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First published online 29 March 2005
doi: 10.1242/jcs.02304

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Assembly pathway of the anastral Drosophila oocyte meiosis I spindle

Helén Nilsson Sköld^*, Donald J. Komma and Sharyn A. Endow

Department of Cell Biology, Duke University Medical Center, Durham, NC 27710, USA

View larger version (127K): [in a new window]   Fig. 1. Meiosis I spindle assembly in a wild-type ncdgfp* oocyte. Images from a time-lapse sequence (Movie 1 in supplementary material) show the steps of spindle assembly in a live oocyte. (A,B) Germinal vesicle breakdown. The margin of the nuclear envelope shows regions of unevenness (arrowhead). The dark spot in the germinal vesicle (arrow) is the karyosome of condensed chromosomes, or endobody. (C,D) Dispersion of the germinal vesicle contents into the ooplasm. The arrows indicate the microtubule foci or asters, one of which is associated with the endobody. (E-G) Nucleation of randomly oriented microtubules at the endobody. (H) Differentiation of the endobody into microtubule-associated bivalent chromosomes. (I) Formation of lateral interactions between microtubule-associated chromosomes. (J-L) Formation and elongation of a bipolar spindle. Time, minutes:seconds. Bar, 5 µm.

View larger version (109K): [in a new window]   Fig. 2. Chromosomes in the meiosis I spindle. The chromosomes in live oocytes were confirmed by comparing fixed and stained oocytes. (A) Mature spindle in a live ncdgfp* oocyte. The dark region in the center of the spindle excludes NcdGFP* and was inferred to correspond to the condensed meiotic chromosomes. (B) Wild-type oocyte (Oregon R) fixed and stained with rhodamine-labeled anti--tubulin antibody (red) and DAPI (green). The small dots at the distal ends of the chromosome mass are the small 4th chromosomes. Bar, 5 µm.

View larger version (36K): [in a new window]   Fig. 3. Movement of chromosomes following meiosis I spindle assembly. Time-lapse images of the same oocyte as in Fig. 1, shown at 5 minute intervals for 45 minutes following the appearance of a polar spindle (see Movie 2 in supplementary material). The dark regions, interpreted to be the meiotic chromosomes, continue moving in the spindle. Time, minutes:seconds. Bar, 5 µm.

View larger version (101K): [in a new window]   Fig. 4. Meiosis I spindles in ncdNKgfp* mutant oocytes. (A) Split spindle with a separated microtubule-associated chromosome. (B) Split, multipolar spindle. (C-E) Partially fused or side-by-side spindles. (F) Apparently normal spindle. Bar, 5 µm.

View larger version (113K): [in a new window]   Fig. 5. Meiosis I spindles in ncd2gfp* mutant oocytes. (A) Immature spindle. (B-F) Immature, multipolar, or multiple spindles. Bar, 5 µm.

View larger version (57K): [in a new window]   Fig. 6. Ncd motor-associated asters at the germinal vesicle. (A) Two large fluorescent asters just outside the germinal vesicle of an ncdNKgfp* oocyte. The karyosome or endobody (arrow), around which microtubule nucleation occurred, is the dark body to the lower right, closest to the fluorescence foci. The other two dark regions are due to uneven dispersion of germinal vesicle contents into the ooplasm. (B) A cluster of large asters near the germinal vesicle of an ncdNKgfp* oocyte. (C) Two large asters in the germinal vesicle of an APL10/+ oocyte; two more large asters are in the ooplasm. The fluorescent asters migrate towards the karyosome of condensed chromosomes and nucleate microtubules at germinal vesicle breakdown. The asters are larger in ncdNKgfp* and APL10/+ or APL10 oocytes, than wild-type ncdgfp* oocytes (see Fig. 1C,D for comparison). Bar, 5 µm.

View larger version (17K): [in a new window]   Fig. 7. Binding by the NcdNKGFP* motor to meiosis I spindle microtubules. The dissociation rate (kd) of the motor from microtubules was estimated for NcdNKGFP* and wild-type NcdGFP* using fluorescence loss in photobleaching (FLIP) experiments. Plots of fluorescence (grey value) as a function of time (s, seconds) were fitted to a single exponential curve to estimate the kd. Representative plots are shown for NcdNKGFP* (crosses and red line, kd=0.035 second–1) and wild-type NcdGFP* (filled circles and black line, kd=0.069 second–1). The mean kd for NcdNKGFP* was 0.042±0.005 second–1 (n=16) and that for wild-type NcdGFP* was 0.065±0.005 second–1 (n=16). Curves are normalized to a starting grey level of 1.000 for comparison.

View larger version (21K): [in a new window]   Fig. 8. Assembly of anastral Drosophila oocyte meiosis I spindles. The model shows seven steps that lead to anastral spindle assembly, based on observation of spindle assembly in live wild-type ncdgfp* oocytes. The blue body represents the karyosome of condensed meiotic chromosomes, or endobody, at which microtubule nucleation is initiated.

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