfected with Flag:hCENP-Etail and control cells. 200 cells were counted in three independent transfections. P < 0.001. Error bars represent standard errors.
	Figure 4. Microtubule capture by CENP-E in vitro produces a ternary complex of microtubule–CENP-E–BubR1 in which CENP-E–activated BubR1 kinase activity is silenced. (A) In vitro kinase assays were performed with combinations of purified GST–BubR1 (lane a), CENP-E (lane b), and 1 μM, 0.1 μM, 10 nM, 1 nM, or 0.1 nM GMPCPP microtubules (lanes 4–8, respectively), 1 μM tubulin (lane 9), or 33 μM GMPCPP (lane 10). (B and C) CENP-E, BubR1, and microtubules form a ternary complex. After centrifugation through a 40% sucrose cushion, BubR1–CENP-E–GMPCPP microtubule complex formation was assayed by (B) immunoblot or (C) triple immunofluorescence for BubR1 (red), CENP-E (green), and microtubules (blue).
	Figure 5. CENP-Etail activation in vitro of BubR1 kinase cannot be silenced by addition of microtubules. (A) In vitro kinase assays were performed with combinations of purified CENP-E, CENP-Etail, and GST–BubR1 in the presence or absence of GMPCPP microtubules (1 μM), as indicated. Histone H1 was added as a substrate. (B) The CENP-Etail–BubR1 complex cannot bind microtubules. After centrifugation through a 40% sucrose cushion, BubR1–CENP-Etail–GMPCPP microtubule complex formation was assayed by triple immunofluorescence for BubR1 (red), CENP-Etail (green), and microtubules (blue). (C) Schematic of silencing BubR1-dependent, mitotic checkpoint signaling upon microtubule capture by CENP-E. (Left) Activities of four kinetochore associated kinases (MapK, Mps1, Bub1, and BubR1) are essential for generation by unattached kinetochores of a “stop anaphase” mitotic checkpoint inhibitor (for detailed review see Cleveland et al., 2003). CENP-E activates the essential BubR1 kinase activity resulting in rapid cycling of Mad2 onto unattached kinetochores and release in a form that is a “stop anaphase” inhibitor. (Right) Spindle microtubule capture by CENP-E silences CENP-E–dependent BubR1 kinase activity without dissociation from BubR1. In the absence of active BubR1 kinase, Mad2 and its stably bound kinetochore tether, Mad1 (Shah et al., 2004; De Antoni et al., 2005), as well as the Rod–Zw10–dynactin–cytoplasmic dynein complex, are released from kinetochores (Kops et al., 2005), thus silencing mitotic checkpoint signaling at kinetochores.

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