A CDK1 phosphorylation site on Drosophila PAR-3 regulates neuroblast polarisation and sensory organ formation
- Molecular, Cell and Developmental Biology, University of Dundee, United Kingdom
- MRC PPU, School of Life Sciences, University of Dundee, United Kingdom
Figures
Figure 1 with 1 supplement
Full inhibition of analog-sensitive CDK1.
(A) Live larval brains expressing Baz::GFP, before and after addition of 1-NA-PP1 10 µM at t0. Arrows point to mitotic neuroblasts. Scale bar: 20 µm. (A’) Cumulative sum chart of NEB events, …
Figure 1—figure supplement 1
Titrating 1-NAPP1 concentration for partial CDK1 inhibition.
(A) Cumulative sum chart of NEB events in cdk1as2 brains, normalized to the total number by the time of 1-NA-PP1 addition (t0). N=141 divisions in 4 brains (0.5 µM), 77 divisions in 4 brains (1 µM), …
Figure 2
Partial inhibition of analog-sensitive CDK1.
(A) Two consecutive divisions of live control and cdk1as2 cycling neuroblasts expressing Baz::GFP, before and after addition of 1-NA-PP1 0.5 µM at t0. Arrow: coalesced Baz crescent in metaphase. …
Figure 3 with 1 supplement
Asymmetric cell division-specific phosphorylation of Baz-S180.
(A) Features of the Baz protein. Consensus motifs suggesting a potential phosphoregulation of Baz by CDK1 are highlighted. (B) Fixed fly brain expressing Baz::GFP (magenta) and stained with a …
Figure 3—figure supplement 1
Localisation of Cyclins in asymmetrically dividing cells and Baz-S180 phosphorylation in imaginal discs.
(A) Leg disk sensory organ precursor in metaphase, at 0 h APF. Bottom panels: orthogonal view. Scale bar: 5 µm. (B) Wing disk columnar epithelium cell in metaphase. Bottom panels: orthogonal view. …
Figure 4
Localisation and function of Baz-S180 phosphomutants in neuroblasts.
(A) Fixed metaphase neuroblasts expressing Baz::mScarlet (red) and stained for Miranda (magenta), with or without Neur-GAL4 driving RFP RNAi and BazWT::GFP (green) expression. Scale bar: 5 µm. (B) …
Figure 5
Baz-S180 phosphorylation regulates sensory organ formation.
(A) Adult nota with or without Pannier-GAL-driven RNAi of baz::mScarlet and/or delta. Scale bar: 100 µm. (B) Control adult notum and baz::mScarlet, delta-depleted nota expressing BazWT::GFP, BazS180A…
Figure 6 with 1 supplement
Baz-S180 phosphomutants localisation during SOP lineage divisions.
In all panels, Neur-GAL4 drives deltaand baz::mScarlet RNAi as well as BazWT/S180A/S180D::GFP expression and live pupal nota are shown. For BazWT: 4 nota analysed in 2 independent experiments. For …
Figure 6—figure supplement 1
In all panels, Neur-GAL4 drives delta RNAi, baz::mScarlet RNAi and BazWT/S180A/S180D::GFP expression and live pupal nota are shown.
All scale bars: 5 µm. (A) SOP lineage from pI to pIIIb division in a case where Baz::GFP (green) is expressed in the entire SOP lineage. Bottom panels: orthogonal view. Arrows: Baz-positive puncta …
Figure 7
Drosophila Baz-S180 and human PARD3-S187 are substrates of CDK1 in vitro.
(A) Domains of Baz/PARD3 (up) and close-ups (down) of the N-Terminal fragments used to test phosphorylation of Baz by CDK1 in vitro by mass spectrometry. Blue bars: residues found phosphorylated in …
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Figure 7—source data 1
CyclinB1/CDK1 phosphorylates a Baz fragment on S180 in vitro.
(File 7B left panel colour) Full western blot corresponding to Figure 7B. left panel. Dual-color LICOR fluorescent image of 800 nm (green, anti-Baz-pS180 antibody) and 700 nm (red, ladder) channels shown in left panel. (File 7B left panel bw) Same image in black and white. (File 7B right panel colour) Full western blot corresponding to Figure 7B. right panel. Membrane stripped and reprobed with anti-GST antibody. Dual-color LICOR fluorescent image of 800 nm (green, anti-GST antibody) and 700 nm (red, ladder). (File 7B left panel bw) same image in black and white.
- https://cdn.elifesciences.org/articles/97902/elife-97902-fig7-data1-v2.zip
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Drosophila melanogaster) | mira::mCherry | CrispR edited locus by Januschke lab; Ramat et al., 2017 | ||
| Gene (Drosophila melanogaster) | baz::mScarlet-I | CrispR edited locus by Januschke lab; Houssin et al., 2021 | ||
| Gene (D. melanogaster) | cdk1as2 | CrispR edited locus by Januschke lab | This study | |
| Gene (D. melanogaster) | Baz::GFP | Bloomginton Stock Center | BDSC 51572 | |
| Gene (D. melanogaster) | ubi-bazΔLB::GFP | Kullmann and Krahn, 2018 | ||
| Gene (D. melanogaster) | ubi-bazΔOD::GFP | Kullmann and Krahn, 2018 | ||
| Gene (D. melanogaster) | ubi-his2av::mRFP | Gonzalez lab | Gift from Cayetano Gonzalez | |
| Gene (D. melanogaster) | UASz-bazWT::GFP | This study | ||
| Gene (D. melanogaster) | UASz-bazS180A::GFP | This study | ||
| Gene (D. melanogaster) | UASz-bazS180D::GFP | This study | ||
| Gene (D. melanogaster) | Neur-GAL4 | Bloomington stock center | BDSC 80575 | |
| Gene (D. melanogaster) | Pnr-GAL4 | Bloomington stock center | BDSC 3039 | |
| Gene (D. melanogaster) | UAS-RFP RNAi | Bloomington stock center | BDSC 67852 | |
| Gene (D. melanogaster) | UAS-dl RNAi | Bloomington stock center | BDSC 28032 | |
| Gene (D. melanogaster) | cycB::GFP | Bloomington stock center | BDSC 51568 | |
| Gene (D. melanogaster) | cycB3::GFP | Bloomington stock center | BDSC 91673 | |
| Gene (D. melanogaster) | Neur-GAL4, UAS-Pon::RFP | Emery et al., 2005 | ||
| Gene (D. melanogaster) | delta::GFP | Bloomington stock center | BDSC 59819 | |
| Chemical compound, drug | 1-NAPP-1 | Sigma Aldrich | 529579 | |
| Antibody | affinity purified Baz-PS180 antibody | sheep | This study | YAGGDS*PERLF – (where S* is phospho-Serine) |
| Antibody | Affinity purified Numb antibody | sheep | This study | GST-full length Numb protein (CG3779-PA) used as antigen |
| Antibody | Affinity purified Mira antibody | sheep | This study | RLFRTPSLPQRLR peptide used as antigen |
| Antibody | Affinity purified Mira antibody | rabbit; Ramat et al., 2017 | ||
| Antibody | rabbit anti-PKCζ | Santa Cruz | sc-17781 | |
| Antibody | Mouse anti-Cyclin A | DSHB | DSHB A12 | |
| Antibody | Mouse anti-Su(H) | Santa Cruz | sc-398453 AF647 | |
| Antibody | Mouse anti-Cut | DSHB | DSHB 2B10 | |
| Recombinant DNA reagent | UASz 1.0 | DGRC | DGRC 1431 |
Author response table 1
| Figure 1 | No change |
|---|---|
| Figure 2 | • Panel A now also shows a control neuroblast (Reviewer 2 request) • Panels after G have been rearranged to put the quantification of the Mira signal (now 2J) next to the Numb signal (now 2K, Reviewer 2 request) |
| Figure S2 | A new panel (B) shows two successive divisions of a cdk1AS2 neuroblast in the absence of 1-NAPP1 (Reviewer 1 request) |
| Figure 3 | No change |
| Figure S3 | No change |
| Figure 4 | New panels (G-H’) now describe the Numb phenotype in the Baz phosphomutants experiments (Reviewer 2 request) |
| Figure 5 |
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| Figure 6 | The previous Figure 6 has been replaced with Figure 7 (see below). This entirely new Figure 6 shows that Baz-S180 phosphorylation affects Baz localisation during the pIIa cell asymmetric cell division in the SOP lineage (Reviewer 1 request) |
| Figure S6 | This entirely new figure describes mosaic expression of UASz-driven Baz within the SOP lineage and how we took advantage of it to analyse the contribution of SOP lineage cells to various pools of cortical Baz. It also describes a new cell non autonomous mechanism controlling the pIIb cell division orientation. |
| Figure 7 | We have replaced the previous Figure 6 (testing full length human PARD3 phosphorylation by CDK1 in vitro) with this entirely new Figure 7. We synthesized Drosophila Baz and human PARD3 N-terminal fragments and showed that CDK1 phosphorylates Baz-S180 and the “equivalent” PARD3-S187 in vitro. |
Additional files
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Supplementary file 1
Phosphopeptides identified from phospho-proteomics of Drosophila Baz and human PARD3 N-terminal fragments in the presence or absence of CDK1/CyclinB1.
Bold text: very good position assignment. Red text: phosphorylation also observed in the absence of CDK1/CyclinB1.
- https://cdn.elifesciences.org/articles/97902/elife-97902-supp1-v2.xlsx
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Supplementary file 2
Genotypes of Drosophila lines used in this study.
- https://cdn.elifesciences.org/articles/97902/elife-97902-supp2-v2.xlsx
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Supplementary file 3
Origin of Drosophila stocks used in this study.
- https://cdn.elifesciences.org/articles/97902/elife-97902-supp3-v2.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/97902/elife-97902-mdarchecklist1-v2.pdf
