Negative feedback couples Hippo pathway activation with Kibra degradation independent of Yorkie-mediated transcription
- Department of Molecular Genetics and Cell Biology, The University of Chicago, United States
- Committee on Development, Regeneration and Stem Cell Biology, The University of Chicago, United States
Figures
Figure 1
Transcriptional feedback alone does not explain Kibra (Kib) upregulation in Mer clones.
(A–G’’’) All tissues shown are living late third instar wing imaginal discs expressing the indicated fluorescent proteins. (A–C) Endogenous Kib::GFP in ex (A and A’) or Mer (B and B’) somatic mosaic …
Figure 2 with 2 supplements
The Hippo (Hpo) pathway regulates Kibra (Kib) levels independently of Yorkie (Yki)-mediated transcription.
(A) A cartoon of the DNA construct used to generate the Ubi>Kib-GFP transgenic fly line. (B–E) Depletion of Hpo pathway components Mer, Sav, Hpo, and Wts by RNAi in the posterior compartment of the …
Figure 2—figure supplement 1
The Hippo pathway regulates Kibra (Kib) levels independently of Yorkie (Yki) transcriptional activity.
(A) Similar to endogenous Kib:GFP (left), Ubi>Kib-GFP (right) accumulates both at the junctional (white arrows) and apical medial cortex (red arrows). Scale bars=5 μm. (B) Size comparison of adult …
Figure 2—figure supplement 2
The Hippo (Hpo) pathway regulates Kibra (Kib) levels independently of Ex.
(A–D) Ex levels are elevated upon Hpo pathway inactivation, with a particularly strong increase upon Hpo or Wts depletion (C and D, respectively). (E) Single sd (GFP-) or hpo (RFP-) somatic mosaic …
Figure 3 with 1 supplement
Slimb regulates Kibra (Kib) abundance via a consensus degron.
(A–A’) Depletion of Slimb in the dorsal compartment of the wing imaginal disc results in increased Kib-GFP levels both apically (A) and basally (A’). (B) Alignment of the fly, mouse, and human Kib …
Figure 3—figure supplement 1
Effect of different E3 ubiquitin ligases involved in the Hippo (Hpo) pathway on Kibra (Kib) levels.
(A–C) Depletion of SCFSlimb E3 ubiquitin ligase components Slimb (A), Cul1 (B), or SkpA (C) in the dorsal compartment of the wing imaginal disc results in increased Ubi>Kib-GFP levels. (D) Depletion …
Figure 4
The Hippo pathway regulates Kibra (Kib) abundance via a putative degron motif.
(A–F’) Mer somatic mosaic clones in wing discs expressing either UASp-Kib-GFP (A-C') or UASp-KibS677A-GFP (D–F’) with the nub>Gal4 driver. Note that wild-type Kib-GFP is significantly elevated in Mer…
Figure 5 with 1 supplement
Kibra (Kib) abundance is regulated independently of Ex.
(A–C’) Depletion of Ex (A and A’), Crumbs (Crb; B and B’), or both Ex and Crb (C and C’) in the posterior wing imaginal disc does not affect Ubi>Kib-GFP abundance. Yellow arrows indicate the …
Figure 5—figure supplement 1
The Hippo (Hpo) pathway controls Kibra (Kib) abundance in a tightly compartmentalized manner.
(A–G) Kib abundance is regulated only by a subset of Hpo pathway components. (H–H’) Validation of RNAi lines against ft (H) and ds (H’) used to deplete these components in (B) and (C), respectively. …
Figure 6 with 2 supplements
The WW domains of Kibra (Kib) are required for Hippo (Hpo) pathway- and Slimb-mediated degradation.
(A) Diagram of Kib truncations generated for this study. (B) Widefield fluorescence images of wing imaginal discs expressing wild-type and WW-domain truncations of Kib-GFP expressed under the …
Figure 6—figure supplement 1
The role of WW domains in Hippo pathway-mediated Kibra (Kib) degradation.
(A–I’) Effect of Hpo depletion in the posterior compartment of the wing imaginal disc on different Kib truncations. Deletion of the WW domains, individually (G–H’) or together (I and I’) stabilizes …
Figure 6—figure supplement 2
Complex formation and Kibra (Kib) degradation.
(A–D) Slimb forms a complex with wild-type Kib and KibΔWW1&2 (A), Mer1-600 (B), Hpo (C), and Wts (D). (E–F) Co-IP of wild-type Kib or KibΔWW1&2 with Mer (E) or Wts (F). All experiments were …
Figure 7 with 1 supplement
Kibra (Kib) degradation is patterned by mechanical tension in the wing pouch to control proportional growth.
(A–B’) Grayscale images of the wing pouch, which produces the adult wing blade, expressing UASp-Kib-GFP (A) or UASp-KibS677A-GFP (B) at identical genomic locations under the nub>Gal4 driver. …
Figure 7—figure supplement 1
Kibra (Kib) degradation is not uniform across the pouch region of the wing imaginal disc.
(A) A grayscale and heatmap image of a wing imaginal disc expressing endogenous Kib::GFP. (B–B’) Additional examples of wing discs expressing Kib-GFP or KibS677A-GFP under UASp control using the nub>…
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Drosophila melanogaster) | kibra | DOI: 10.1016/j.devcel.2009.12.012 DOI: 10.1016/j.devcel.2009.12.011 DOI: 10.1016/j.devcel.2009.12.013 | FLYB: FBgn0262127 | |
| Genetic reagent (D. melanogaster) | Kib::GFP | DOI: 10.1016/j.devcel.2017.02.004 | ||
| Genetic reagent (D. melanogaster) | Mer4 19AFRT | LaJeunesse et al., 1998 | ||
| Genetic reagent (D. melanogaster) | exe1 40AFRT | PMID: 8269855 | ||
| Genetic reagent (D. melanogaster) | 19AFRT sd47M | Wu et al., 2008 | ||
| Genetic reagent (D. melanogaster) | hpoBF33 42DFRT | Jia et al., 2003 | ||
| Genetic reagent (D. melanogaster) | ban3-GFP | DOI: 10.1242/dev.070367 | ||
| Genetic reagent (D. melanogaster) | UAS-Mer RNAi | DOI: 10.1016/j.devcel.2017.02.004 | ||
| Genetic reagent (D. melanogaster) | UAS-sav RNAi | Bloomington Drosophila Stock Center | BL 28006 | |
| Genetic reagent (D. melanogaster) | UAS-hpo RNAi | Vienna Drosophila Resource Center | VDRC 104169 | |
| Genetic reagent (D. melanogaster) | UAS-wts RNAi | Vienna Drosophila Resource Center | VDRC 106174 | |
| Genetic reagent (D. melanogaster) | UAS-ex RNAi | Vienna Drosophila Resource Center | VDRC 109281 | |
| Genetic reagent (D. melanogaster) | UAS-crb RNAi | Vienna Drosophila Resource Center | VDRC 39177 | |
| Genetic reagent (D. melanogaster) | UAS-yki RNAi (III) | Vienna Drosophila Resource Center | VDRC 40497 | |
| Genetic reagent (D. melanogaster) | UAS-slimb RNAi | Bloomington Drosophila Stock Center | BL 33898 | |
| Genetic reagent (D. melanogaster) | UAS-Cul1 RNAi | Bloomington Drosophila Stock Center | BL 29520 | |
| Genetic reagent (D. melanogaster) | UAS-SkpA RNAi | Bloomington Drosophila Stock Center | BL 32870 | |
| Genetic reagent (D. melanogaster) | UAS-mahj RNAi | Bloomington Drosophila Stock Center | BL 34912 | |
| Genetic reagent (D. melanogaster) | UAS-Nedd4 RNAi | Bloomington Drosophila Stock Center | BL 34741 | |
| Genetic reagent (D. melanogaster) | UAS-POSH RNAi | Bloomington Drosophila Stock Center | BL 64569 | |
| Genetic reagent (D. melanogaster) | UAS-POSH | Bloomington Drosophila Stock Center | BL 58990 | |
| Genetic reagent (D. melanogaster) | UAS-Su(dx) RNAi | Bloomington Drosophila Stock Center | BL 67012 | |
| Genetic reagent (D. melanogaster) | UAS-Herc4 | DOI: 10.1371/journal.pone.0131113 | ||
| Genetic reagent (D. melanogaster) | UAS-Smurf RNAi | Bloomington Drosophila Stock Center | BL 40905 | |
| Genetic reagent (D. melanogaster) | UAS-Fbxl7 RNAi | Vienna Drosophila Resource Center | VDRC 108628 | |
| Genetic reagent (D. melanogaster) | UAS-ft RNAi | Bloomington Drosophila Stock Center | BL 34970 | |
| Genetic reagent (D. melanogaster) | UAS-ds RNAi | Vienna Drosophila Resource Center | VDRC 36219 | |
| Genetic reagent (D. melanogaster) | UAS-dachs-V5 | DOI: 10.1242/dev.02427 | ||
| Genetic reagent (D. melanogaster) | UAS-Tao1 RNAi | Vienna Drosophila Resource Center | VDRC 17432 | Previously used in DOI: 10.1016/j.devcel.2011.08.028 |
| Genetic reagent (D. melanogaster) | UAS-mats RNAi | Bloomington Drosophila Stock Center | BL 34959 | |
| Genetic reagent (D. melanogaster) | UAS-Pez RNAi | Bloomington Drosophila Stock Center | BL 33918 | |
| Genetic reagent (D. melanogaster) | Ey>Flp 19AFRT Ubi-GFP; Ubi-RFP 42DFRT | DOI: 10.1016/j.devcel.2013.04.021 | ||
| Genetic reagent (D. melanogaster) | Ft-GFP | VDRC 318477 | ||
| Genetic reagent (D. melanogaster) | Ds:GFP | Brittle et al., 2012 | ||
| Genetic reagent (D. melanogaster) | Ubi-Kib-GFP-FLAG 86Fb | This paper | See Materials and methods section | |
| Genetic reagent (D. melanogaster) | UASp-Kib-GFP-FLAG 86Fb | This paper | See Materials and methods section | |
| Genetic reagent (D. melanogaster) | UASp-KibS677A-GFP-FLAG 86Fb (this study) | This paper | See Materials and methods section | |
| Genetic reagent (D. melanogaster) | Ubi-Kib-GFP-FLAG VK37 | This paper | See Materials and methods section | |
| Genetic reagent (D. melanogaster) | Ubi-KibΔWW1-GFP-FLAG VK37 | This paper | See Materials and methods section | |
| Genetic reagent (D. melanogaster) | Ubi-KibΔWW2-GFP-FLAG VK37 | This paper | See Materials and methods section | |
| Genetic reagent (D. melanogaster) | Ubi-KibΔWW1 and 2-GFP-FLAG VK37 | This paper | See Materials and methods section | |
| Genetic reagent (D. melanogaster) | Ubi-Kib1-857-GFP-FLAG VK37 | This paper | See Materials and methods section | |
| Genetic reagent (D. melanogaster) | Ubi-Kib484-1288-GFP-FLAG VK37 | This paper | See Materials and methods section | |
| Genetic reagent (D. melanogaster) | Ubi-Kib858-1288-GFP-FLAG VK37 | This paper | See Materials and methods section | |
| Genetic reagent (D. melanogaster) | Ubi-KibΔCC1-GFP-FLAG VK37 | This paper | See Materials and methods section | |
| Genetic reagent (D. melanogaster) | Ubi-KibΔCC2-GFP-FLAG VK37 | This paper | See Materials and methods section | |
| Antibody | anti-Ex (Guinea pig polyclonal) | DOI: 10.1016/j.cub.2006.02.063 | RRID:AB_2568722 | Tissue staining (1:5000) |
| Antibody | anti-FLAG (Mouse monoclonal) | Sigma Aldrich | Cat#F1804; RRID:AB_262044 | IB (1:20,000) |
| Antibody | anti-Sd (Guinea pig polyclonal) | Guss et al., 2013 | RRID:AB2567874 | Tissue staining (1:1000) |
| Antibody | anti-GFP (Guinea pig polyclonal) | DOI: 10.1091/mbc.E19-07-0387 | NA | IP (1:1250) |
| Antibody | anti-GFP (Rabbit polyclonal) | Michael Glotzer (University of Chicago) | NA | IB (1:5000) |
| Antibody | anti-Hpo (mouse polyclonal) | DOI: 10.1016/j.devcel.2017.02.004 | NA | IB (1:5000) |
| Antibody | anti-HA (Rabbit polyclonal) | Santa Cruz | Cat#sc-805; RRID:AB_631618 | IB (1:5000) |
| Antibody | anti-Myc 9B11 (Mouse monoclonal) | Cell Signaling | Product #2276 | IP (1:1000) IB (1:40,000) |
| Antibody | anti-V5 (Mouse monoclonal) | GenScript | Cat# A01724-100 | IB (1:2500) |
| Antibody | anti-alpha tubulin (Mouse monoclonal) | Sigma Aldrich | Cat# T 9026 | IB (1:2500) |
| Cell line (D. melanogaster) | S2-DGRC | Cherbas Lab, Indiana University | RRID:CVCL_TZ72 | https://dgrc.bio.indiana.edu/product/View?product=6 |
Additional files
-
Supplementary file 1
Primers used in this study with the corresponding sequences.
Note the highlighted G residue in KibS677A For primer corresponds to the substitution that will result in the mutation of serine-677 to alanine.
- https://cdn.elifesciences.org/articles/62326/elife-62326-supp1-v2.docx
-
Transparent reporting form
- https://cdn.elifesciences.org/articles/62326/elife-62326-transrepform-v2.docx
