Cha BJ et al. (1998), XMAP230 is required for the assembly and organi...

XB-ART-14532

J Cell Sci 1998 Aug 01;111 ( Pt 16):2315-27. doi: 10.1242/jcs.111.16.2315.

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XMAP230 is required for the assembly and organization of acetylated microtubules and spindles in Xenopus oocytes and eggs.

Cha BJ, Error B, Gard DL.

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Species referenced: Xenopus
Genes referenced: clstn2 map4 pax3

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	Fig. 1. XMAP230 is present in Xenopus oocytes, eggs, embryos, and some adult tissues. Coomassie Blue-stained (CBB) SDS-PAGE gels (A) and immunoblots with anti- XMAP230 (B) revealed that XMAP230 was enriched in the MT fractions (CS1 and CS2) and the heat-stable MAP fraction (HSS) from Xenopus eggs (arrowheads indicate XMAP230). Little or no XMAP230 was detectable in the warm supernatants (WS1 and WS2) by CBB or immunoblotting. XMAP230 was detected by specific antibody in the cytoplasmic extract (B; Extr). Non-immune rabbit serum did not recognize any proteins in the cytoplasmic extract (B; NRS). Asterisks in A and B denote a proteolytic fragment of XMAP230. (C) Comparable amounts of soluble protein from oocytes, eggs, and early embryos (stage 7, mid-blastula; stage 12, early neural plate; stage 24, early tailbud; stage 41, tadpole) were separated by SDS-PAGE and blotted with anti-XMAP230 (XMAP230; top panel) or anti-a-tubulin (bottom panel). (D) Comparable amounts of total soluble protein from adult tissues were separated by SDS-PAGE and blotted with anti-XMAP230 or monoclonal anti-a-tubulin. XMAP230 was readily detected in egg, brain, and testis. Longer exposures of the autoradiogram revealed lesser amounts of XMAP230 in intestine and lung (not shown; see text).
	Fig. 2. XMAP230 antibodies stain MTs during early oogenesis. (A) The MT network of interphase oogonia was brightly stained by anti- XMAP230 (arrow points to the centrosome). (B) XMAP230 staining was concentrated in the cytoplasm (asterisk) of post-mitotic (stage 0) oocytes, but the density of the MT array prevented resolution of individual MTs (see text). (C) However, staining of individual MTs with anti-XMAP230 was readily apparent in stage 0 oocytes subjected to MT disassembly and regrowth (see text; arrow denotes the centrosome; N, nucleus). (D) MTs in an early stage I oocyte (50-75 mm in diameter) were faintly stained with XMAP230 antibody (GV denotes the germinal vesicle, or oocyte nucleus). (E) MTs of late stage I oocytes (>100 mm in diameter) were not detectably stained by anti-XMAP230. (F) Anti-a-tubulin revealed extensive MT arrays in oocytes of this size. (G) Radiallyoriented MTs were apparent in stage III oocytes stained by anti-a-tubulin. (H) MTs in stage III oocytes were not detectably stained by anti-XMAP230. Note that follicle cells surrounding stage I-III oocytes in D, E, and H were intensely stained by anti-XMAP230 (arrows in E and H). Bars: 5 mm (AC), 10 mm (D), and 25 mm (E-H).
	Fig. 3. XMAP230 is associated with MTs in stage VI oocytes. (A-C) This stage VI oocyte was stained with both anti-XMAP230 (A; green channel in C) and monoclonal anti-a-tubulin (B; red channel in C). Many individual MTs were stained with both anti-XMAP230 and antitubulin, and appear yellow in the merged image (arrows in C). Close examination reveals the punctate distribution of anti-XMAP230 along some MTs, which exhibit patches of red in the merged image (arrows in insets; insets 1 and 2 are enlargements of the region within the dotted rectangle in C, and are projections of 2 and 4 optical sections, respectively). (D-F) Localization of XMAP230 along acetylated MTs was also apparent in oocytes stained with anti-XMAP230 (D and green channel in F) and antibodies specific for acetylated a-tubulin (E and red channel in F). XMAP230 was distributed in patches of variable length along some acetylated MTs. The inset in F shows the region boxed with a dotted line at higher magnification. Acetylated MTs with patches of XMAP230 are indicated with arrows. A small number of MTs stained by anti- XMAP230 were not detectably stained by anti-acetylated a-tubulin (large arrowheads in D-F). Figures are projections of 4-5 serial optical sections collected at 0.5 mm intervals. Bars, 25 mm.
	Fig. 4. XMAP230 is associated with the MTOC-transient MT array and meiotic spindles during oocyte maturation, but not with cytoplasmic MTs. The MTOC-TMA complex of this Xenopus oocyte was stained by antibodies to both anti- XMAP230 (A, arrows) and acetylated a-tubulin (B). The second meiotic spindle was brightly stained by anti- XMAP230 (C). The brightly stained bundles of MTs in the central spindle (arrows in C) probably correspond to kinetochore fibers. Note the lack of staining of cytoplasmic MTs. Anti-a-tubulin stained both the second meiotic spindle (D) and surrounding cytoplasmic MTs (arrowheads). (E-G) A substantial population of cytoplasmic MTs coexists with the MTOC-TMA and spindles during oocyte maturation (E-G are from early meiosis I). These cytoplasmic MTs were readily apparent in oocytes stained with anti-a-tubulin (E), but were not stained with either anti- XMAP230 (F) or anti-acetylated a-tubulin (G). Bars: 25 mm (A,B,E,F,G); 10 mm (C and D).
	Fig. 5. Microinjection of anti-XMAP230 antibodies locally depletes cytoplasmic XMAP230 in stage VI oocytes. (A-B) Immunofluorescence microscopy of XMAP230 antibody-injected oocytes stained with anti-XMAP230 antibodies and fluorescent secondary antibody revealed that XMAP230 antibodies locally depleted cytoplasmic XMAP230 near the injection site (A; asterisk). XMAP230 and the injected antibody form multiple insoluble aggregates (arrows in A), and no MT staining is observed. MT-bound XMAP230 is apparent outside of the region occupied by the injected antibody (arrowheads in A), and in the uninjected side (B) of the same oocyte. (C-D) MT-bound XMAP230 is apparent in both the injected (C) or uninjected (D) sides of another oocyte injected with non-immune rabbit IgG. Bars, 25 mm.
	Fig. 6. Microinjection of anti- XMAP230 disrupts MT organization and acetylation in stage VI oocytes. (A) A low magnification view of an oocyte fixed 12 to 18 hours after microinjection of affinitypurified anti-XMAP230, and stained with monoclonal anti-a- tubulin (red channel) to reveal MTs and fluoresceinconjugated anti-rabbit IgG (green channel) to detect the injected antibody. The injection site () was easily recognizable due to the presence of insoluble antibody-antigen complexes (seen better in B; arrows) and a ‘ring’ of injected antibody bound to MTs. (B) Numerous disordered MTs were apparent in the injected side of another oocyte stained for a-tubulin and injected antibody. Near the periphery of the injection site, individual MTs were decorated with the injected anti- XMAP230 (arrowheads), and thus appeared yellow. Arrows denote brightly-stained aggregates of injected antibodies. (C) The MT array of the uninjected side of the same oocyte appeared unaffected. (D) A low magnification view of an oocyte fixed 12 hours after injection of anti- XMAP230, and stained to reveal acetylated MTs (red channel) and injected XMAP230 antibody (green channel). Staining of acetylated MTs (red) on the injected side () was visibly reduced in intensity compared to the uninjected side. (E) The staining intensity and number of acetylated MTs was visibly reduced in the region of injected antibody (*). Near the margin of the injection site, individual MTs stained by anti-acetylated a-tubulin were also decorated with the injected XMAP230 antibodies (arrowheads), and thus appeared yellow. (F) The organization and number of acetylated MTs in the uninjected side of the same oocyte appeared unaffected. Bars, 25 mm.
	Fig. 7. Injection of anti-XMAP230 into stage VI oocytes inhibits MT acetylation during recovery from cold-induced MT depolymerization. Stage VI oocytes injected with anti-XMAP230, were incubated at 0-2°C for 2 hours to depolymerize MTs, and were allowed to recover for 18 hours at room temperature prior to fixation for immunofluorescence microscopy. (A and B) Substantial MT assembly was apparent in both the uninjected (A) and injected (B) sides of an oocyte stained with anti-a-tubulin. (C) Substantial numbers of acetylated MTs were apparent in the uninjected side of an oocyte stained with 6-11B-1. (D) The intensity of staining and number of acetylated MTs stained by 6-11B-1 was visibly reduced on the injected side of the same oocyte (* denotes the region of injected antibody; the approximate extent of antibody diffusion is denoted by the dotted line). Bars, 25 mm.
	Fig. 8. Injection of anti- XMAP230 disrupts the assembly of the transient MT array and meiotic spindles during oocyte maturation. (A and B) Injection of nonimmune rabbit IgG had little or no affect on the organization of the MTOCTMA (A) or first meiotic spindle (B; shown in transverse orientation, see Gard, 1992). The injected non-immune rabbit IgG did not decorate either the MTOCTMA or the meiotic spindle. (C) Injection of anti- XMAP230 disrupted the organization of the MTOCTMA, often resulting in the apparent splitting of the MTOC (arrowhead) or the formation of large MT aggregates (arrow). (D) Injected oocytes subsequently assembled poorly organized MT aggregates or severely distorted spindles that were heavily stained by the injected antibodies. Oocytes in C and D were stained with anti-acetylated a-tubulin (red channel) and fluorescein anti-rabbit (green channel) to show the injected antibody. Bars: 25 mm (A and C); 10 mm (B and D).