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, normalized to the total number by the time of 1-NA-PP1 addition (t0). N=324 divisions in 4 brains (cdk1+) and 75 divisions in 5 brains (cdk1as2), 3 experiments. (B) Loss of Baz::GFP cortical localisation in a cycling cdk1as2 neuroblast at prophase, after addition of 1-NA-PP1 10 µM at t0. Observed in 12 cases across 8 experiments. Scale bar: 5 µm. (C) Live neuroblasts metaphase-arrested by exposure to Colcemid 100 µM, expressing Baz::GFP (green) and His::RFP (magenta), before and after addition of 1-NA-PP1 10 µM at t0. Scale bar: 5 µm. Arrows: decondensed DNA after neuroblasts exit mitosis. (C’) Number of metaphase-arrested neuroblasts, normalised to their number at the time of 1-NA-PP1 10 µM addition (t0). N=53 neuroblasts in 5 brains (cdk1+) and 58 neuroblasts in 5 brains (cdk1as2), 2 experiments. (D) Mitotic slippage of a metaphase-arrested (Colcemid 100 µM) cdk1as2 neuroblast after addition of 1-NA-PP1 10 µM at t0. Scale bar: 5 µm.
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), and 100 divisions in 3 brains (2 µM), 1 experiment. (B) Two successive divisions of a cdk1AS2 neuroblast in the absence of 1-NA-PP1 followed by one division in the presence of 1-NA-PP1.
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. Arrowhead: non-coalesced crescent. Scale bar: 5 µm. (A’) Ratio between the apical Baz signal at metaphase in the presence of 1-NA-PP1 0.5 µM and the Baz signal during the previous metaphase, in the absence of 1-NA-PP1. In control neuroblasts, 1-NA-PP1 0.5 µM addition does not reduce the intensity of Baz crescents compared to the previous cell cycle (loss of 5.3 ± 24%, n=39). In cdk1as2 neuroblasts, 1-NA-PP1 addition reduces the intensity of Baz crescents compared to the previous cell cycle (loss of 34.2 ± 17%, n=36). Statistical test: two-tailed Mann–Whitney U test. For this boxplot and every following boxplot: cross: maximal and/or minimal outliers (beyond 1.5×interquartile range); grey circle: average; red dots: individual measurements; centre line, median; box limits, upper and lower quartiles; whiskers, 1.5×interquartile range. 5 brains per condition across 3 experiments. (B) Two consecutive divisions of cycling baz815-8, cdk1as2 neuroblasts expressing BazΔLB::GFP or BazΔOD::GFP, before and after exposure to 1-NA-PP1 0.5 µM. Scale bar: 5 µm. n=92 divisions in the presence of 1-NA-PP1 in 7 brains (BazΔLB) and 75 divisions in 5 brains (BazΔOD), 2 experiments. (C) Two consecutive divisions of cycling cdk1as2 embryonic neuroblasts expressing Baz::GFP and His::RFP, before and after exposure to 1-NA-PP1 0.5 µM. (C’) Ratio between the apical Baz signal at metaphase in the presence of 1-NA-PP1 0.5 µM and the Baz signal during the previous metaphase, in the absence of 1-NA-PP1 0.5 µM in cdk1as2 embryonic neuroblasts. Statistical test: two-tailed Mann–Whitney U test. n=37 successive divisions in 31 neuroblasts across 5 embryos, 3 experiments. (D) Cycling neuroblasts at metaphase. Scale bar: 5 µm. (D’) Control cycling neuroblasts maintain stable Baz levels throughout metaphase (+0.4 ± 6.1% in 75 seconds, n=52 neuroblasts, 4 brains) in the presence of 1-NA-PP1 0.5 µM. In CDK1as2 cycling neuroblasts, Baz levels decrease during metaphase in the presence 1-NA-PP1 0.5 µM (–9.8 ± 7.3% in 75 s, n=49, 6 brains). Statistical test: two-tailed Mann–Whitney U test. Two experiments. (E) cdk1as2 neuroblast first treated with 1-NA-PP1 0.5 µM for 1 hr, and then treated with Colcemid 100 µM. Neuroblasts stay polarized during metaphase (Baz crescents were maintained in 40/40 neuroblasts arrested in metaphase for at least 45’, 6 brains, 2 experiments). t0: NEB. Scale bar: 5 µm. (F) cdk1as2 neuroblast first treated with 1-NA-PP1 0.5 µM for 1 hr, then treated with Colcemid 100 µM for 30 min, after which 1-NA-PP1 was washed out (in this case 16.5’ after NEB). Some metaphase-arrested neuroblasts undergo Baz crescents coalescence following 1-NA-PP1 washout (n=19/51 neuroblasts, 7 brains, 3 experiments). t0: NEB. Scale bar: 5 µm. (G) Cycling neuroblasts fixed at metaphase, exposed to 1-NA-PP1 0.5 µM, expressing Baz::GFP (green) and stained for aPKC (blue) and Miranda (magenta). Scale bar: 5 µm. (H) Cycling neuroblasts fixed at metaphase, exposed to 1-NA-PP1 0.5 µM, expressing Baz::GFP (green) and stained for Numb (magenta). Scale bar: 5 µm. (I) Apical aPKC signal and (J) basal Mira signal in metaphase neuroblasts exposed to 1-NA-PP1 0.5 µM. n=23 neuroblasts in 5 brains (cdk1+) and 27 neuroblasts in 4 brains (cdk1as2), 2 experiments. (K) Basal Numb signal in metaphase neuroblasts exposed to 1-NA-PP1 0.5 µM. n=55 neuroblasts in 6 brains (cdk1+) and 30 neuroblasts in 13 brains (cdk1as2), 2 experiments. Statistical test: two-tailed Mann–Whitney U test. (L) In control neuroblasts, an apical Baz crescent (green) assembles in prophase and coalesces (green arrows) into a narrower, brighter crescent at NEB. Its intensity is maintained throughout metaphase. Upon partial inhibition of CDK1, an apical Baz crescent (green) assembles in prophase but fails to coalesce at NEB and its intensity decreases during metaphase. Basal polarity proteins (magenta) form more intense crescents than in controls.
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 phospho-specific antibody against Baz-pS180 (green). Arrows: mitotic neuroblasts. Arrowheads: mitotic neuroepithelial cells. Scale bar: 20 µm. (C) Left set of panels: cell cycle stages of fixed neuroblasts. Right set of panels: metaphase neuroepithelial cell. Scale bar: 5 µm. A Baz-pS180 signal was observed in 118/118 metaphase neuroblasts and 0/31 metaphase neuroepithelial cells (13 brains, 3 experiments). (D) Left: notum microchaete sensory organ precursor in metaphase, at 16 hr APF. Right: notum epidermal cell in metaphase, at 16 hr APF. Bottom panels: orthogonal view. Scale bar: 5 µm.
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. Scale bar: 5 µm. (C) Metaphase SOP expressing Pon::RFP (red) under the control of Neur-GAL4, stained for CycA (magenta) and Baz-pS180 (green). (D) Left panels: fixed larval brain neuroblasts expressing Baz::GFP (magenta) and stained for Cyclin A (green). Middle and right panels: live larval brain neuroblasts expressing endogenously expressed CycB3::GFP or CycB::GFP protein traps. (E) Fixed neuroblasts depleted of endogenous Baz::mScarlet and expressing BazWT::GFP or BazS180A::GFP. Scale bar: 5 µm.
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) Live neuroblasts depleted of endogenous Baz::mScarlet and expressing BazWT::GFP, BazS180A::GFP or BazS180D::GFP. Scale bar: 5 µm. (C) Intensity of the apical Baz::GFP signal in cycling neuroblasts depleted of endogenous Baz::mScarlet, normalized to the intensity of the cytoplasmic Baz signal in interphase. t0: NEB. n=18 divisions (BazWT::GFP), 23 divisions (BazS180A::GFP) and 21 divisions (BazS180D::GFP). 5 brains for all conditions, 2 experiments. Error bars: standard deviation. (D) Live metaphase-arrested neuroblasts depleted of endogenous Baz::mScarlet and expressing BazWT::GFP, BazS180A::GFP or BazS180D::GFP. T0: NEB. Scale bar: 5 µm. (E) Fixed neuroblasts depleted of endogenous Baz::mScarlet and expressing BazWT::GFP, BazS180A::GFP or BazS180D::GFP, stained for Miranda (green) and DAPI (blue). Two cases are displayed for BazS180D::GFP in metaphase, one showing polarized cortical Miranda (left) and the other mostly cytoplasmic Miranda (right). Scale bar: 5 µm. (F) Ratio between the basal Miranda cortical signal and the cytoplasmic signal. n=29 prophases, 128 metaphases, 44 anaphases in 17 brains (BazWT::GFP), 23 prophases, 100 metaphases, 42 anaphases in 18 brains (BazS180A::GFP), and 40 prophases, 119 metaphases, 47 anaphases in 18 brains (BazS180D::GFP). Statistical test: two-tailed Mann–Whitney U test. Box plots: cross: maximal and/or minimal outliers (beyond 1.5×interquartile range); grey circle: average; red dots: individual measurements; centre line, median; box limits, upper and lower quartiles; whiskers, 1.5×interquartile range. 2 experiments. (G) Fixed neuroblasts in metaphase, depleted of endogenous Baz::mScarlet and expressing BazWT::GFP, BazS180A::GFP or BazS180D::GFP (green), stained for Miranda (blue), Numb (magenta) and DAPI (blue). Two cases are displayed for BazS180D::GFP in metaphase, one showing polarized cortical Miranda (high Mira cortical/cytoplasmic ratio, left) and the other mostly cytoplasmic Miranda (low Mira c/c ratio, right). Scale bar: 5 µm. (G’) Proportions of cases with basal or uniform localisation of Numb in metaphase. (H) Fixed neuroblasts in metaphase, depleted of endogenous Baz::mScarlet and expressing BazWT::GFP, BazS180A::GFP or BazS180D::GFP, stained for Numb (green) and DAPI (blue). Scale bar: 5 µm. (H’) Proportions of cases with basal or uniform localisation of Numb in anaphase.
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::GFP or BazS180D::GFP. Scale bar: 100 µm. (C) Pupal notum stained for Su(H) (blue). Pannier-GAL4 drives both the depletion of endogenously expressed Baz::mScarlet and the expression of BazS180D::GFP. Arrows: dorso-central macrochaetes. Arrowheads: scutellar macrochaetes. Scale bar: 100 µm. (D) Quadrants defined for macrochaete bristles counting within the Pannier-GAL4 expression domain. The two upper quadrants include dorso-central macrochaetes and the two lower quadrants include scutellar macrochaetes (blues asterisks). Other macrochaetes (red asterisks) outside of the Pannier-GAL4 expression domain were ignored. Scale bar: 100 µm. (D’) Number of individual bristles per quadrant in baz::mScarlet, delta-depleted adult nota, with or without expression of Baz::GFP transgenes. Statistical test: two-tailed Mann–Whitney U test. Box plot: cross: maximal and/or minimal outliers (beyond 1.5×interquartile range); grey circle: average; red dots: individual measurements; centre line, median; box limits, upper and lower quartiles; whiskers, 1.5×interquartile range. n=332 quadrants in 83 flies (no rescue), 308 quadrants in 77 flies (BazWT::GFP), 264 quadrants in 66 flies (BazS180A::GFP) and 152 quadrants in 38 flies (BazS180D::GFP). (E) Pupal notum stained for Cut (green) and Su(H) (magenta). Asterisks: individual SOP clusters. Scale bar: 50 µm. (F) Number of individual SOP clusters per quadrant in baz::mScarlet, delta-depleted pupal nota expressing Baz::GFP transgenes. Statistical test: two-tailed Mann–Whitney U test. Box plot: cross: maximal and/or minimal outliers (beyond 1.5×interquartile range); grey circle: average; red dots: individual measurements; centre line, median; box limits, upper and lower quartiles; whiskers, 1.5×interquartile range. N=34 quadrants in 9 nota (BazWT::GFP), 24 quadrants in 6 nota (BazS180A::GFP), and 41 quadrants in 11 nota (BazS180D::GFP). 4 experiments. (G) Percentage of cases of Socket cell to Shaft cell transformation cases in baz::mScarlet, delta-depleted pupal nota expressing Baz::GFP transgenes. n=140 SOP clusters in 9 nota (BazWT::GFP), 127 SOP clusters in 6 nota (BazS180A::GFP), and 212 SOP clusters in 11 nota (BazS180D::GFP). 4 experiments. Statistical test: two-tailed Z score calculation of population proportions. (H) 8 h APF notum expressing Delta::GFP (magenta) and Baz::mScarlet (blue) stained for Baz-pS180 (green). Right panels: close-ups of the boxed areas in left panels. Scale bars: 50 µm (left) and 10 µm (right). Arrows: Delta-positive stripes. Arrowhead: scutellar macrochaete.
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 BazS180A: 3 nota in 2 experiments. For BazS180D: 3 nota in 2 experiments. (A) SOP lineage. Diverging arrows: asymmetric cell divisions. (B) SOP cluster at the two-cell (pIIb/pIIa) stage. Arrow: Baz::mScarlet (magenta) is not detectable at the pIIb/pIIa interface. (C) Pupal notum during SOP lineage divisions. Arrows: SOP clusters starting to express Baz::GFP during the imaging session. Scale bar: 50 µm. (D) Orthogonal view of pI division at metaphase. Apical is up and anterior is left. Scale bar: 5 µm. Boxed areas: cortical areas measured. (D’) Cortical/cytoplasmic BazWT/S180A/S180D::GFP signal during pI division at different locations of the cortex. Statistical test: two-tailed Mann–Whitney U test. Box plot: cross: maximal and/or minimal outliers (beyond 1.5×interquartile range); grey circle: average; red dots: individual measurements; centre line, median; box limits, upper and lower quartiles; whiskers, 1.5×interquartile range. (E) Orthogonal view of pIIa division at metaphase in cases where BazWT/S180A/S180D::GFP is not expressed in the pIIb lineage. Apical is up and anterior is left. Scale bar: 5 µm. Boxed areas: cortical areas measured. (E’) Cortical/cytoplasmicBazWT/S180A/S180D::GFP signal during pIIa division at different locations of the cortex. Statistical test: two-tailed Mann–Whitney U test. Box plot: cross: maximal and/or minimal outliers (beyond 1.5×interquartile range); grey circle: average; red dots: individual measurements; centre line, median; box limits, upper and lower quartiles; whiskers, 1.5×interquartile range.
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 at the pIIb/pIIa interface. Arrowheads: Baz-positive cortical patch at the pIIIb/pIIa interface during pIIa division. (B) SOP lineage from pI to pIIa division in a case where Baz::GFP (green) is only expressed in the pIIb lineage. Bottom panels: orthogonal view and schematic depiction were Baz is expressed in the lineage. Arrow: intense Baz-positive cortical patch anterior to pIIa during its division. (C) SOP lineage from pI to pIIa division in a case where Baz::GFP (green) is only expressed in the pIIa lineage. Bottom panels: Orthogonal view and schematic depiction of where Baz is expressed in the lineage. (D) Proportions of the different cases of mosaic Baz::GFP expression within the SOP lineage (n=108 SOP clusters from 4 BazWT nota, 3 BazS180A nota and 3 BazS180D nota, 2 independent experiments). (E) pIIb interphase in a case where Baz::GFP (green) is only expressed in the pIIb lineage. Arrows: Baz-positive punctae at the pIIb/pIIa lateral interface. (F) pIIa interphase in a case where Baz::GFP (green) is only expressed in the pIIa lineage. Arrows: Baz-positive punctae at the pIIb/pIIa lateral interface. (G) Contribution of pIIb/pIIIb and pIIa to cortical Baz pools at their interface. Arrows: puncta present in both pIIa and pIIb. Arrowhead: cortical patch during pIIa division, exclusively present in pIIa. (H) pIIb and pIIIb divisions when Baz::GFP is expressed in both pIIb and pIIa (left panels) or exclusively in pIIb (right panels). Bottom panels: orthogonal view. Dashed line: pIIb division axis. (H’) Quantification of the angle ɑ between the pIIb division axis and the plane of the epithelium when Baz::GFP is expressed or not in pIIa. Statistical test: two-tailed Z score calculation of population proportions. (I) pIIb tends to divide perpendicularly to the plane of the epithelium when Baz::GFP (green) is expressed in pIIa, and within the plane of the epithelium when Baz::GFP is not expressed in pIIa. Cases pooled from 4 BazWT nota, 3 BazS180A nota and 3 BazS180D nota, 2 independent experiments.
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 the absence or presence of CDK1/CyclinB. Orange bars: residues found phosphorylated only in the presence of CDK1/CyclinB. Solid bars: unambiguous position. Dashed bars: ambiguous position. Positions between parenthesis: residues found phosphorylated in only one of the two repeats. (B) Western blot showing phosphorylation of a purified GST-Baz2-320 fragment on Serine180 following incubation with human CDK1/CyclinB. Left panel: Baz-pS180 antibody (Supplementary file 1). Right panel: reprobing with an anti-GST antibody. (C) Alignment of the Baz sequence around S180 with human PARD3. Orange brackets: consensus CDK phosphosites. The western blots were repeated once yielding the same result.
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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 |
|
| 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
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A CDK1 phosphorylation site on Drosophila PAR-3 regulates neuroblast polarisation and sensory organ formation
eLife 13:e97902.
https://doi.org/10.7554/eLife.97902
