<i>cul-5</i> mutant ovarioles form aberrant egg chambers.

<p>Ovarioles from control (A) and <i>cul-5^EY21463</i> mutant (B–L) females, labelled for Vas (white) and DNA (blue). (A, B) Control (A) and many <i>cul-5</i> mutant (B) ovarioles show normal morphology. In a substantial fraction of <i>cul-5</i> mutant ovarioles (C–J), one or several egg chambers contain more than 16 germ line cells (aberrant egg chambers). (C, D) Cysts in region III of <i>cul-5</i> mutant germaria are often irregularly shaped and fail to take up a oval or round shape as wild type cysts do. (E) Frequently, several follicles containing excess germ cells are observed in a single ovariole. (F) More rarely, <i>cul-5</i> mutant egg chambers contain less than 16 germ line cells. (G) More mature aberrant egg chambers can develop relatively normally. (H) Aberrant egg chambers are also observed in a different allelic combination. (I) In some <i>cul-5</i> mutant ovarioles, individual egg chambers are not separated by a stalk, but by two layers of follicular epithelium. (J) A fraction of <i>cul-5</i> ovarioles has germaria that do not contain germ cells. (K, L) In aged flies, a fraction of ovarioles undergoes morphogenetic catastrophe and lose the highly ordered structure of normally developing ovarioles. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009048#pone-0009048-t001" target="_blank">Table 1</a> for quantification of this phenotype.</p

Follicular morphogenesis in aberrant <i>cul-5</i> mutant egg chambers.

<p>(A–H) Wild type (A–B) and <i>cul-5^EY21463</i> mutant (C–H) egg chambers were labelled for Vas (red) and FasIII (A, C–F) or EyA (B, G–H) (green in A–C, E, G or white in D, F, H). (A–C, E, G) are single confocal planes, while (D, F, H) are projections of Z-stacks spanning the entire corresponding egg chamber. (A) In wild type, FasIII accumulates in two pairs of polar cells at the anterior and posterior pole of the follicle (arrows). (B) EyA accumulates in a complementary pattern, and is excluded from the anterior and posterior polar cells while it accumulates in the nuclei of all other follicle cells. (C–D) In most aberrant egg chambers in <i>cul-5</i> mutants, only the normal two groups of polar cells are specified. (E–H) Only in small fraction of aberrant <i>cul-5</i> follicles, more than two groups of cells have upregulated FasIII (E–F) or downregulated EyA (G–H).</p

<i>cul-5</i> mutant follicles do not contain 2ⁿ germ line cells.

<p>The graph plots the number of germ cells (Y-axis) in 20 randomly chosen aberrant follicles (X-axis). The green and red lines indicate the normal 16 (green) germ cells per follicles, or multiples of 16 (red). For each follicle, we counted the number of germ line cells three times (error bars denote ±SD). The number at the bottom of the column indicates the number of Orb-positive cells (oocytes) found in that particular follicle.</p

<i>cul-5</i> phenotypes as a function of age.

<p>All numbers are percentages except for n, the number of ovarioles examined.</p>1<p>Unless otherwise indicated these phenotypes were observed in ovarioles that also contained ≥1 tumorous follicle, so they were included in the counts for that category.</p>2<p>Observed adjacent to a follicle containing >16 germ cells.</p>3<p>Observed in ovarioles without follicles containing >16 germ cells, therefore included in the category ‘others’ in the upper section.</p>4<p>Phenotypes observed at very low penetrance (<1%) include binuclear germ cells, multilayered follicle cells, or unusually long stalks.</p>5<p>This phenotype was only scored in 54/173 ovaries.</p

Germ line clusters overproliferate in the germarium of <i>cul-5</i> mutant ovarioles.

<p>(A–E) Wild type (A) and <i>cul-5^EY21463</i> mutant ovarioles (B–E) were stained for Vas (blue) and phospho-histone H3 (white). (A) In wild type ovarioles, small numbers of germ cells divide synchronously in the germarium. (B–E) In some <i>cul-5</i> mutant ovarioles, large numbers of germ line cells undergo mitosis in a germarium, while we never observe germ cell divisions in the distal germarium or in the vitellarium (B). (D–E) are projections of a Z-series taken through (C). The asterisk indicates dividing cells of somatic origin in the projections. (F) In wild type egg chambers, each oocyte (arrow) contains exactly four ring canals enriched in F-actin (labelled with Phalloidin). (G) In aberrant egg chambers in <i>cul-5</i> mutants, more than four ring canals are observed in oocytes (arrow) and nurse cells. (H–K) <i>cul-5</i> mutant germaria (J–K) feature large fusomes (labelled with mAb 1b1) spanning more than 16 cyst cells that are never observed in wild type (H–I) germaria. (H, J) are single 0.5 µm planes, while (I, K) are 3D reconstructions of corresponding Z-stacks spanning the width of the germaria. The bracket in K indicates a single polyfusome that is traceable through the entire Z-stack.</p

DUB3 Deubiquitylating Enzymes Regulate Hippo Pathway Activity by Regulating the Stability of ITCH, LATS and AMOT Proteins

<div><p>The YAP and TAZ transcriptional coactivators promote oncogenic transformation. Elevated YAP/TAZ activity has been documented in human tumors. YAP and TAZ are negatively regulated by the Hippo tumor suppressor pathway. The activity and stability of several Hippo pathway components, including YAP/TAZ, is regulated by ubiquitin mediated protein turnover and several ubiquitin ligase complexes have been implicated in human cancer. However, little is known about the deubiquitylating enzymes that counteract these ubiquitin ligases in regulation of the Hippo pathway. Here we identify the DUB3 family deubiquitylating enzymes as regulators of Hippo pathway activity. We provide evidence that DUB3 proteins regulate YAP/TAZ activity by controlling the stability of the E3 ligase ITCH, the LATS kinases and the AMOT family proteins. As a novel Hippo pathway regulator, DUB3 has the potential to act a tumor suppressor by limiting YAP activity.</p></div

DUB3 mediates ITCH, LATS1/2 and AMOT proteins to regulate Hippo activity.

<p>(A) A schematic view of DUB3-mediated regulation of Hippo signaling. DUB3 de-ubiquitylates ITCH, LATS1/2 and AMOT to promote their stability. In the presence of stabilized AMOT, ITCH promotes YAP degradation. (B) Luciferase reporter assays showing the effects of ITCH overexpression on YAP/TAZ activity. HEK293T cells were transfected to express the luciferase reporters together with a control or ITCH expression vector in the presence of a mixture of siRNAs targeting AMOT, AMOTL1 and AMOTL2, a mixture siRNAs targeting LATS1 and LATS2 or a scrambled siRNA control. Data represent the average of three independent transfection experiments ± SD. (C) Luciferase reporter assays showing the effects of DUB3 siRNAs on YAP/TAZ activity. HEK293T cells were transfected to express the luciferase reporters together with a control or DUB3 siRNA in the presence of ITCH siRNA, a mixture of siRNAs targeting ITCH and NEDD4 or a scrambled siRNA control. Data represent the average of three independent transfection experiments ± SD.</p

LATS and AMOT proteins are required for DUB3-mediated regulation of Hippo signaling.

<p>(A) Immunoblots showing the effect of DUB3 expression on LATS kinases, AMOT and YAP. HEK293T cells were transfected with a vector expressing Flag-DUB3 or a control vector together with a mixture of siRNAs targeting AMOT, AMOTL1 and AMOTL2 or a scrambled siRNA control. Blots were probed with antibodies against Flag, AMOT, AMOTL1, LATS1, LATS2 and YAP. Anti-Actin was used to control for loading. Samples were run on the same SDS-acrylamide gels with intervening lanes removed. (B) Immunoblots showing the effect of DUB3 depletion on LATS kinases, AMOT and YAP. HEK293T cells were transfected with a vector expressing Flag-DUB3 or a control vector together with a mixture of siRNAs targeting AMOT, AMOTL1 and AMOTL2 or a scrambled siRNA control. Blots were probed with antibodies against DUB3, AMOT, LATS1, LATS2 and YAP. Anti-Actin was used to control for loading. Samples were run on the same gels with intervening lanes removed. (C) Immunoblots showing the effect of DUB3 depletion on ITCH, LATS kinases and AMOT. HEK293T cells were transfected with a control or DUB3 siRNA in the presence of LATS1 and LATS2 siRNAs or a scrambled siRNA control. Blots were probed with antibodies against DUB3, ITCH, AMOT, LATS1, LATS2 and YAP. Anti-Actin was used to control for loading. (D) Luciferase reporter assays showing the effects of DUB3 siRNAs on YAP/TAZ activity. HEK293T cells were transfected to express the luciferase reporters together with a control or DUB3 siRNA in the presence of a mixture of siRNAs targeting AMOT, AMOTL1 and AMOTL2, a mixture of siRNAs targeting LATS1 and LATS2 or a scrambled siRNA control. Data represent the average of three independent transfection experiments ± SD. P values were determined using Student’s T-test (2-tailed, unequal variance).</p

DUB3 interacts with ITCH and mediates its stability.

<p>(A) Immunoblots showing the effect of DUB3 expression on ITCH. HEK293T cells were transfected with a vector expressing Flag-DUB3, its C89S DUB3 or a control vector. Blots were probed with anti-Flag, anti-ITCH, anti-NEDD4 and anti-SMURF1 antibodies. Anti-Actin was used to control for loading. (B) Immunoblots showing the effect of DUB3 siRNAs on ITCH. HEK293T cells were transfected with independent siRNAs against DUB3 or a scrambled siRNA. Blots were probed with antibodies against ITCH or Actin for loading control. (C) Immunoprecipitation assays showing interaction between DUB3 and ITCH. HEK293T cells were transfected to express Flag-tagged DUB3 and Myc-tagged ITCH as indicated. Transfected cells were treated with MG132 5μM overnight before being harvested for immunoprecipitation with anti-Flag or anti-Myc-conjugated beads. Blots were probed with anti- Flag to detect DUB3 or anti-Myc to detect ITCH. (D) Ubiquitylation assay showing the effect of DUB3 on ITCH ubiquitylation. HEK293T cells were co-transfected with a vector expressing Myc-ITCH and a vector expressing Flag-tagged DUB3, Flag-tagged DUB3 C89S or a control vector. Transfected cells were treated with MG132 5μM overnight before being harvested for immunoprecipitation with anti-Myc-or isotype IgG-conjugated beads in PLC buffer freshly supplemented with 10mM of NEM. Immunoblots were probed with antibodies against HA, Flag and Myc.</p

DUB3 regulates Hippo activity by mediating YAP turnover.

<p>(A) Luciferase reporter assays showing the effects of DUB3 shRNAs on YAP/TAZ activity. HEK293T cells were transfected to express the 8XGTIIC_luc YAP/TAZ reporter, which contains 8 TEAD binding sites to control the expression of firefly luciferase and a vector expressing CMV-Renilla luciferase to normalize for transfection efficiency, together with independent shRNA vectors to deplete DUB3 or with a control shRNA. shRNA to deplete LATS2 was used as a positive control. Data represent the average of three independent transfection experiments ± SD. P values were determined using Student’s T-test (2-tailed, unequal variance). (B) Luciferase reporter assays showing the effects of DUB3 siRNAs on YAP/TAZ activity. HEK293T cells were transfected to express the luciferase reporters together with independent siRNAs to deplete DUB3 or with a control scrambled siRNA. Data represent the average of three independent transfection experiments ± SD. P values were determined using Student’s T-test (2-tailed, unequal variance). (C) Luciferase reporter assays showing the effect of DUB3 on YAP/TAZ activity. HEK293T cells were transfected to express the luciferase reporters together with a vector expressing Flag-DUB3, the C89S mutant form of DUB3 or relevant controls vectors. shRNA to deplete LATS2 was used as a positive control. Data represent the average of three independent replicates ± SD. P values were determined using Student’s T-test (2-tailed, unequal variance). (D) Immunoblots showing the effect of DUB3 expression on YAP protein. HEK293T cells were transfected with a vector expressing Flag-DUB3, the C89S mutant form of DUB3 or a control vector. Blots were probed with anti-YAP antibody and anti-Flag. Anti-Actin was used to control for loading. (E) Immunoblots showing the effect of DUB3 depletion on the YAP expression level. HEK293T cells were transfected with independent DUB3 siRNAs or a scrambled siRNA control. Blots were probed with anti-YAP antibody and anti-Flag. Anti-Actin was used to control for loading. (F) Effect of DUB3 siRNAs on the expression of YAP transcriptional targets. HEK293T cells were transfected with DUB3 or control siRNAs. mRNA expression of DUB3, Cyr61, ANKRD1 was measured by RT-PCR. GAPDH mRNA was used for normalization and TBP was used as an additional control gene. Data represent the average of 3 independent experiments ± SD. * indicates p <0.01, compared to the relevant controls (Student’s T-test; 2-tailed, unequal variance). (G) Effect of DUB3 on cell growth. Human primary fibroblast BJ cells were engineered to express hTert, H-Ras^G12V and to deplete p53 and p16, or with the addition or YAP^S127A/S397A. BJ^{p53kd/p16kd/HRas} and BJ^{p53kd/p16kd/HRas/YAPS127A/S397A} cells were virally transduced and selected to stably express DUB3, its inactive C89S mutant form or with an empty vector as a control. Cells were counted at 24-hour intervals for a period of 72h. The assay was performed in triplicate. Data represent the average ± SD. * indicate p <0.05, compared to the relevant controls (Student’s T-test; 2-tailed, unequal variance).</p