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Fig.1. Xenopus NEDD1 (XNEDD1) localizes to centrosomes and spindle microtubules. (Aa) Western blot of mitotic (M) and interphase (I) Xenopus egg extracts probed with antibodies raised against the C-terminal half of NEDD1 (amino acids 301-655). The position of molecular mass markers is indicated on the left. (b) Western blots of mitotic or interphase extracts treated with (+) alkaline phosphatase (AP) or control buffer (–). (B) Immunofluorescence micrographs of `cycled' microtubule asters (top panels) or spindles (bottom panels) induced by addition of sperm chromatin to egg extracts. Overlays show XNEDD1 in green and α-tubulin in red. Scale bar, 10 μm. (C) Immunofluorescence micrographs of sperm tips (top panels) incubated in egg extract in the presence of nocodazole to prevent microtubule assembly, or sperm-induced asters (bottom panels) triple-labeled with antibodies against XNEDD1 or γ-tubulin, or stained for DNA, as indicated above the panels. Scale bars, 1 μm (top panels) and 10 μm (bottom panels). Overlays show XNEDD1 in green, γ-tubulin in red and DNA in blue. (D,E) Xenopus tissue culture cells at various cell cycle stages (indicated on the left) triple labeled for XNEDD1, α-tubulin or γ-tubulin and DNA, as indicated above the panels. Overlays in D show XNEDD1 in green, α-tubulin in red, DNA in blue. Overlays in E show γ-tubulin in green, XNEDD1 in red, DNA in blue. Scale bars, 10 μm.
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Fig.2. Xenopus NEDD1 (XNEDD1) depletion from Xenopus egg extract leads to disruption of microtubule structures. (A) Western blot analysis to determine the extent of XNEDD1 depletion. Relative amounts of mock-depleted and XNEDD1-depleted extract loaded are indicated above the lanes. (B) Schematic diagram of the constructs (XNEDD1-FL, XNEDD1-N and XNEDD1-C) used in this study. (C) Examples of sperm-induced asters assembled in mock-depleted (top panels) or XNEDD1-depleted extracts (all other panels). XNEDD1-depleted extracts were supplemented with buffer or full-length XNEDD1, XNEDD1-N or XNEDD1-C, as indicated. All extracts were supplemented with small amounts of rhodamine-tubulin to visualize microtubules (red in overlays). Signals in the rhodamine channel (left panels) or UV to visualize DNA (middle panels) were acquired separately and digitally overlaid. Scale bar, 10 μm. (D,E) Quantification of the (D) fluorescence intensity and (E) asters assembled around sperm centrioles for the experiments shown in C, expressed as percent of mock-depleted control. Error bars give the standard error (± s.e.). (F) Examples of spindles assembled in mock-depleted (top two rows of panels) or XNEDD1-depleted extracts (all other panels). Scale bar, 10 μm. (G) Quantification of the relative abundance of bipolar spindles, spindles with at least one disorganized pole and spindles with greatly reduced microtubule numbers in mock-depleted (light bars) and XNEDD1-depleted (dark bars) egg extracts. Error bars give the standard error (± s.e.).
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Fig.3. Addition of recombinant Xenopus NEDD1 (XNEDD1) to egg extracts disrupts microtubule structures. (A) Micrographs of microtubule asters formed in egg extracts in the presence of buffer, 300 nM full-length XNEDD1, XNEDD1-N or XNEDD1-C (all expressed and purified as GST fusion proteins; the GST portion was proteolytically removed prior to the experiment), as indicated. Microtubules (red in the overlays) and DNA (blue) are visualized as described for Fig. 2. Scale bar, 10 μm. (B) Micrographs of spindles formed in the egg extracts in the presence of buffer, 300 nM full-length XNEDD1 or XNEDD1-C, as indicated on the left. Microtubules (red in the overlays) and DNA (blue) are visualized as described for Fig. 2. Scale bar, 10 μm. (C) Quantification of aster intensity for the experiment shown in A, expressed as percent of mock-depleted control. Error bars give the standard error (± s.e.). (D) Micrographs of sperm heads incubated in egg extracts with buffer (top panels) or 300 nM XNEDD1-C (bottom panels) and labeled with antibodies against γ-tubulin (green in the overlays) or acetylated tubulin (to mark the position of the sperm centrioles; red in the overlays), and stained to visualize the DNA (blue in the overlays). Scale bar, 1 μm. (E) Quantification of the amount of γ-tubulin immunofluorescence associated with sperm heads for the experiment shown in D, expressed as percent of mock-depleted control. Error bars give the standard error (± s.e.).
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Fig.4. Xenopus NEDD1 (XNEDD1) depletion reduces the amount of γ-tubulin that associates with the centrosome but does not abolish γ-tubulin recruitment. (A,B) Micrographs of γ-tubulin staining at sperm heads incubated in buffer (top panels), mock-depleted extract (middle panels), or XNEDD1-depleted extract (bottom panels). Signals from the individual fluorescein (γ-tubulin, left column; green in overlay), rhodamine (α-tubulin, center column; red in overlay), or UV (DNA stain; blue in overlay) are shown as overlays in the right column. Scale bar, 1 μm. (B) Quantification of the amount of γ-tubulin associated with sperm centrioles for the experiment shown in A, normalized against the γ-tubulin immunofluorescence for sperm tips incubated in mock-depleted extract. Error bars, SE. (C) Drosophila centrosomes were rendered inactive by treatment with KI, and were then incubated with mock-depleted (top row) or XNEDD1-depleted (bottom row) extract (see text for detail). The extract was then washed off and reconstituted centrosomes were incubated with a solution of pure tubulin (containing a small amount of rhodamine-tubulin) to assay their ability to nucleate microtubules. Microtubules were fixed, and visualized under the microscope. Centrosomes reconstituted with mitotic (left panels) or interphase (right panels) extract are shown. Scale bar, 5 μm. (D) Quantification of γ-tubulin recruited to reconstituted centrosomes treated as described in C, except that they were fixed and stained for γ-tubulin immunofluorescence instead of being tested in the microtubule nucleation assay. γ-tubulin immunofluorescence intensity is reported as percent of mock-depleted control. Error bars give the standard error (± s.e.). (E) Western blot showing the extent of XNEDD1 depletion (top panel) and lack of co-depletion of γ-tubulin (bottom panel). (F) Microtubule asters assembled in mock-depleted (top panel) or XNEDD1-depleted (bottom panel) extracts are stained for γ-tubulin (left row; green in overlay), α-tubulin (second row; red in overlay) or DNA (third row; blue in overlay). (G) Quantification of γ-tubulin immunofluorescence relative to the amount of α-tubulin fluorescence for the experiment shown in F. Error bars give the standard error (± s.e.).
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Fig.5. Xenopus NEDD1 (XNEDD1) interacts with the γTuRC in egg extracts. (A) Beads coated with recombinant GST, full-length GST-XNEDD1, GST-XNEDD1-N, GST-XNEDD1-C, or γ-tubulin antibodies were incubated with pure tubulin to assay their ability to nucleate microtubules. Beads were either used directly (left panels; not exposed to extract) or were first incubated in extract (middle panels, exposed to extract) or in γ-tubulin-depleted extract (right panels, exposed to extract but γTuRC-δ). Representative pictures are shown for each condition. Beads were detected by autofluorescence; microtubules appear as spiky halos surrounding the beads. Scale bar, 10 μm. (B) Western blot of proteins that associate with the beads coated as in A and incubated in extract. The blot was probed with antibodies directed against the γTuRC subunits Xgrip210, Xgrip109 or γ-tubulin, as indicated on the right. Protein `baits' are indicated above the lanes. Extr, extract control; GST, GST-coated beads; FL, full-length GST-XNEDD1; N, GST-XNEDD1-N; C, GST-XEDD1-C; IP, beads coated with antibodies against γ-tubulin. (C) Western blot of imunoprecipitations from egg extracts using antibodies directed against γ-tubulin or XNEDD1, as indicated above the blot. Input represents 1.5 μl of extract; immunoprecipitations were from 100 μl of extract.
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Fig.6. Xenopus NEDD1 (XNEDD1) and γ-tubulin exist in distinct complexes. (A) Coomassie-Blue-stained gel of proteins that co-purify with control (NR, lane 1), XNEDD1 (lane 2) or γ-tubulin (lane 3) immunoprecipitations. Positions of γTuRC subunits (Xgrips) are indicated on the right, positions of molecular mass markers are indicated on the left. Asterisks denote proteins unique to the XNEDD1 immunoprecipitation. (B) Western blots of 5-40% sucrose-gradient fractions probed for XNEDD1 (upper panel) or γ-tubulin (lower panel). Positions of molecular mass markers (ovalbumin (3.5 S), rabbit muscle aldolase (7.35 S), bovine liver catalase (11.3 S) and ferritin (17.6 S); run on a parallel gradient) are indicated below the blot. (C) Graphical representation of the western blots shown in (B). The intensity of western blot bands were measured and are shown as fraction of maximum for XNEDD1 and γ-tubulin, as indicated. The main peaks for XNEDD1 and γ-tubulin do not overlap. (D) Western blots of sucrose-gradient fractions of XNEDD1-depleted extracts probed for XNEDD1 (upper panel) or γ-tubulin (lower panel).
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Fig.7. Xenopus NEDD1 (XNEDD1)-disruption inhibits microtubule organization in centrosome-independent aster assembly. (A) XNEDD1 and α-tubulin immunofluorescence of centrosome-independent asters assembled in Xenopus egg extracts after addition of RanGTP. XNEDD1 localizes to the center of centrosome-independent asters and along their microtubules. Scale bar, 5 μm. (B) Microtubule structures assembled in XNEDD1-depleted extracts in response to RanGTP. Scale bar, 10 μm. (C) Microtubule structures assembled in response to RanGTP in extracts to which recombinant XNEDD1 (expressed and purified as a GST fusion protein; the GST portion was proteolytically removed prior to the experiment) had been added, as indicated. Scale bar, 10 μm.
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Fig.8. Xenopus NEDD1 (XNEDD1) bundles microtubules in vitro and localizes to kinetochore fibers in Xenopus tissue culture cells. (A) Recombinant XNEDD1-C (without the GST used to purify the protein) added to in vitro microtubule polymerization reactions causes microtubule bundling. Recombinant proteins (as indicated) were added to the reaction at the indicated concentrations. Scale bar, 10 μm. (B) Recombinant XNEDD1-C binds to microtubules along their length. (a) GST-XNEDD1-C was allowed to bind to microtubules, and the samples were processed for immunofluorescence with antibodies against GST (XNEDD1-C; green in overlay) or α-tubulin (red in overlay). Scale bar, 5 μm. (b) Microtubule-pelleting assay using a constant amount of recombinant XNEDD-C and increasing amounts of taxol-stabilized microtubules. Supernatant (S) and pellet (P) of each sample were loaded on a gel and stained with Coomassie Blue. Only the part of the gel corresponding to XNEDD1 (upper panels) or tubulin (lower panels) are shown. (C) Recombinant XNEDD1-C bundles preformed microtubules. Recombinant XNEDD1-C (without the GST used to purify the protein) was incubated with taxol-stabilized microtubules. An aliquot of the reaction mixture was spotted onto a microscope slide and viewed under the microscope. Microtubules appear highly bundled in the presence of 300 nM XNEDD1-C. Scale bar, 10 μm. (D) XNEDD1 binds to kinetochore fibers. (Upper panels) Immunofluorescence shows that XNEDD1-positive fibers end at kinetochores stained by Bub1. XNEDD1, green; Bub1, red; DNA, blue. Scale bar, 5 μm. (Lower panels) Immuno-localization of XNEDD1 at kinetochore fibers. XNEDD1, green; kinetochore fibers, red; DNA, blue. Scale bar, 5 μm. (E) Higher magnification image of the boxed area shown in D.
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