Abstract
The small guanosine triphosphatase Ran loaded with GTP (RanGTP) can stimulate assembly of the type V intermediate filament protein lamin B into a membranous lamin B spindle matrix, which is required for proper microtubule organization during spindle assembly. Microtubules in turn enhance assembly of the matrix. Here we report that the isolated matrix contains known spindle assembly factors such as dynein and Nudel. Using spindle assembly assays in Xenopus egg extracts, we show that Nudel regulates microtubule organization during spindle assembly independently of its function at kinetochores. Importantly, Nudel interacts directly with lamin B to facilitate the accumulation and assembly of lamin-B-containing matrix on microtubules in a dynein-dependent manner. Perturbing either Nudel or dynein inhibited the assembly of lamin B matrix. However, depleting lamin B still allowed the formation of matrices containing dynein and Nudel. Therefore, dynein and Nudel regulate assembly of the lamin B matrix. Interestingly, we found that whereas depleting lamin B resulted in disorganized spindle and spindle poles, disrupting the function of Nudel or dynein caused a complete lack of spindle pole focusing. We suggest that Nudel regulates microtubule organization in part by facilitating assembly of the lamin B spindle matrix in a dynein-dependent manner.
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Acknowledgements
We thank Dr Reimer Stick for LB3 monoclonal antibody, Dr Robert Goldman for the full-length LB3 construct, Ms Shufang He and Ms Xiaoyan Wang for help with the Biacore experiments, Mr Dan Ducat and Mr Ben Goodman for help with egg extracts, Ms Ona Martin for technical support, and Dr Max Guo and the members of the Zheng laboratory for critical comments. The work was supported by the National Institutes of Health (GM56312 to Y.Z.; MH067880 and P41 RR011823 to J.R.Y.), the Chinese Academy of Sciences (CXTD-S2005-3 to X.Z.), the Ministry of Science and Technology of China (2007CB914501 and 2005CB522703 to X.Z.), the National Science Foundation of China (30721065, 30623003 and 30830060 to X.Z.) and a Cystic Fibrosis Foundation Therapeutics, inc. computational fellowship (BALCH05X5 to B.L.). Y.Z. is an investigator of the Howard Hughes Medical Institute.
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Y.Z. and X.Z. planned the project. L.M. and Y.Z. designed the experiments and analysed most of the data. L.M. performed most of the experiments with assistance from R.C. M.Y.T. isolated the spindle matrix (Fig. 2a and Supplementary Information, Table 1). S.W. analysed the effects of Nudel on lamin B loading onto microtubules (Supplementary Information, Fig. S7). B.L. and J.R.Y. conducted the mass-spectrometry analyses of the spindle proteome. Y.Z. wrote the manuscript with L.M. and X.Z.
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Ma, L., Tsai, MY., Wang, S. et al. Requirement for Nudel and dynein for assembly of the lamin B spindle matrix. Nat Cell Biol 11, 247–256 (2009). https://doi.org/10.1038/ncb1832
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DOI: https://doi.org/10.1038/ncb1832
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