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Direct binding of NuMA to tubulin is mediated by a novel sequence motif in the tail domain that bundles and stabilizes microtubules

Wellcome Trust Centre for Cell Biology, Institute of Cell and Molecular Biology, University of Edinburgh, King's Buildings, Edinburgh, EH9 3JR, UK

View larger version (62K): [in a new window]   Fig. 3. NuMA tail IIA co-localizes with microtubules in interphase cells and induces the formation of straight and stable microtubule bundles. (A) Cells expressing GFP-NuMA tail fragments (tail I, tail II or tail IIA), fixed and processed for immunofluorescence of tubulin (left column), corresponding fluorescence of the GFP-tag (middle column), and merged fluorescence (right column). Red, tubulin; green, GFP; blue, chromosomes stained with DAPI. Bar, 20 µm. (B) Cell expressing GFP-NuMA tail IIA, fixed and processed for fluorescence microscopy of GFP (upper left panel), followed by electron microscopy. Lower left panel: low magnification electron micrograph of the same cell, upper and lower right panels: high magnification views showing microtubule bundles in areas 1 and 2, as indicated by arrows in the GFP fluorescence micrograph. Bar, 1 µm. (C,D) Cells expressing GFP-NuMA tail IIA (green), fixed and processed for immunofluorescence of endogenous NuMA (C, red), or fluorescence of actin using rhodamine-phalloidin (D, red). Bars, 20 µm (C,D). (E) Diagram of the various human NuMA constructs used for transfection experiments. Amino acid positions are indicated.

View larger version (28K): [in a new window]   Fig. 1. Taxol-stabilized microtubules bind to NuMA from Xenopus egg extracts, irrespective of dynactin or dynein inhibition. (A) Anti-NuMA immunoblot of egg extract, and of supernatants (sup.) and pellets from extracts containing taxol-stabilized microtubules. Extracts were either untreated (left), or treated with recombinant dynamitin (middle), or monoclonal antibody against dynein intermediate chain (right). The cell cycle does not affect binding of NuMA to microtubules. (B) Anti-NuMA immunoblot; comparison of taxol microtubule pellets and supernatants from metaphase-arrested egg extract with equivalent material from extract released into interphase by calcium chloride. The taxol-treated extracts were supplemented with additional microtubules to increase the pelleting efficiency. Identical percentages of supernatants and pellets were loaded. NuMA in phosphatase-treated extract still binds to microtubules. (C) Anti-NuMA immunoblot of egg extract (extract), supernatant (taxol S.) and pellet (taxol P.) after centrifugation of taxol-treated extract, and the same pellet treated with either control buffer (b.contr.) or lambda phosphatase (-pptase). The phosphatase treatment consistently led to an increased immunoreactivity of NuMA on western blots (identical amounts were loaded for phosphatase treatment and controls, as verified by Ponceau staining in multiple experiments). The phosphatase-treated pellet was subsequently resuspended and re-centrifuged. The resulting supernatant (S.2) and pellet (P.2) are shown.

View larger version (49K): [in a new window]   Fig. 2. A region within the distal half of the NuMA tail domain binds directly to tubulin. (A) Immunoblot for tubulin, showing Xenopus egg extract, and eluates of Niagarose beads after incubation in untreated egg extract (beads only), or egg extract supplemented with hexahistidine-tagged Xenopus NuMA tail I (beads+tail1) or NuMA tail II (beads+tail2). For comparison, phosphocellulose-purified tubulin (tubulin) is shown. (B) Top: Coomassie-stained gel showing purified tubulin (tub.), hexa-histidine tagged SNAP (SNAP), and hexahistidine-tagged fusion proteins of Xenopus NuMA head domain (N. head), a 425-residue fragment of the Xenopus NuMA rod domain (N. rod), the proximal half of the Xenopus NuMA tail (N. tail1), and the distal half of the Xenopus NuMA tail (N. tail2). Middle: immunoblot for tubulin, showing a binding assay of soluble tubulin mixed with hexa-histidine-tagged fusion proteins as shown on the Coomassie-stained gel, and adsorbed to magnetic Niagarose beads. Supernatants (left) and bead eluates (Ni++ beads eluate, right) are shown. `no prot.' indicates a control of soluble tubulin, binding to Ni-beads only; `tub.' indicates purified tubulin only. The relative amounts of tubulin bound in each reaction were measured with a phosphoimager and noted underneath; the NuMA tail2 sample, showing the strongest binding, was set to 100%. Bottom: an identical immunoblot, probed with antibody against the peptide sequence of hexa-His-Gly (anti 6xHis-G). The lack of reactivity against the SNAP fusion protein is due to cloning in a pQE-9 vector, encoding hexa-histidine without glycine. All other fusion proteins were cloned in pRSET, leading to immunoreactive fusion proteins containing hexa-His-Gly. (C) Coomassie stained gel, showing supernatants and pellets of bovine serum albumin (BSA), Xenopus NuMA tail II (NuMA t.2), taxol-stabilized microtubules (tub.), and taxol-stabilized microtubules incubated with bovine serum albumin (BSA+tub.) or Xenopus NuMA tail II (Nu.t2+tub.). (D) Microtubule assembly from phosphocellulose-purified tubulin mixed with rhodamine-labelled tubulin, without additions (left), or with added Xenopus NuMA tail I (middle), or tail II (right). Bar, 20 µm. (E) Scatchard plot, showing Xenopus NuMA tail II binding at increasing concentrations (in mol/l) to taxol-stabilized microtubules. (F) The percentage of bound Xenopus NuMA tail II to taxol-stabilized microtubules, quantifying unphosphorylated protein (open squares), or counts of NuMA tail II phosphorylated with recombinant cdc2 kinase/cyclinB and radioactive ATP (open diamonds).

View larger version (67K): [in a new window]   Fig. 4. NuMA tail IIA stabilises microtubules. Cells expressing GFP-NuMA tail IIA (green) were incubated on ice for 1 hour, fixed and processed for immunofluorescence of tubulin (A, red) or incubated at room temperature and stained for acetylated tubulin (B, red). Chromosomes are stained with DAPI (blue). Note the absence of a microtubule network in the untransfected cell in (A) Bar, 20 µm.

View larger version (54K): [in a new window]   Fig. 5. NuMA tail IIA localises to spindle poles in mitotic cells and induces microtubule asters, as well as multipolar and asymmetric spindles. (A) Cells expressing GFP-NuMA tail fragments (tail I, tail II, tail IIA or tail IIB, green) fixed in mitosis and processed for immunofluorescence of tubulin (red). Chromosomes are stained with DAPI (blue). The frequencies of individual phenotypes are indicated on the right (also see text). (B) Mitotic cell expressing GFP-NuMA tail IIA (green), immunofluorescence staining of endogenous NuMA (red). (C) Bacterially expressed hexa-histidine-tagged fusion proteins of NuMA tail II (left) or tail IIA (right) induce microtubule asters when incubated in Xenopus egg extracts. Bars, 10 µm (A,B); 20 µm (C).

View larger version (55K): [in a new window]   Fig. 6. Full-length NuMA requires tail IIA to associate with tubulin when expressed in the cytoplasm of interphase cells. Cells expressing GFP-NuMA NLS, tail II or tail IIA+NLS (green), fixed and processed for immunofluorescence of tubulin (red). Chromosomes are stained with DAPI (blue). (A) Interphase cells. Note the presence of tubulin aggregates co-localizing with GFP-NuMA NLS (top row), and the absence of tubulin in protein aggregates of the mutants lacking tail II or tail IIA. Bar, 20 µm. (B) Mitotic cells. Bar, 10 µm.

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