XB-IMG-123493
Xenbase Image ID: 123493
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Figure 6. CaMKII triggers spindle disassembly in anaphase independently of APC/C activation. Spindles were preassembled in CSF extracts (Meta), and anaphase was induced by the addition of calcium or CaMKII* in the absence or presence of XErpND. (A) Spindles were visualized by direct fluorescence of Cy3-tubulin (red) and DAPI (blue) after 20 min. Out of n imaged spindles, the mean overall fluorescence intensity was determined, and an average image was generated using the Matlab macro. The pole (P) to pole distance (spindle size) was measured as well as the corresponding mean fluorescence distribution plotted along the pole to pole axis in a 1.5-μm-wide area (intensity distribution; see blue lines in average images). Intensity and spindle size are indicated in relative terms (percentage). (B) Immunoblots were used to determine the amounts of cyclin B, an autoradiograph was used to monitor amounts of exogenously added in vitro translated (IVT) securin, and an autoradiograph displaying a histone H1 kinase assay was used to determine Cdk1 activity under the conditions used in A. The black line indicates that intervening lanes have been spliced out. (C) Anaphase was induced in preassembled spindles by low doses of CaMKII* in the presence of cyclinBΔ90, and samples were fixed after 40 min. Spindles were analyzed as in A. (D) Determination of cyclin B by immunoblotting under the conditions used in C. (E) Spindles were first treated with CaMKII* and XErpND and mixed with extracts treated without (−) or with (+) CaMKII* to activate APC/C. (top) Immunoblotting was used to determine the amounts of cyclin B and histone H1 kinase assay to measure the Cdk1 activity. (bottom) Representative fluorescence images of structures after 40 min. Tubulin, red; DAPI (DNA), blue. MT, microtubules; Meta, extracts after spindle assembly. Bars, 5 μm. Image published in: Reber S et al. (2008) © 2008 Reber et al. Creative Commons Attribution-NonCommercial-ShareAlike license Larger Image Printer Friendly View |