Zyłkiewicz E et al. (2011), The N-terminal coiled-coil of Ndel1 is a regula...

XB-ART-42766

J Cell Biol 2011 Feb 07;1923:433-45. doi: 10.1083/jcb.201011142.

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The N-terminal coiled-coil of Ndel1 is a regulated scaffold that recruits LIS1 to dynein.

Zyłkiewicz E , Kijańska M , Choi WC , Derewenda U , Derewenda ZS , Stukenberg PT .

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Ndel1 has been implicated in a variety of dynein-related processes, but its specific function is unclear. Here we describe an experimental approach to evaluate a role of Ndel1 in dynein-dependent microtubule self-organization using Ran-mediated asters in meiotic Xenopus egg extracts. We demonstrate that extracts depleted of Ndel1 are unable to form asters and that this defect can be rescued by the addition of recombinant N-terminal coiled-coil domain of Ndel1. Ndel1-dependent microtubule self-organization requires an interaction between Ndel1 and dynein, which is mediated by the dimerization fragment of the coiled-coil. Full rescue by the coiled-coil domain requires LIS1 binding, and increasing LIS1 concentration partly rescues aster formation, suggesting that Ndel1 is a recruitment factor for LIS1. The interactions between Ndel1 and its binding partners are positively regulated by phosphorylation of the unstructured C terminus. Together, our results provide important insights into how Ndel1 acts as a regulated scaffold to temporally and spatially regulate dynein.

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Species referenced: Xenopus
Genes referenced: arhgef7 aurka cdk1 cdk5 dctn2 dnai1 isyna1 nde1 ndel1 pafah1b1 ran

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	Figure 1. Ndel1 function is required for microtubule aster formation. (a) Affinity-purified α-Ndel1 antibodies recognize a 39-kD band in Xenopus egg extracts. (b) Schematic of putative role of dynein and Ndel1 in Ran aster formation in egg extracts. (c) Ndel1 colocalizes with dynein in the central focus of asters. (d) Addition of p50/dynamitin, α-dynein intermediate chain (DIC) or α-Ndel1 antibodies disrupts formation of microtubule asters. (e) Quantification of aster formation in the experiment described in panel d. Over 100 microtubule structures were quantified in three independent antibody addition experiments; asterisks indicate statistically significant differences (Student’s t test with P < 0.05; indicates P < 0.005; * indicates P < 0.0005). Bar, 20 µm.
	Figure 2. The entire N-terminal coiled-coil of Ndel1, but not the L IS1-binding domain alone, is sufficient to rescue microtubule aster formation in Xenopus egg extracts. (a) Schematic of truncated mouse Ndel1 (mNdel1) proteins used in rescue experiments. (b) Ndel1 proteins were immunodepleted greater than 90%. CSF-arrested extracts were diluted to the specified amount or control ΔIgG and ΔxNdel1 extracts were immunoblotted with affinity-purified polyclonal α-Ndel1 antibodies. (b′) Representative images of the predominant microtubule structures observed after rescuing xNdel1-depleted extracts (ΔxNdel1) with the indicated recombinant proteins. Structures such as those in ΔxNdel1+1–174 (mNdel11–174), ΔxNdel1+8–192 (mNdel18–192), and ΔNdel1+8–310 (mNdel18–310) would be classified as asters, whereas those seen in ΔxNdel1+BSA and ΔxNdel1+8–99 (mNdel18–99) are unfocused structures. (b′′) Quantification of three independent experiments after Ndel1 depletion and rescue with truncated mouse Ndel1 proteins; for each depletion and rescue condition over 100 microtubule structures were quantified; asterisks indicate statistically significant differences (Student’s t test with P < 0.05; * indicates P < 0.0005). (c) Ndel1 coimmunoprecipitates LIS1, but not dynein and dynactin in Xenopus egg extracts, and α-Ndel1 antibodies do not deplete LIS1. α-Ndel1 antibodies were used to immunoprecipitate proteins from Xenopus CSF-arrested extracts. The resulting precipitates (IPs) and extracts from which the IP was performed (SUPS) were immunoblotted for indicated proteins. (d) Level of Ndel1 depletion from CSF-arrested egg extract as measured by immunoblot. (d′) LIS1-binding domain mNdel188–192 (88–192) is not sufficient to rescue aster formation in Ndel1-depleted extracts. (d′′) Quantification of microtubule structures from three independent experiments where Ndel1-depleted extracts were rescued with the indicated mNdel1 variants. Over 100 microtubule structures were quantified, asterisks indicate statistically significant differences (Student’s t test with P < 0.05; * indicates P < 0.0005). Bar, 20 µm.
	Figure 3. Dynein interacts with polar residues within the coiled-coil domain of Ndel1. (a) Immunoblot analysis of dynein IC (DIC) and LIS1 pulled down from CSF-arrested Xenopus egg extract by the indicated recombinant mouse Ndel1 proteins. (b) Multiple conserved glutamic acids extend form the coiled-coil backbone of Ndel1; residues shown in color were mutated, residues shown in red are the most critical for dynein binding; image created in Pymol (http://www.pymol.org/) based on the structure of mNdel18–192 (Protein Data Bank accession no. 2v71). (c) Immunoblot analysis of dynein IC (DIC) and LIS1 pulled down from mitotic Xenopus egg extract by mutated mNdel18–192 and mNdel18–310 proteins. (d) Level of Ndel1 depletion from CSF-arrested egg extract with affinity-purified polyclonal α-Ndel1 antibodies analyzed by Western blot. (e) mNdel18–192 E48A/E52A (8–192 E48A/E52A) and mNdel18–310 E48A/E52A (8–310 E48A/E52A) proteins do not significantly rescue aster formation. Representative images of predominant phenotypes from each condition are shown. (f) Quantification of over 100 microtubule structures from three independent experiments after Ndel1 depletion and rescue with mouse Ndel1 variants; asterisks indicate statistically significant differences (Student’s t test with P < 0.05; *** indicates P < 0.0005). Bar, 20 µm.
	Figure 4. The N terminus of Nde1 can focus microtubules into asters. (a) Immunoblot analysis of dynein IC (DIC) and LIS1 pulled down from CSF-arrested Xenopus egg extract by the indicated recombinant mouse Nde1 proteins. (b) Level of Ndel1 depletion from CSF-arrested egg extracts with affinity-purified polyclonal α-Ndel1 antibody analyzed by Western blot. (c) Addition of mNde17–170 to xNdel1-depleted extracts rescues aster formation. (d) Quantification of microtubule structures from three independent experiments where Ndel1-depleted extracts were rescued with the indicated mouse Nde1 variants; over 100 microtubule structures were quantified from each experiment; asterisks indicate statistically significant differences (Student’s t test with P < 0.05; *** indicates P < 0.0005). Bar, 20 µm.
	Figure 5. Ndel1 recruits L IS1 to drive aster formation, and a mutant that cannot bind L IS1 generates microtubule bundles. (a) Depletion level quantification for experiment shown in panel d. (b) The role of Ndel1 is to recruit LIS1 to drive aster formation. The interaction between LIS1 and Ndel1 is required for aster formation, but Ndel1 also has a LIS1-independent function. Representative images of the predominant microtubule structures assembled in Ndel1-depleted extracts rescued with the indicated recombinant protein(s). Note that the Ndel1 proteins with mutated LIS1-binding residues are unable to form asters, but the resulting structures are more organized than in Ndel1-depleted extracts (bundles). Addition of 350 nM exogenous LIS1 to Ndel1-depleted extracts partly rescues microtubule focusing, whereas addition of 350 nM LIS1 and 100 nM Ndel18–192 E119A/R130A (8–192 E119A/R130A) rescues significantly better. (c) Quantification of microtubule structures from three independent experiments where Ndel1-depleted extracts were rescued with the indicated mouse Ndel1 proteins and/or LIS1; over 100 microtubule structures were quantified; asterisks indicate statistically significant differences in percentage of focused asters (Student’s t test with P < 0.05; indicates P < 0.005; * indicates P < 0.0005). (d) Representative image of a hollow aster. Image is shown at different contrast than in panel b to emphasize presence of a hollow center of the aster. Bar, 20 µm.
	Figure 6. Proposed role for the regulation of Ndel1 ability to act as a scaffold to promote the interaction between dynein and L IS1. (a) Ndel1 mutants in Aurora A consensus site (S251A) and Cdk1/Cdk5 sites (T219 and T245) abolish the interaction with dynein in a pull-down assay. (b, top) A ribbon model of a nascent Ndel1 tetramer where one dimeric coiled-coil is green and the second is yellow. Note that the structure of the C terminus has not been solved and is presented in cartoon format. The asterisks indicate potential phosphorylation sites. (b, bottom) Space-filling model colored to show charge distribution of Ndel1 in the presumptive autoinhibited form. The C terminus has a pI of 9.8 and is therefore depicted schematically as blue. We propose that in the unphosphorylated state the two Ndel1 dimers interact through the LIS1-binding region, as seen in the crystal structure. In this conformation the highly basic C-terminal tail inhibits the interaction between Ndel1 and dynein by binding the highly acidic (pI 4.5) dynein interaction region on the coiled-coil as shown. (c) Proposed model of the tripartite interaction between dynein, LIS1, and the N terminus of Ndel1. The proteins are all drawn approximately to scale, although their precise locations are not known.

References [+] :

Bradshaw, NDE1 and NDEL1: multimerisation, alternate splicing and DISC1 interaction. 2009, Pubmed