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Biol Open
2012 May 15;15:422-9. doi: 10.1242/bio.2012596.
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Dgp71WD is required for the assembly of the acentrosomal Meiosis I spindle, and is not a general targeting factor for the γ-TuRC.
Reschen RF
,
Colombie N
,
Wheatley L
,
Dobbelaere J
,
St Johnston D
,
Ohkura H
,
Raff JW
.
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Dgp71WD/Nedd1 proteins are essential for mitotic spindle formation. In human cells, Nedd1 targets γ-tubulin to both centrosomes and spindles, but in other organisms the function of Dgp71WD/Nedd1 is less clear. In Drosophila cells, Dgp71WD plays a major part in targeting γ-tubulin to spindles, but not centrosomes, while in Xenopus egg extracts, Nedd1 acts as a more general microtubule (MT) organiser that can function independently of γ-tubulin. The interpretation of these studies, however, is complicated by the fact that some residual Dgp71WD/Nedd1 is likely present in the cells/extracts analysed. Here we generate a Dgp71WD null mutant lacking all but the last 12 nucleotides of coding sequence. The complete loss of Dgp71WD has no quantifiable effect on γ-tubulin or Centrosomin recruitment to the centrosome in larval brain cells. The recruitment of γ-tubulin to spindle MTs, however, is severely impaired, and spindle MT density is reduced in a manner that is indistinguishable from cells lacking Augmin or γ-TuRC function. In contrast, the absence of Dgp71WD leads to defects in the assembly of the acentrosomal female Meiosis I spindle that are more severe than those seen in Augmin or γ-TuRC mutants, indicating that Dgp71WD has additional functions that are independent of these complexes in oocytes. Moreover, the localisation of bicoid RNA during oogenesis, which requires γ-TuRC function, is unperturbed in Dgp71WD(120) mutants. Thus, Dgp71WD is not simply a general cofactor required for γ-TuRC and/or Augmin targeting, and it appears to have a crucial role independent of these complexes in the acentrosomal Meiosis I spindle.
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Fig. 1. Dgp71WD120 is a null mutant.(A) Schematic view of the Dgp71WD gene. Coding sequences are indicated in green and UTRs in blue; the inverted triangle represents the insertion site of the Dgp71WDGE30807 P-element. The dotted line represents the region deleted in the Dgp71WD120 mutant. Coloured arrows refer to PCR primer pairs used for the PCRs shown in (B) - red = reaction (i), purple = reaction (ii). (B) Agarose gel of the PCR products obtained using the indicated primer pairs (the final reaction is a positive control) using either WT or Dgp71WD120 genomic DNA. (C) Western blots of WT and Dgp71WD120 3rd instar larval brain extracts at two different exposure levels. In WT extracts a double band can be seen below the 84 kDa marker. The higher band is nonspecific-background whereas the lower band represents Dgp71WD. The lower band is absent in the mutant, indicating that Dgp71WD120 is likely a protein null. (D) WT and Dgp71WD120 3rd instar larval brain cells stained with anti-Cnn (green) and anti-Dgp71WD (red) antibodies; DNA (blue). Scale bar represents 2.5 µm.
Fig. 2. Centrosomal recruitment of Cnn and γ-tubulin is unperturbed in Dgp71WD120 and Dgrip75175 mutant brain cells.(A,B) Images show metaphase 3rd instar larval Ganglion Mother cells (GMCs) from WT, Dgp71WD120, and Dgrip75175 brains that were stained with anti-Cnn (A) (green) or anti-γ-tubulin (B) (red) antibodies; DNA (blue). (C) Images show living metaphase 3rd instar larval NBs from WT, Dgp71WD120, and Dgrip75175 brains expressing γ-tubulin GFP. (D) Quantification of the total amount of centrosomal Cnn, γ-tubulin or γ-tubulin-GFP in brain cells. Centrosomal γ-tubulin or Cnn levels in fixed cells were quantified in at least 7 cells in each of 5 brains. Centrosomal γ-tubulin-GFP levels in living cells were quantified in at least 9 NBs of each genotype. Significance testing was conducted using a Student's t-test. Error bars are S.E.M. Scale bar represents 2.5 µm (A,B) and 5 µm (C).
Fig. 3. Dgp71WD120, Dgrip75175 and wac NBs have severe spindle defects, yet successfully complete bipolar asymmetric divisions.(A–D) WT and mutant 3rd instar larval neuroblasts (NBs) expressing Jupiter-GFP were filmed from before NEB to the onset of cytokinesis. Representative WT (A), Dgp71WD120 (B), Dgrip75175 (C) and wac (D) NBs are shown. Time is shown as min:sec. Time point −00:20 represents one time point before NEB; the asterisk (*) marks the time of the metaphase to anaphase transition. Mutant spindles at the metaphase-anaphase transition consist of only a few k-fibre bundles unlike WT cells, where spindle density is higher (third row). Central spindle formation is strongly impaired in mutant spindles (red arrowheads) compared to WT spindles (white arrowheads). (E) Spindle length was measured over time from NEB to the onset of cytokinesis in WT (n = 11), Dgp71WD120 (n = 9), Dgrip75175 (n = 10), and wac (n = 7) NBs. Data was aligned to Anaphase B onset (time = 0 on the graphs). Mutant cells take significantly longer to undergo division, and have significantly longer spindles than WT NBs at Anaphase-B onset. (F) The density of Jupiter-GFP (as a proxy for MT density) was calculated at spindles in WT (n = 11), Dgp71WD120 (n = 9), Dgrip75175 (n = 8) and wac (n = 5) NBs. Spindle Jupiter-GFP intensity is significantly lower in mutant neuroblasts. Significance testing was conducted using a Student's t-test. Scale bar represents 5 µm.
Fig. 4. bicoid mRNA localises normally in Dgp71WD120 oocytes.(A–C)
bcd mRNA fluorescent in situ hybridisation in representative stage 11/12 WT, Dgp71WD120 and Dgrip75175 oocytes. bcd mRNA localises normally to the anterior end of the oocyte in WT and Dgp71WD120 mutants but is mislocalised towards the posterior in Dgrip75175 mutants (white arrowhead). (D) Quantification of bcd mRNA localisation defects in WT, Dgp71WD120 and Dgrip75175 oocytes. Oocytes were scored as having bcd localised either at <25% or >25% egg length. WT, n = 30; Dgp71WD120, n = 36, Dgrip75175, n = 17. Significance testing was conducted using a Chi-squared test. Scale Bar = 50 µm.
Fig. 5. Dgp71WD120 mutant oocytes have defective Meiosis I spindles.(A) WT (n = 38) and hemizygous Dgp71WD120/Df(2L)ED119 Meiosis I spindles (n = 40) from non-activated oocytes were fixed and stained with anti-DTACC (red) and anti-α-tubulin (green) antibodies; DNA (blue). (B)
Dgp71WD120 meiosis I spindles have a significantly reduced spindle area and width compared to WT spindles. (C)
Dgp71WD120 meiosis I spindles do not have any significant alteration in spindle length or chromosome positioning. (D) Quantification of the percentage of WT or Dgp71WD120 spindles lacking DTACC polar staining or showing only very weak staining. Significance testing was conducted using Student's t-tests (B,C) and a Chi-squared test (D). Error bars are SEM. Scale bar represents 5 µm. (E) Schematic showing how spindle length and chromosome distance were measured in (C).
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