Localised dynactin protects growing microtubules to deliver oskar mRNA to the posterior cortex of the Drosophila oocyte
- University of Cambridge, United Kingdom
- Medical Research Council, Laboratory of Molecular Biology, United Kingdom
- University of Cambridge, Centre for Mathematical Sciences, United Kingdom
Figures
Figure 1
The 4D2 mutation disrupts the localisation of oskar mRNA at the oocyte posterior.
(A) A schematic diagram of a stage 9 Drosophila egg chamber showing oskar mRNA transport. (B–C) Confocal images of stage 9 egg chambers from a wild-type ovary (B) and from a 4D2 homozygous germline …
Figure 2
The 4D2 mutation does not affect the anchoring of Staufen and oskar at the posterior cortex.
(A–D) Confocal images of stage 9 (A and B) and stage 10b egg chambers (C and D) from wild-type (A and C) and 4D2 germline clone ovaries (B and D) stained for Staufen (A–D) and Oskar (A’–D’) …
Figure 3 with 2 supplements
arp14D2 affects the delivery of p150Glued, the dynein heavy chain and kinesin-βgal to the posterior.
(A–B) Wild-type (A) and arp14D2 mutant (B) stage 9 egg chambers stained for kinesin-βgal. (C–D) Wild-type (C–C’’) and arp14D2 (D–D’’) mutant stage 9 egg chambers stained for p150Glued (C–D) and the …
Figure 3—figure supplement 1
Mapping 4D2 to the Arp1 region.
(A) Diagram of the right arm of chromosome 3 showing the visible markers used for recombination mapping (cu sr e ca) and the position of the Arp1 locus. (B) Diagram showing the extent of the …
Figure 3—figure supplement 2
Transheterozygous combinations of arp1 alleles disrupt the localisation of GFP-Staufen to the posterior cortex.
(A–D) Confocal images of GFP-Staufen (green) and DNA (DAPI; blue) in arp14D2/+ (A), an arp14D2 germ line clone (B), arp14D2/ arp11 (C) and arp14D2/ arp12 (D) stage 9 egg chambers. Scale bar 20 μm.
Figure 4
The arp14D2 mutation is predicted to disrupt the interaction between the Arp1 rod and the p150Glued/dynamitin/p24 shoulder domain in the Dynactin complex.
(A) A ClustalX alignment of part of subdomain 2 (amino acids 26–78) in the Arp1 orthologues from different animal species and Drosophila Actin 5C. The orthologues shown are Homo sapiens (human), Ratt…
Figure 5
The arp14D2 allele does not disrupt oocyte determination, mRNA transport from the nurse cells to the oocyte or bicoid mRNA localisation.
(A–A”) A confocal image of a germarium from an arp14D2/+heterozygote stained for the oocyte marker Orb (white in A; red in A’) and expressing nls-GFP (white in A’; green in A’). (B–B”) A confocal …
Figure 6
The arp14D2 mutation reduces the frequency and the directional bias of oskar mRNA movements near the posterior of the oocyte.
(A) A graph showing the relative frequency of oskar mRNA movements at different distances from the posterior pole in arp14D2/+ (n = 528 tracks) and arp14D2 mutant oocytes (n = 410). The posterior …
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Figure 6—source data 1
The number of oskar mRNA tracks at the specified distances from the posterior pole.
- https://doi.org/10.7554/eLife.27237.015
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Figure 6—source data 2
Direction of oskar mRNA tracks at the specified distances from the posterior pole.
- https://doi.org/10.7554/eLife.27237.016
Figure 7 with 1 supplement
Dynactin is required for microtubule growth to the posterior cortex.
(A) A histogram showing the average velocities (µm/sec) of EB1 comets on growing microtubule plus ends in the centre of the oocyte (15–30 µm from the posterior) and near the posterior cortex (0–15 …
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Figure 7—source data 1
Velocities of EB1-GFP comets at the specified distances from the posterior pole.
- https://doi.org/10.7554/eLife.27237.019
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Figure 7—source data 2
Distances of EB1-GFP tracks from the posterior cortex.
- https://doi.org/10.7554/eLife.27237.020
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Figure 7—source data 3
Lifespan of EB1-GFP comets near the posterior cortex.
- https://doi.org/10.7554/eLife.27237.021
Figure 7—figure supplement 1
Kinesin 1 is required for microtubule growth to the posterior cortex.
(A) A histogram showing the average velocities (µm/sec) of EB1 comets on growing microtubule plus ends in the centre of the oocyte (15–30 µm from the posterior) and near the posterior cortex (0–15 µm…
Figure 8
Microtubules continue growing for twice as long at the posterior cortex than at the lateral cortex.
(A) A confocal image of microtubules in a wild-type stage 9 oocyte indicating the regions of the posterior and lateral cortex (yellow lines) from which the kymographs in (C) were collected. (B) A …
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Figure 8—source data 1
Dwell time of EB1-GFP comets at the lateral and posterior cortex
- https://doi.org/10.7554/eLife.27237.023
Figure 9
A model of the kinesin1/dynactin/microtubule positive feedback loop that increases the length and directional bias of microtubules near the posterior cortex.
A series of diagrams showing the steps in the positive feedback loop that ensures that microtubules reach the posterior cortex to deliver oskar mRNA. The distribution of cortical ncMTOCs along the …
Tables
Table 1
Quantification of oskar mRNA particle movements.
https://doi.org/10.7554/eLife.27237.010| Phenotype | Average speed, μm/s | Tracks to Posterior, % | Mobile fraction, % |
|---|---|---|---|
| WT | 0.36 ± 0.01 (N=1075) | 65 ± 0.8 (N=989) | 12.0 ± 2.1 (N = 358) |
| arp14D2 | 0.37 ± 0.03 (N=296) | 62 ± 0.7 (N=353) | 15.4 ± 2.5 (N = 361) |
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Values shown are means plus and minus the SEM. In all cases, the differences between wild-type and the arp1[4D2] mutant were statistically insignificant, p>0.05.
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Table 1—source data 1
Speed of oskar mRNA particles.
- https://doi.org/10.7554/eLife.27237.011
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Table 1—source data 2
Direction of oskar mRNA particles.
- https://doi.org/10.7554/eLife.27237.012
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Table 1—source data 3
Mobile fraction of oskar mRNA particles.
- https://doi.org/10.7554/eLife.27237.013
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.27237.025
