Localised dynactin protects growing microtubules to deliver oskar mRNA to the posterior cortex of the Drosophila oocyte

  1. Ross Nieuwburg
  2. Dmitry Nashchekin
  3. Maximilian Jakobs
  4. Andrew P Carter
  5. Philipp Khuc Trong
  6. Raymond E Goldstein
  7. Daniel St Johnston  Is a corresponding author
  1. University of Cambridge, United Kingdom
  2. Medical Research Council, Laboratory of Molecular Biology, United Kingdom
  3. University of Cambridge, Centre for Mathematical Sciences, United Kingdom
9 figures, 1 table and 1 additional file

Figures

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 …

https://doi.org/10.7554/eLife.27237.003
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’) …

https://doi.org/10.7554/eLife.27237.004
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 …

https://doi.org/10.7554/eLife.27237.005
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 …

https://doi.org/10.7554/eLife.27237.006
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.

https://doi.org/10.7554/eLife.27237.007
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…

https://doi.org/10.7554/eLife.27237.008
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 …

https://doi.org/10.7554/eLife.27237.009
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 …

https://doi.org/10.7554/eLife.27237.014
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
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 …

https://doi.org/10.7554/eLife.27237.017
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
Figure 7—source data 2

Distances of EB1-GFP tracks from the posterior cortex.

https://doi.org/10.7554/eLife.27237.020
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…

https://doi.org/10.7554/eLife.27237.018
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 …

https://doi.org/10.7554/eLife.27237.022
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
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 …

https://doi.org/10.7554/eLife.27237.024

Tables

Table 1
Quantification of oskar mRNA particle movements.
https://doi.org/10.7554/eLife.27237.010
PhenotypeAverage speed, μm/sTracks to Posterior, %Mobile fraction, %
WT0.36 ± 0.01 (N=1075)65 ± 0.8 (N=989)12.0 ± 2.1 (N = 358)
arp14D20.37 ± 0.03 (N=296)62 ± 0.7 (N=353)15.4 ± 2.5 (N = 361)
  1. 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.

Table 1—source data 1

Speed of oskar mRNA particles.

https://doi.org/10.7554/eLife.27237.011
Table 1—source data 2

Direction of oskar mRNA particles.

https://doi.org/10.7554/eLife.27237.012
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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