Overexpressed Traf4 affects Adherens Junctions and promotes JNK activity

A. Schematic of Traf4-PA structure with N-terminal zinc finger domains (ZnF - yellow boxes), and C-terminal MATH domain (orange box). The C-terminal GFP tag is represented in green. B-C. Staining of 3rd instar wing imaginal disc with overexpressed Traf4-PA:GFP in the posterior compartment showing the Traf4-PA:GFP overexpression domain (GFP, green in B&C, white in B’), the Adherens Junctions components Armadillo (Arm, red in B, white in B’’) and E-Cadherin (E-Cad, blue in B&C, white in B’’’), and cleaved Caspase 3 (Cas3, red in C, white in C’). D. Staining of 3rd instar wing imaginal disc in the puc-LacZ/+ genetic background with overexpressed Traf4-PA:GFP in the posterior compartment showing Traf4-PA:GFP (GFP, green in D), E-Cadherin (blue in D), and JNK activation (β-gal, red in D, white in D’). E-G. Female adult wings of the indicated genotypes showing the smaller posterior compartment after Traf4-RA overexpression (E&F) and the loss of posterior wing disc material in the puc-LacZ/+ background (G).

Overexpressed Traf4 promotes cell delamination

A-D. Staining of 3rd instar wing imaginal disc overexpressing either GFP (A&B) or Traf4-RA:GFP (C&D) and showing the overexpression domain (GFP, green in A-D, white in A’-D’), E-Cadherin (blue in A-D), and cleaved Caspase 3 (Cas3, red in A-D, white in A’’-D’’). Discs are shown along the xy axis in A&C and along the z axis in B&D. Note the accumulation of GFP+/Cas3+ cells on the basal side of the discs (yellow arrow, opposite to E-Cadherin) after Traf4-RA overexpression at 29°C.

Traf4-induced cell delamination requires caspase activity

A-D. Staining of 3rd instar wing imaginal disc overexpressing Traf4-RA:GFP at 29°C either with GFP (A&B) or with the caspase inhibitor P35 (C&D) and showing the overexpression domain (GFP, green in A-D, white in A’-D’), E-Cadherin (blue in A-D), and cleaved Caspase 3 (Cas3, red in A-D, white in A’’-D’’). Discs are shown along the xy axis in A&C and along the z axis in B&D. E-H. Staining of 3rd instar wing imaginal disc overexpressing Traf4-RA:GFP at 25°C either in wild type background (E&F) or with one mutant copy for armadillo (arm2/+; G&H) and showing the overexpression domain (GFP, green in E-H, white in E’-H’), E-Cadherin (blue in E-H), and cleaved Caspase 3 (Cas3, red in E-H, white in E’’-H’’). Discs are shown along the xy axis in E&G and along the z axis in F&H.

E(spl)m4 and E(spl)m2 small proteins interact with Traf4

A. Yeast two-hybrid assay using Traf4-RA as a bait and the different Bearded proteins as preys. Upper panel: selection of dicaryonic yeast cells containing both bait and prey vectors on media lacking leucine and tryptophan (-leu -trp). Lower panel: interaction assay through adenosine auxotrophy rescue by growing the dicaryons on media lacking leucin, tryptophan, and adenosine (- leu -trp -ade). Empty vectors are used as negative controls testing for potential self-activation of either the baits or the preys. Here the Traf4-RA prey vector is self-activating. B-I. Predicted structures and molecular dynamics simulations (MDS) of Traf4-E(spi)-m2 and Traf4-E(spi)-m4 complexes B-C. Alphafold3 (AF3) structural model of Traf4-E(spl)-m2 (B) and Traf4-E(spl)-m4 (C), colored using the standard AF3 confidence (pLDDT) color scale (blue = very high, cyan = confident, yellow = low). D-E. Superimposition of Traf4-E(spl)-m2 (D) and Traf4-E(spl)-m4 (E) structural models at the beginning and at t=1µs of the MDS trajectory. The starting model is colored in dark grey, while the t=1µs snapshot is colored by chain (green for Traf4 MATH domain and orange and magenta for E(spl)-m2 and E(spl)-m4, respectively). F. Interface buried surface area of the Traf4-E(spl)-m2 (black curve) and Traf4-E(spl)-m4 complexes (red curve) as a function of simulation time. G. Interface root mean square deviation (iRMSD) of the Traf4-E(spl)-m2 (black curve) and Traf4-E(spl)-m4 complexes (red curve) as a function of simulation time. H-I. Top (H) and side (I) views of an AF3-predicted structural model of trimeric Traf4 superimposed with the predicted Traf4-E(spl)-m4 complex, highlighting the steric incompatibility of the two interactions. The Traf4 trimer and E(spl)-m4-bound MATH domain are colored using the standard AF3 color scale, and E(spl)-m4 is colored in magenta.

E(spl)m4 suppresses the effects of Traf4 overexpression

>A-B. Staining of 3rd instar wing imaginal disc in the puc-LacZ/+ genetic background and co-overexpressing Traf4-PA:GFP in the posterior compartment either with GFP (A) or E(spl)m4 (B). Discs show the Traf4-PA:GFP overexpression domain (GFP, green in A&B), JNK activation (β-gal, red in A-B, white in A’-B’), and E-Cadherin (E-Cad, blue in A-B, white in A’’-B’’). C-E. Female adult wings after Traf4-RA overexpression in the puc-LacZ/+ background either with GFP (C), E(spl)m4 (D), or Tom (E).

Overexpressed E(spl)m4 mimics some Traf4 loss of function phenotypes

A. Upper panel: Traf4 genetic locus adapted from the JBrowse from Flybase and showing the location of the P element EP578 used from generating new Traf4 alleles, and the sequences targeted by the Traf4-RNAi KK107398. Lower panel: diagram showing the extent of the Traf4ex111 and Traf4L2 deletion mutants; interrupted lines represents the lacking DNA; black lines represent the DNA still present; orange lines represent regions of uncertainty due to the PCR-based mapping. Traf4L2 mapping is based on published data. B-G. Wing margin bristles in the indicated genotypes showing defects in spacing (red stars). (B) Low magnification image of control wing. (C-G) High magnification images corresponding to the region highlighted by the yellow box in B. H-K. Staining of 3rd instar wing imaginal disc with mosaic tissues to assay for cell competition in which clones of GFP marked clones of mutant tissues were generated and marked positively by GFP. Clones were either wild-type (no cell competition, H), lacking one copy of RpS3 (RpS3-/+ loser clones being eliminated, I), or Rps3-/+ and overexpressing either an RNAi for Traf4 (J) or E(spl)m4 (K). Discs show nuclei (DAPI, blue), the extent of clone growth (GFP, green), and apoptotic cells (Dcp-1, red). L. Quantification of the clone area from (H-K) shown as total clone area in the wing pouch normalized by total wing pouch area. n = 6-15 discs. Error-bars show standard error of the mean (sem). One-way ANOVA statistical test. **** p < 0.0001, ** p < 0.01, * p < 0.05 M. Model for Traf4 inhibition by E(spl)m4.

BDSC: Bloomington Drosophila Stock Center; VDRC: Vienna Drosophila Resource Center

Overexpressed Traf4 and apico-basal polarity markers (related to Fig. 1).

A-B. Staining of 3rd instar wing imaginal disc with overexpressed Traf4-PA:GFP in the posterior compartment showing the Traf4-PA:GFP overexpression domain (GFP, green in A&B), the subapical compartment marker aPKC (red in A, white in A’), the Septate Junctions marker Discs Large (Dlg, red in B, white in B’) and E-Cadherin (E-Cad, blue in A&B, white in A’’&B’’).

Interaction between Traf4 and E(spl)m2 and E(spl)m4 (related to Fig. 4).

A. Alignment of the different Bearded proteins using the Uniprot portal and highlighting similarity. The red boxes represent previously identified motifs, where motif 1 corresponds to an amphipathic alpha helix, and motif 2 is the domain mediating the interaction between Brd proteins (except E(spl)m2) and the E3-Ubiquitin Ligase Neuralized. This study identifies the motif 3 as the interface with Traf4. B. Comparison of Traf4-E(spl)m4 predicted interaction interface with interfaces observed on other TRAF family members complexed with various proteins and peptides. Pdb codes 1QSC, 1CA9, 1F3V, 4GHU and 1KZZ were used.

E(spl)m4 suppresses Traf4-induced cell delamination (related to Fig. 5).

A-D. Staining of 3rd instar wing imaginal disc overexpressing Traf4-RA:GFP at 29°C either with GFP (A&B) or with E(spl)m4 (C&D) and showing the overexpression domain (GFP, green in A-D, white in A’-D’), E-Cadherin (blue in A-D), and cleaved Caspase 3 (Cas3, red in A-D, white in A’’-D’’). Discs are shown along the xy axis in A&C and along the z axis in B&D.