Reconstruction of Par-dependent polarity in apolar cells reveals a dynamic process of cortical polarization
- Kyoto University, Japan
- RIKEN Center for Biosystems Dynamics Research, Japan
- The University of Tokyo, Japan
Figures
Figure 1
S2 cells polarize due to elevated Par3 expression.
(A) Immunostaining of endogenous aPKC, Par6, and Par3 in S2 cells 2 days following transfection of the empty vector. Blue indicates DAPI staining. Images in A-D were at the equatorial plane of …
Figure 2
Par3 localization requires Lgl in S2 cells.
(A) Endogenous expression of Lgl in S2 cells stained with anti-Lgl and DAPI at 2 days post-transfection of the empty vector. (B) Par3 and endogenous Lgl localize complementarily in 71% of cells (n = …
Figure 3
Quantification of cell polarity and 3D imaging.
(A) Definition of the asymmetric index (ASI). ASI is defined by the maximum difference in the cumulative intensity of fluorescence (such as Par3-mKate2 and Par6-GFP) between a half cell perimeter …
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Figure 3—source data 1
Source data for the histogram in Figure 3.
- https://doi.org/10.7554/eLife.45559.005
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Figure 3—source data 2
The individual histogram in each experiment in Figure 3.
- https://doi.org/10.7554/eLife.45559.006
Figure 4 with 1 supplement
The localization of Par6, Par3 and Cdc42 in the Par-islands.
(A–D) An image of a S2 cell expressing both myc-Par3-mKate2 and Par6-GFP, immunostained for myc, GFP and Cdc42. (A) Triple staining image for myc-Par3-mKate2, Par6-GFP and Cdc42. (B-D) Images …
Figure 4—figure supplement 1
Temporal pattern of Par6 expression level.
Temporal pattern of the fluorescence intensity of Par6-GFP expressed in S2 cells transfected with pAct-Par6-GFP. The fluorescence intensity of Par6-GFP was measured for 6 cells every 1 hr from 2 …
Figure 5
Temporal pattern of Par complex clustering.
(A and B) Time-lapse images of S2 cells inducing Par3-mKate2 expression via the Metallothionein promoter, leading to polarized (A) or non-polarized (B) Par3 distribution. Time 0 (h: min) was at the …
Figure 6 with 1 supplement
Temporal changes in cell polarity toward the two steady states.
(A–D) Temporal pattern of the asymmetric index (A), fluorescence intensity of Par3-mKate2 (B), its rate of change (B) and the ratio of Par6-GFP/Par3-mKate2 (D) of S2 cells that were transfected with …
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Figure 6—source data 1
Source data for the temporal change of S2 cells.
- https://doi.org/10.7554/eLife.45559.014
Figure 6—figure supplement 1
Comparison in the expression level between the endogenous Par3 and the induced Par3.
The expression levels were compared between Par3-induced by the MT promoter and the endogenous Par3 by western blotting. S2 cells were transfected with pMT-myc-Par3-mKate2, and induced for 8 hr …
Figure 7
Steady state dynamics of polarized Par complex.
(A) The distribution of ASI among cells with memGFP (left) driven by the Act5C promoter and Par-3-mKate2 (right) induced MT promoter. ASI was measured for the equatorial plane of cells 8 hr after …
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Figure 7—source data 1
Source data for the histogram in the steady state.
- https://doi.org/10.7554/eLife.45559.016
Figure 8
Cell autonomous formation of the Par complex polarization.
(A) Time-lapse imaging of Par3 distribution during mitosis. Time indicates hr:min after CuSO4 addition. Images of equatorial plane (upper panels), and the max projection of the whole cell (lower …
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Figure 8—source data 1
Source data for the position of centrosome.
- https://doi.org/10.7554/eLife.45559.021
Figure 9
Curvature of Par-islands.
(A) Cell membrane regions along the equatorial contour were visualized by memGFP and classified into three regions, Par-islands, neighboring regions, and non-neighboring regions. A Par-island region …
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Figure 9—source data 1
Source data for the membrane curvature.
- https://doi.org/10.7554/eLife.45559.023
Figure 10
SRRF-processed confocal images reveals a unit-like segment in Par-islands.
(A) SRRF-processed confocal images of cells expressing both Par3-mKate2 and Par6-GFP. Scale bar, 5 μm. (B) Left. the average of 200 confocal images of Par3-mKate2 distribution in a cell that …
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Figure 10—source data 1
Source data for the contour line length of SRRF images.
- https://doi.org/10.7554/eLife.45559.025
Figure 11
Structual analysis of the assembly state of the Par-complex using STED microscopy.
(A) STED image of a cell that expresses Par3-GFP. The distribution of GFP was detected via indirect immunofluorescence staining. Scale bar, 5 μm for (A–D). (B) Deconvoluted image of the …
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Figure 11—source data 1
Source data for the length of segment of STED images.
- https://doi.org/10.7554/eLife.45559.027
Figure 12 with 2 supplements
CR1 domain of Par3 is required for the formation of large regular meshwork of Par-islands.
(A) Schematic representation of the functional domains of Par3 and ΔCR1 mutant constructs. (B) Time-lapse imaging of the distribution of Par6-GFP in cells inducing the expression of Par3ΔCR1-mKate2 …
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Figure 12—source data 1
Source data for the ASI and island size of Par3ΔCR1 mutant.
- https://doi.org/10.7554/eLife.45559.032
Figure 12—figure supplement 1
The suppression of the endogenous Par3 by RNAi.
The estimation of knock down efficiency for the endogenous Par3 by western blotting. S2 cells were transfected with the empty vector. Western blotting was performed two-days post-transfection and …
Figure 12—figure supplement 2
Expression level of myc-Par3sm-GFP and flag-Par3ΔCR1sm-GFP, and effect of RNAi.
(A) The estimation of knock down efficiency for the endogenous Par3 by western blotting. S2 cells were transfected with the empty vector. Western blotting was performed 2-days post-transfection and …
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Figure 12—figure supplement 2—source data 1
Source data for the Western blotting image.
- https://doi.org/10.7554/eLife.45559.031
Figure 13
Role of the phosphorylation of Par3 and membrane binding.
(A) Schematic representation of the functional domains of Par3 and S980A and ΔMAR mutant constructs used in this study. (B–C) Time-lapse imaging of the distribution of Par6-GFP in cells where …
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Figure 13—source data 1
Source data for the ASI of Par3S980A mutant.
- https://doi.org/10.7554/eLife.45559.034
Videos
Video 1
3D time-lapse movie of a polarized S2 cell monitored by Par6-GFP 2 days after transfection of pAct-Gal4 >UAS-myc-Par3-mKate2, pAct-Par6-GFP, and pAct-aPKC.
Par-islands are clustered with dynamic movements.
Video 2
3D time-lapse movie of a S2 cell monitored by Par6-GFP following induction of myc-Par3-mKate2 from Metallothionein promoter.
Induction started at time 0 by the addition of CuSO4 2 days after transfection of pMT-myc-Par3-mKate2, pAct-Par6-GFP, and pAct-aPKC.
Video 3
3D time-lapse movie of a polarized S2 cell monitored by Par6-GFP at 8 hr induction of myc-Par3-mKate2 with the co-expression of pAct-Par6-GFP and pAct-aPKC.
https://doi.org/10.7554/eLife.45559.017
Video 4
3D movie of a polarized S2 cell stained for myc-Par3-mKate2 and Lgl at 8 hr induction of myc-Par3-mKate2 with the co-expression of pAct-Par6-GFP and pAct-aPKC.
https://doi.org/10.7554/eLife.45559.018
Video 5
3D movie of a nonpolarized S2 cell stained for myc-Par3-mKate2 and Lgl at 8 hr induction of myc-Par3-mKate2 with the co-expression of pAct-Par6-GFP and pAct-aPKC.
A part of the adjacent cell is included in the movie.
Video 6
3D time-lapse movie of a S2 cell monitored by Par6-GFP, 3 min after the addition of Latrunculin B, at 8 hr induction of myc-Par3-mKate2, following 2 days transfection of pMT-myc-Par3-mKate2, pAct-Par6-GFP, and pAct-aPKC.
https://doi.org/10.7554/eLife.45559.035Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (D. melanogaster) | y, w, Baz-GFP | Bloomington Drosophila Stock Center | BDSC:51572; FLYB:FBtc0099880 | FlyBase symbol: P{PTT-GC}baz CC01941 |
| Cell line (D. melanogaster) | S2 | Invitrogen, Thermo Fisher Scientific | Invitrogen:R69007 | |
| Antibody | Rabbit polyclonal anti-aPKC | Santa Cruz | Santa_Cruz:sc-216 | IHC (1:1000) WB (1:3000) |
| Antibody | Mouse monoclonal anti-Par3 | Ohshiro et al. (2000) | IHC (1:100) | |
| Antibody | Rabbit polyclonal anti-Par3 | Ohshiro et al. (2000) | IHC (1:1000) WB (1:3000) | |
| Antibody | Rabbit polyclonal anti-Par6 | Izumi et al. (2004) | IHC (1:1000) | |
| Antibody | Chicken polyclonal anti-Myc | Bethyl | Bethyl:A190-103A | IHC (1:1000) |
| Antibody | Mouse monoclonal anti-Miranda | Ohshiro et al. (2000) | IHC (1:100) | |
| Antibody | Rabbit polyclonal anti-Lgl | Ohshiro et al. (2000) | IHC (1:1000) WB (1:1000) | |
| Antibody | Guinea pig polyclonal anti-Cdc42 | Ulrich Tepass (University of Tronto) | IHC (1:100) | |
| Antibody | Chicken polyclonal anti-GFP | aves | aves:GFP-1020 | IHC (1:1000) |
| Antibody | Mouse monoclonal anti-GFP | chemicon | chemicon:MAB3580 | IHC (1:1000) |
| Antibody | Rabbit polyclonal anti-GFP | MBL | MBL:598 | IHC for Larva brain (1:1000) WB (1:2000) |
| Antibody | Mouse monoclonal anti-Flag | Sigma | Sigma:F3165 | IHC (1:1000) |
| Antibody | Alexa 488, Cyanin3, Cyanin5 secondaries | Jackson Immuno Research | IHC for S2 cells (1:4000) IHC for Larva brain (1:200) | |
| Antibody | anti-alpha tubulin | sigma | Sigma:T9026 | WB (1:15000) |
| Chemical compound, drug | Latrunclin B | Wako | Wako:129–05101 | final concentration, 1 µM |
| Chemical compound, drug | CuSO4 | Wako | Wako:034–04445 | final concentration, 1 mM |
| Recombinant DNA reagent | pMT/V5-His B (Gateway vector) | Invitrogen, Thermo Fisher Scientific | Invitrogen:V412020 | |
| Recombinant DNA reagent | pAc5.1/V5-His B (Gateway vector) | Invitrogen, Thermo Fisher Scientific | Invitrogen:V411020 | |
| Recombinant DNA reagent | pAc-Gal4 (Gateway vector) | Addgene | Addgene:24344 | provided from Liqun Luo (Stanford University) |
| Recombinant DNA reagent | pDAMCS (Gateway vector) | this paper | Progenitors: pAct5C0 plasmids (BglII-XhoI fragment); Gateway vector pUC19 | |
| Recombinant DNA reagent | pUAST | Brand and Perrimon (1993) | ||
| Recombinant DNA reagent | Par6-GFP (plasmid) | this paper | Progenitors: Par6 and GFP (cDNA); Gateway vector pAc5.1/V5-His B | |
| Recombinant DNA reagent | aPKC-GFP (plasmid) | this paper | Progenitors: aPKC and GFP (cDNA); Gateway vector pAc5.1/V5-His B | |
| Recombinant DNA reagent | myc-Par3-mKate2 (plasmid) | this paper | Progenitors: Par3 and mKate2 (cDNA); Gateway vector pMT/V5-His B, pAc-Gal4 or pDAMCS | |
| Recombinant DNA reagent | myc-Par3-GFP (plasmid) | this paper | Progenitors: Par3 and GFP (cDNA); Gateway vector pMT/V5-His B, pAc-Gal4 or pDAMCS | |
| Recombinant DNA reagent | membrane-GFP | this paper | Progenitors: myristoylation tag from Fyn and GFP (cDNA); Gateway vector pAc5.1/V5-His B | |
| Software, algorithm | Huygens Professional | Scientific Volume Imaging | ver. 17.10 | |
| Other | DAPI | nacalai tesque | nacalai_tesque: 11034–56 | IHC for S2 cells (1:4000) IHC for Larva brain (1:200) |
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.45559.036
