Becker BE et al. (2003), XMAP215, XKCM1, NuMA, and cytoplasmic dynein ar...

XB-ART-4660

Dev Biol 2003 Sep 15;2612:488-505. doi: 10.1016/s0012-1606(03)00330-0.

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XMAP215, XKCM1, NuMA, and cytoplasmic dynein are required for the assembly and organization of the transient microtubule array during the maturation of Xenopus oocytes.

Becker BE , Romney SJ , Gard DL .

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During the maturation of Xenopus oocytes, a transient microtubule array (TMA) is nucleated from a novel MTOC near the base of the germinal vesicle. The MTOC-TMA transports the meiotic chromosomes to the animal cortex, where it serves as the precursor to the first meiotic spindle. To understand more fully the assembly of the MTOC-TMA, we used confocal immunofluorescence microscopy to examine the localization and function of XMAP215, XKCM1, NuMA, and cytoplasmic dynein during oocyte maturation. XMAP215, XKCM1, and NuMA were all localized to the base of the MTOC-TMA and the meiotic spindle. Microinjection of anti-XMAP215 inhibited microtubule (MT) assembly during oocyte maturation, disrupting assembly of the MTOC-TMA and subsequent assembly of the first meiotic spindle. In contrast, microinjection of anti-XKCM1 promoted MT assembly throughout the cytoplasm, disrupting organization of the MTOC-TMA and meiotic spindle. Finally, microinjection of anti-dynein or anti-NuMA disrupted the organization of the MTOC-TMA and subsequent assembly of the meiotic spindles. These results suggest that XMAP215 and XKCM1 act antagonistically to regulate MT assembly and organization during maturation of Xenopus oocytes, and that dynein and NuMA are required for organization of the MTOC-TMA.

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Species referenced: Xenopus
Genes referenced: ckap5 dnai1 dync1li1 kif2c numa1 tuba4b
???displayArticle.antibodies??? Ckap5 Ab3 Ckap5 Ab4 Dnai1 Ab1 Kif2c Ab3 Numa1 Ab1 Tuba4b Ab14 Tuba4b Ab2 ckap5 Ab1

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	Fig. 1. Three XMAP215 peptide antisera recognize a common 250-kDa protein in Xenopus egg extracts. Total soluble protein (30 g) from a Xenopus egg extract was separated by SDS-PAGE and blotted with TSDY, SNTS, or DDLK anti-XMAP215 antisera. All three peptide antibodies recognized a common 250-kDa protein that comigrated with purified XMAP215 which was not detected by nonimmune rabbit IgG (RIgG). The antibodies also detected several smaller proteins (asterisks), which are likely proteolytic degradation fragments of XMAP215. Sizes of molecular weight markers are shown to the left of the blot.
	Fig. 2. XMAP215 antibodies stained the MTOC-TMA and meiotic spindles during oocyte maturation. Maturing oocytes were fixed and stained with anti- -tubulin (A, D, and G), a cocktail of three XMAP215 peptide antibodies (B, C, E, and H), and TO-PRO-3 to visualize chromosomes (F, I). (A) At WSF, MTs were observed extending from an MTOC near the vegetal surface of the GV into the nucleoplasm. (B) XMAP215 antibodies stained the base of the MTOC-TMA, and faintly stained MTs extending into the nucleoplasm (a projection of 15 optical sections). (C) XMAP215 antibodies stained the base of a late MTOC-TMA as it approached the animal cortex (0–15 min after WSF, a single optical section). (D–F) XMAP215 co-localized with MTs of the compacted meiotic spindle 15–30 min after WSF (a projection of 16 optical sections). (G–I) XMAP215 colocalized with MTs throughout the first meiotic spindle (30–45 min after WSF, a projection of 13 optical sections). Scale bars are 50 m (A and B) and 10 m (C–I).
	Fig. 3. XKCM1 and NuMA are localized to the MTOC-TMA and meiotic spindles during maturation. Maturing oocytes were fixed and stained with anti- -tubulin (A, D), anti-XKCM1 (B, C, E, G, I), anti-NuMA (J–L), and TO-PRO-3 to visualize chromosomes (F, H). (A) At WSF, MTs of the MTOC-TMA were observed extending from the transient MTOC near the vegetal surface of the GV into the nucleoplasm. (B) Anti-XKCM1 stained the base of the MTOC-TMA and faintly stained MTs of the TMA extending into the nucleoplasm (a projection of 11 optical sections). (C) XKCM1 antibodies stained the base of a late MTOC-TMA as it approached the animal cortex (0–15 min after WSF, a single optical section). (D–F) XKCM1 colocalized with MTs of the compacted meiotic spindle (15–30 min after WSF, a projection of 14 optical sections). XKCM1 antibodies also stained foci associated with condensed meiotic chromosomes that may correspond to the centromeres (arrows). (G–I) XKCM1 is localized to centromeres and MTs of the first meiotic spindle at 30–45 min after WSF (arrows, projection of 17 optical sections). (J) Anti-NuMA stains the base of the MTOC-TMA at 0–20 min after WSF in this single optical section. (K) NuMA is localized to the compacting meiotic spindle 15–30 min after WSF (a projection of 10 optical sections). (L) Antibodies against NuMA stain the poles of the first meiotic spindle 30–45 min after WSF (a projection of 7 optical sections). Scale bars are 50 m (A, B, and J), 10 m (C–H, K, and L), and 2 m (I).
	Fig. 4. Microinjection of XMAP215 antibodies disrupted the assembly of the MTOC-TMA and meiotic spindles during maturation. Stage VI oocytes were injected with a cocktail of three XMAP215 peptide antisera, matured with progesterone, fixed 0–15 min after WSF (A–E) or 45–60 min after WSF (F–I), and stained with anti- -tubulin to visualize MTs (A, B, D, F, G), anti-rabbit IgG to visualize the injected antibodies (C, E, H), and TO-PRO-3 to visualize the condensed meiotic chromosomes (I). (A) An oocyte exhibiting numerous MTs and asters surrounding the region of the GV (a projection of 15 optical sections). Closer examination (B and C) revealed that many of the asters were associated with or nucleated by aggregates of XMAP215 and the injected antibody (arrowheads, a projection of 27 optical sections). (D) This oocyte exhibited an MTOC with reduced number of shorter MTs extending into the nucleoplasm. (E) Anti-rabbit IgG revealed aggregates of injected antibody within the MTOC (arrows, a projection of 11 optical sections). (F) Oocytes injected with nonimmune rabbit IgG antibodies exhibited normal meiotic spindles (a projection of 14 optical sections). (G) Numerous small asters were apparent in the animal cytoplasm of this anti-XMAP215-injected oocyte. (H) Anti-rabbit IgG revealed that each aster was associated with or nucleated by an aggregate of injected antibody (arrowheads). Smaller antibody aggregates did not organize MT asters (arrows). (I) TO-PRO-3 revealed condensed meiotic chromosomes associated with each of the large MT asters (arrowheads, a projection of 15 optical sections). Scale bars are 100 m (A), 50 m (B–E), 10 m (F), and 20 m (G–I).
	Fig. 5. Microinjection of XKCM1 antibodies disrupted the assembly of the MTOC-TMA and meiotic spindles during maturation. Oocytes were microinjected with anti-XKCM1 antisera, matured with progesterone, fixed 0–15 min (A–C) or 45–60 min (D–G) after WSF, and stained with anti- -tubulin to visualize MTs (A–E and G) or TO-PRO-3 to visualize chromosomes (F). (A) An oocyte injected with nonimmune rabbit IgG exhibited a normal MTOC-TMA (a projection of 10 optical sections). (B) This anti-XKCM1-injected oocyte exhibited an atypical MTOC-TMA, in which MTs appeared to nucleate from an extensive region of the GV surface and radiate into the underlying cytoplasm (a projection of 7 optical sections). (C) In this anti-XKCM1-injected oocyte, MTs appear to be nucleated from lateral regions of the GV surface. Note the increased assembly of MTs in the cytoplasm surrounding the atypical MTOC-TMA (arrow, a projection of 7 optical sections). (D) Low magnification view of the animal hemisphere of this oocyte revealed two large MT “spheres” (white arrows), two large asters (white arrowheads), and numerous small asters (black arrow). This image is a projection of 10 optical sections. (E and F) Optical cross sections of a MT sphere reveal its “hollow” appearance and a cluster of condensed chromosomes. The appearance of chromosomes in (F) results from bleed through of the TO-PRO-3 into the MT channel. These images are a projection of 21 optical sections. (G) A higher magnification view of a large aster (a projection of 28 optical sections). Scale bars are 100 m (A and C), 50 m (B), 150 m (D), and 20 m (E–G).
	Fig. 6. Microinjection of anti-XMAP215 and anti-XKCM1 has opposite effects on the assembly of cytoplasmic MTs during oocyte maturation. Oocytes were microinjected with either nonimmune rabbit IgG, a cocktail of three XMAP215 peptide antisera, or anti-XKCM1, mature with progesterone, fixed 0–15 min after WSF, and stained with anti- -tubulin to visualize MTs. (A) Rabbit IgG-injected oocytes exhibited a dense network of cytoplasmic MTs surrounding the MTOC-TMA (a projection of 41 optical sections). (B) Cytoplasmic MTs were absent in this oocyte injected with XMAP215 antibodies (a projection of 35 optical sections). (C) The number of cytoplasmic MTs was increased in this anti-XKCM1-injected oocyte (a projection of 23 optical sections). Scale bar is 20 m.
	Fig. 7. Microinjection of antibodies against cytoplasmic dynein and NuMA disrupted the organization of the MTOC-TMA and first meiotic spindle during oocyte maturation. Oocytes were microinjected with either anti-DIC (A–C and F–H) or anti-NuMA (D–E and I–K), matured with progesterone, fixed, and stained with anti- -tubulin to visualize MTs. (A) Lateral view of an oocyte injected with anti-DIC and fixed 0–10 min after WSF revealed expansion of the MTOC-TMA to the lateral surface of the GV (a single optical section). (B) In another oocyte fixed 0–10 min after WSF, the MTOC surrounds the entire GV with MTs filling the nucleoplasm (a single optical section). (C) A split MTOC-TMA near the animal cortex of an anti-DIC-injected oocyte fixed 0–10 min after WSF (a projection of 10 optical sections). (D) This lateral view of an oocyte injected with anti-NuMA and fixed 0–15 min after WSF revealed an expansion of the MTOC-TMA laterally around the GV (a projection of 31 sections). (E) A split and expanded MTOC is observed in this anti-NuMAinjected oocyte fixed 0–15 min after WSF (a projection of 44 optical sections). (F–H) Anti-DIC-injected oocytes fixed 30–45 min after WSF exhibited examples of abnormal spindles including “starburst” spindles (F, a projection of 25 optical sections), bipolar spindles with protruding MT bundles (G, a projection of 30 optical sections), and multiple monopolar spindles (arrows) and loose bundles of MTs (H, a projection of 18 optical sections). (I, J) A variety of abnormal spindles including multipolar spindles (I, a projection of 16 optical sections), multiple monopolar and bipolar spindles (J, a projection of 20 optical sections, arrows show bipolar spindles), and multiple asters and/or monopolar spindles (K, a projection of 26 optical sections) were observed in oocytes injected with anti-NuMA and fixed 45–60 min after WSF. Scale bars are 50 m (A–F and J–K) and 20 m (G–I).
	Fig. 8. Chromosomes were associated with the aberrant MT structures in anti-DIC-injected oocytes during oocyte maturation. Oocytes were microinjected with anti-DIC, matured with progesterone, fixed 30–45 min after WSF, and stained with anti- -tubulin to visualize MTs (red) and YO-PRO-1 to visualize chromosomes (green). (A) This oocyte injected with anti-DIC antibodies exhibited abnormal “spindles,” including “starbursts” (white arrows), monopolar spindles (white arrowhead), and loose bundles of MTs (black arrow). Chromosomes were associated with distal ends of MT bundles and monopolar spindles. Extrachromosomal rDNAs (black arrowheads) were not associated with MT “spindles.” This image is a projection of 12 optical sections. (B) Higher magnification view of a “starburst” showing that chromosomes were associated with the distal ends of MT “arms” (a projection of 12 optical sections). Scale bars are 20 m.