Dynein-mediated transport and membrane trafficking control PAR3 polarised distribution
- Institut Jacques Monod, CNRS, UMR 7592, Paris Diderot University, Sorbonne Paris Cité, France
Figures
Figure 1 with 3 supplements
Dynamic PAR3 distribution along the oocyte anterior posterior axis.
(A) Description of the quantification Fiji Macro. After selecting three points in the oocyte (yellow stars) and delimitation of oocyte perimeter, the macro allows us to obtain different data about …
-
Figure 1—source data 1
Quantification of TubGal4; UASp PAR3-GFP egg chambers during oogenesis.
- https://doi.org/10.7554/eLife.40212.007
Figure 1—figure supplement 1
Membrane et developmental stage definitions.
(A) Egg chamber expressing PAR3-GFP under its own promoter (green/w, P[w+, FRT9-2]18E, f, baz[815.8], P{CaryP, PB[BAC Baz- sfGFP2]attP18}) and nuclear RFP (red) both in somatic and germinal cells. Wh…
Figure 1—figure supplement 2
PAR3 distribution without total signal normalisation.
(A) PAR3 density from stage 8 to stage 10. The quantity of PAR3 has been normalised by the membrane length of the domain but not by the total oocyte intensity. Stage 8, n = 8; stage 9A, n = 9; stage …
Figure 1—figure supplement 3
Control experiments of PAR3 quantification process.
(A) Localisation of GFP-PAR3 (left) and PAR1-GFP (right) under control of maternal Tub67c-GAL4 in stage 9B egg chambers. Note the weak density of PAR3 and the strong density of PAR1 in the PPM …
-
Figure 1—figure supplement 3—source data 1
Proportion of anterior PAR3 (APM) in oocytes versus in adjacent nurse cells.
- https://doi.org/10.7554/eLife.40212.006
Figure 2 with 2 supplements
Cytoskeleton involvement in PAR3 polarity.
(A–D) Role of cytoskeleton on PAR3 distribution. Flies are nourished with latrunculin (cyan) for 48 h, colchicin (yellow) for 24 h, or only yeast paste (control). (A) Representative distribution of …
-
Figure 2—source data 1
PAR3 posterior exclusion ratio in oocyte of flies fed with latrunculin, colchicin, or without drugs.
(Figure 2B).
- https://doi.org/10.7554/eLife.40212.013
-
Figure 2—source data 2
PAR3 asymmetry ratio in oocyte of flies fed with latrunculin, colchicin, or without drugs.
(Panel C).
- https://doi.org/10.7554/eLife.40212.014
-
Figure 2—source data 3
PAR3 quantity (cytoplasm intensity/total oocyte intensity) in oocyte of flies fed with latrunculin, colchicin, or without drugs.
(Figure 2D).
- https://doi.org/10.7554/eLife.40212.015
Figure 2—figure supplement 1
Cytoskeleton involvement in PAR3 distribution.
(A) Quantification of PAR3-GFP density between stage 8 and stage 10 upon latrunculin (cyan) and colchicin (yellow) treatment. These data were used to obtain the graph in Figure 2B–D. Error bars …
-
Figure 2—figure supplement 1—source data 1
Quantification of PAR3-GFP density (normalised) between stage 8 and stage 10 upon latrunculin and colchicin treatment.
- https://doi.org/10.7554/eLife.40212.010
Figure 2—figure supplement 2
Effect of colchicin on distribution of PAR3 present at an endogenous level.
(A) Localisation of PAR3-GFP (Trap line) at a pre-vitellogenic stage egg chamber. We note PAR3 localised at the anterior cortex and also in some small dots in the oocyte cytoplasm (arrowhead). We …
-
Figure 2—figure supplement 2—source data 1
Density of cytoplasmic fraction of PAR3-GFP (trap line) at stage 9B upon latrunculin and colchicin treatment.
(Panel D).
- https://doi.org/10.7554/eLife.40212.012
Figure 3
By producing PI(4,5)P2, SKTL controls PAR3 APM accumulation and PPM exclusion.
(A–F) Distribution of PAR3 in response to SKTL activity. PAR3-GFP is expressed in germinal cells at stage 9B under control conditions, or with overexpression (OE) of Myc-SKTL, Myc-SKTLDNRQ, or in …
-
Figure 3—source data 1
Quantification of PAR3 density (normalised) at each plasma membrane domain in sktl mutant or SKTL overexpressed (OE) contexts in stage 9B oocytes.
- https://doi.org/10.7554/eLife.40212.017
-
Figure 3—source data 2
Quantification of PAR3 asymmetry ratio in sktl mutant or control at stage 9B.
(Panel E).
- https://doi.org/10.7554/eLife.40212.018
-
Figure 3—source data 3
Quantification of PAR3 posterior exclusion ratio in sktl mutant or SKTL overexpressed contexts at stage 9B.
(Panel F).
- https://doi.org/10.7554/eLife.40212.019
Figure 4 with 1 supplement
SKTL-dependent PAR3 posterior exclusion bypasses regulation by PAR1.
(A–C) Representative distribution of PAR3-GFP in stage 9B oocyte in a control situation (A), in RNAi PAR1 context (B), or when PAR3 phosphorylation sites by PAR1 are mutated (C). In the control …
-
Figure 4—source data 1
Quantification of PAR3 posterior exclusion in response to PAR1 at stage 9B.
(Panel D).
- https://doi.org/10.7554/eLife.40212.022
-
Figure 4—source data 2
Quantification of PAR3 posterior exclusion in response to PAR1 at stage 9B in combination with SKTL.
(Panel E).
- https://doi.org/10.7554/eLife.40212.023
Figure 4—figure supplement 1
Validation of RNAi PAR1 efficiency.
Flies Tub67c-GAL4 or Tub67c-GAL4; UASp RNAi par1 were put on fly cages to lay eggs on agar plates. The plates were collected every hour to produce the embryonic extracts revealed after western blot. …
Figure 5 with 1 supplement
PAR3 asymmetry depends on RAB5.
(A) PAR3-GFP (A’), (A’’’ green) expressed in germline is present occasionally in RAB5-positive early endosomes (A’’), (A’’’) magenta). A’, A’’ and A’’’ are magnifications of A (white frame). …
-
Figure 5—source data 1
Quantification of PAR3 density at each plasma membrane domain of stage 9B oocytes in response to RAB5 activity impairment.
(Panel E).
- https://doi.org/10.7554/eLife.40212.026
-
Figure 5—source data 2
Quantification of PAR3 posterior exclusion in different RAB5 mutant contexts at stage 9B.
(Panel F).
- https://doi.org/10.7554/eLife.40212.027
Figure 5—figure supplement 1
Validation of RAB5DN effect on endocytosis.
Endocytosis uptake of FM4-64 dye was performed on control (A, Tub67c-GAL4) or on RAB5DN(S43N) expressing egg chambers (B, Tub67c-GAL4; UASp RAB5DN). In panel (A), the dye is internalised in dotted …
Figure 6 with 2 supplements
Role of RAB11 on PAR3 asymmetrical localisation.
(A) Colocalisation of PAR3 (PAR3-GFP maternally expressed) with vesicular trafficking markers. To quantify the colocalisation, we measured the Pearson’s coefficient on stage 9B oocytes and results ar…
-
Figure 6—source data 1
Quantification of PAR3 colocalisation with vesicular compartment.
(Panel A).
- https://doi.org/10.7554/eLife.40212.031
-
Figure 6—source data 2
Quantification of PAR3 asymmetry ratio in rab11 mutant clones.
(Panel C).
- https://doi.org/10.7554/eLife.40212.032
-
Figure 6—source data 3
Quantification of PAR3 posterior exclusion ratio in rab11 mutant clones.
(Panel D).
- https://doi.org/10.7554/eLife.40212.033
Figure 6—figure supplement 1
PAR3 and vesicular compartments.
(A–C’’) Characterisation of the components of PAR3-GFP vesicular structures. PAR3-GFP (green, (A, B, C) is expressed in the germline under control of the maternal driver Tub67c-GAL4. By …
Figure 6—figure supplement 2
PAR3 colocalises with PI(4,5)P2 and RAB11 in front of the ring canal.
(A, B) Localisation of PAR3-GFP near a ring canal between a nurse cell (NC) plasma membrane and the oocyte (Oo) APM. PAR3-GFP is expressed in the germline under the control of the maternal driver Tub…
Figure 7 with 2 supplements
Dynein regulates PAR3 asymmetry.
(A–C) Distribution of PAR3 in response to dynein activity decrease. PAR3-GFP is expressed in germinal cells at stage 9B in control (osk-GAL4; UASp PAR3-GFP) or dhc64 knockdown contexts (osk-GAL4; UAS…
-
Figure 7—source data 1
Quantification of PAR3 density at each plasma membrane domain in Dhc64 knockdown at stage 9B oocytes (Panel B).
- https://doi.org/10.7554/eLife.40212.037
-
Figure 7—source data 2
Quantification of PAR3 posterior exclusion ratio in Dhc64 knockdown at stage 9B oocytes (Panel C).
- https://doi.org/10.7554/eLife.40212.038
Figure 7—figure supplement 1
Control experiments of PAR3 antibody specificity and charge control of immunopurification of GFP-SKTL.
(A) Role of dynein on PAR3 cytoplasmic accumulation (quantification of PAR3 proportion in the cytoplasm related to the whole oocyte intensity). Control (stage 9B, n = 10); RNAi dhc64c (stage 9B, n = …
Figure 7—figure supplement 2
Validation of PAR3 detection by antibody By western blot with anti-PAR3 antibody, we revealed numerous bands corresponding to several isoforms of PAR3 and to post-transcriptionally modified forms.
We hypothesised that the higher band (>250 kD) corresponds to an oligomer form of PAR3. (A) To confirm that this higher band is PAR3, we tried to decrease its intensity by PAR3 RNAi. We …
Figure 8
PAR3 recovery after APM photobleaching.
(A) PAR3-GFP expressed in ovarian follicle (Tub67c-GAL4; UASp PAR3-GFP) is photobleached in all the nurse cells and at the APM (yellow area, A1). The fluorescence recovery was followed for around …
-
Figure 8—source data 1
PAR3 quantity variation (grey levels) at the anterior and the posterior of oocyte after anterior FRAP experiment.
This experiment was performed on three oocytes for each condition (panel B).
- https://doi.org/10.7554/eLife.40212.040
-
Figure 8—source data 2
PAR3 quantity of each zone before FRAP was normalised to 1, and recovery of the fluorescence was observed.
- https://doi.org/10.7554/eLife.40212.041
Figure 9 with 2 supplements
IKKε regulates PAR3 microtubule unloading and APM accumulation.
(A–C) Distribution of PAR3 in response to IKKε knockdown. (A) IKKε knockdown affects the localisation of PAR3 and leads to an accumulation of circular actin clumps (ACs) enriched in PAR3. (B) …
-
Figure 9—source data 1
Quantification of PAR3 density at each plasma membrane domain in IKKe knockdown at stage 9B oocytes (Panel B).
- https://doi.org/10.7554/eLife.40212.047
-
Figure 9—source data 2
Quantification of PAR3 proportion in the cytoplasm related to the whole oocyte intensity in IKKe knockdown context.
(panel C).
- https://doi.org/10.7554/eLife.40212.048
Figure 9—figure supplement 1
Effect of IKKε knockdown.
(A) In an IKKε knockdown oocyte, ACs (A, A’ green) are accumulated around the nucleus (red). The oocyte nucleus position is indicated by an ‘N’ in A. (A’) is a magnification of the oocyte presented …
Figure 9—figure supplement 2
Alteration of microtubule network in IKKε knockdown.
(A) Dynein is trapped in ACs. Dynein is visualised thanks to DLIC-GFP transgene (green) and actin (red) is visualised after staining with phalloidin. (B–C) Effect of IKKε on MT minus ends. MTs minus …
-
Figure 9—figure supplement 2—source data 1
Quantification of PAR3 in the cytoplasm related to the whole oocyte intensity in IKKe knockdown context.
(panel C).
- https://doi.org/10.7554/eLife.40212.046
Figure 10
Speculative model of PAR3 localisation regulation.
(A) In Drosophila oocytes, PAR3 asymmetrical localisation proceeds in at least two steps. The first step (C) occurs at the posterior plasma membrane and leads to the PAR3 exclusion starting from …
Author response image 1
Our results show that the mutant form is more stable than wild type.
Thus, the absences of effect of DNRQ form cannot be explain by protein instability.
Author response image 2
Author response image 3
Videos
Video 1
Recovery of fluorescence after photobleaching of PAR3 at the APM domain.
Representative FRAP experiment on PAR3-GFP (Tub67c-GAL4; UASp PAR3-GFP) in Drosophila egg chamber.
Additional files
-
Source code 1
Oocyte analysis source code.
- https://doi.org/10.7554/eLife.40212.050
-
Transparent reporting form
- https://doi.org/10.7554/eLife.40212.051
