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Circumstantial evidence has suggested the possibility of microtubule-associated protein (MAP) kinase's involvement in spindle regulation. To test this directly, we asked whether MAP kinase was required for spindle assembly in Xenopus egg extracts. Either the inhibition or the depletion of endogenous p42 MAP kinase resulted in defective spindle structures resembling asters or half-spindles. Likewise, an increase in the length and polymerization of microtubules was measured in aster assays suggesting a role for MAP kinase in regulating microtubule dynamics. Consistent with this, treatment of extracts with either a specific MAP kinase kinase inhibitor or a MAP kinase phosphatase resulted in the rapid disassembly of bipolar spindles into large asters. Finally, we report that mitotic progression in the absence of MAP kinase signaling led to multiple spindle abnormalities in NIH 3T3 cells. We therefore propose that MAP kinase is a key regulator of the mitotic spindle.
Figure 1. The MEK inhibitor UO126 blocks spindle assembly in Xenopus mitotic egg extracts. Spindle assembly reactions were assessed in the presence of 50 μM U0126 or 0.5% DMSO. Aliquots of extract were collected at 75 min for (A) biochemical analysis of active MAP kinase (p-MAP kinase), Cdc2 H1 kinase activity, and active JNK1 (p-JNK1); (B) monitoring spindle structures formed in control- (0.5% DMSO) (i) and UO126-treated extracts (ii, iii, and iv); and (C) quantitation of spindle structures in DMSO- and U0126-treated extracts. Results are representative of at least three independent experiments. Note: spindle assembly was never observed in U0126-treated extracts monitored at 10-min intervals from 30â120 min. Cdc2 H1 kinase assays were performed as described (Guadagno and Ferrell, 1998). Bar, 10 μm.
Figure 2. MAP kinase, not Rsk, is required for spindle assembly in Xenopus egg extracts. (A) Immunoblot analysis of endogenous MAP kinase protein and activity (p-MAPK antibody). (B) The efficiency of spindle assembly. 500 spindle structures were counted per sample. The data represent the average ± SEM obtained from three independent experiments. (C) Representative spindle structures. Bar, 10 μm. (D) immunoblots of Rsk1 and Rsk2. (E) Metaphase spindles assembled in mock- and Rsk1/2-depleted extracts. Bar, 10 μm. From three independent experiments the efficiency of spindle formation was â¼50% in mock- and Rsk1/2-depleted extracts (>100 spindle structures assessed per extract). Note: no statistical difference in microtubule density, spindle length, and spindle width was found between spindles formed in mock- or Rsk-depleted extracts (unpublished data).
Figure 3. Depletion MAP kinase in Xenopus M phase egg extracts leads to an increase in the length and polymerization of microtubules. (A) Immunoblot of phospho-MAP kinase. Reprobing the blot for MAP kinase protein shows similar results. (B) Typical microtubule asters. Bar, 10 μM. (C) Immunoblots of α-tubulin for two independent experiments representing the relative abundance of polymerized microtubules in mock, MAP kinaseâdepleted, and MAP kinase add-back extracts. A two- to sixfold increase in total polymerized tubulin was measured in MAP kinaseâdepleted extracts.
Figure 4. Spindle stability requires persistent MAP kinase activation in Xenopus mitotic extracts. Bipolar spindles were assembled in Xenopus egg extracts cycled into mitosis with nondegradable cyclin B. At time 0, extracts were either processed for immunofluorescence with phosphoâMAP kinase antibodies (A), or treated with the MEK inhibitor U0126 (50 μM final) or recombinant Xenopus GST-MKP-1 (40 ng/μl final). (B) Immunoblot analysis of active-MAP kinase at indicated time points. (C) Typical spindle or aster-like structures observed by epifluorescence at selected times following treatments. 100 spindle structures monitored for each sample per time point. Bar, 10 μm.
Figure 5. Inhibition of MAP kinase in NIH 3T3 cells leads to aberrant spindles. (A) Time course of mitotic index and experimental scheme. Cells collected for immunoblot analysis of active-ERK and total ERK protein (B) or fixed with 4% paraformaldehyde (C) and stained with α-tubulin antibodies and DAPI to visualize spindle microtubules (green) and chromosomes (blue), respectively. (Left) Control-treated cells in metaphase and late anaphase. (Right photos) Abnormal spindles observed in MAP kinaseâinhibited cells at mitosis (typical from ten independent experiments). White arrows show unattached chromosomes. Images were collected using a 100X oil objective lens. Bar, 2 μm. (D) Quantitation of abnormal spindle phenotypes in U0126 treated NIH 3T3 cells. The data represent the average ± standard deviation of three independent experiments where 120 abnormal mitotic figures were analyzed for each experiment. (E) Proposed model for MAP kinase pathway in spindle regulation.
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