Evi5 promotes collective cell migration through its Rab-GAP activity

Abstract

Membrane trafficking has well-defined roles during cell migration. However, its regulation is poorly characterized. In this paper, we describe the first screen for putative Rab-GTPase-activating proteins (GAPs) during collective cell migration of Drosophila melanogaster border cells (BCs), identify the uncharacterized Drosophila protein Evi5 as an essential membrane trafficking regulator, and describe the molecular mechanism by which Evi5 regulates BC migration. Evi5 requires its Rab-GAP activity to fulfill its functions during migration and acts as a GAP protein for Rab11. Both loss and gain of Evi5 function blocked BC migration by disrupting the Rab11-dependent polarization of active guidance receptors. Altogether, our findings deepen our understanding of the molecular machinery regulating endocytosis and subsequently cell signaling during migration.

Figure 1.

Rescue experiments demonstrate that CG11727 requires its GAP activity in vivo. (A) Schematic representation of Drosophila egg chambers of stage 9 and 10. Red represents the actin staining, blue represents the nucleus, and green represents the BCs. BCs migrate from the anterior tip toward the posterior oocyte, left to the right in all images. Arrows point the BC clusters. Asterisks mark the oocytes. (B) Schematic representation of the Evi5 gene and its human orthologues EVI5 and EVI5-like. The sequence similarity in the TBC domain between Evi5 and its human orthologue and the proposed targets of EVI5 and EVI5-like are depicted. The position of the catalytic arginine residue in Evi5 mutated for an alanine is illustrated. (C–F) Representative examples of BC migration in a stage 10 egg chamber under the indicated conditions using the C306-Gal4 driver. Control (C) or RNAi(Evi5) (D) is represented. Representative examples of BCs expressing RNAi(Evi5) and in which Evi5 was reintroduced by expression of Evi5-mcherry (E) or Evi5^RA-mcherry (F). BCs are stained with phalloidin (red) and DAPI (blue). Arrows point to BC clusters. Bar, 20 µm. (G) The percentage of migration is expressed as the migration index (M.I.) and completion index (C.I.) for the indicated conditions above (35 < n < 161). Error bars are standard error of the mean.

Figure 2.

Evi5 acts as a Rab11-GAP in vitro. (A–H) Representative images of S2 cells transfected with Evi5-mcherry and the indicated GFP- or YFP- tagged form of Rab. Rab proteins are either expressed in their wild-type (WT) or CA form. A grayscale image of both the green and red channel is shown for every image. The insets show higher magnifications of the regions marked by dashed line squares. Arrows point to structures where Evi5 and Rab11 colocalize. (I) S2 cells were cotransfected with the wild-type, DN, or CA forms of GST-Rab11 together with the indicated GFP constructs. Pull-downs (Pd) using glutathione beads were performed on lysates. Proteins bound to GST-Rab11 were detected by Western blotting (WB). (J) YFP-Rab11^CA was coexpressed with Evi5^RA-mcherry in S2 cells. Insets show higher magnification. (K) Effector pull-down assays performed with lysates from S2 cells cotransfected with GST-Rab11^WT (left) or GST-Rab11^CA (right) together with GFP-Rip11 with or without HA-Evi5. Pull-down of GST-Rab11 and protein analysis were performed as in I. (L) Quantification of the total pulled down GFP-Rip11 normalized to the control on three (GST-Rab11^CA) or four (GST-Rab11^WT) independent experiments. (***, P < 0.05; t test). Ctl, control. Error bars are standard error of the mean. Bars, 5 µm.

Figure 3.

Evi5 genetically interacts with Rab11. (A–C) Representative examples of egg chambers expressing Evi5-mcherry (A), Evi5^RA-mcherry (B), and Rab11^CA using the slbo-Gal4 driver (the arrowheads indicate BCs). (D) M.I. and C.I. for the indicated conditions (51 < n < 161). The dotted line serves as a visual separator between conditions in which Rab11 should be preferential in an inactive (left) and active (right) conformation. (E–G) Representative images of c306-Gal4, Rab11^ex1/+ (E), c306-Gal4, Evi5-mcherry/+ (F), and c306-Gal4, Rab11^ex1/ +, Evi5-mcherry (G) egg chambers. (H) M.I. for the indicated conditions. The red bar represents the difference between the expected indexes if the phenotype were only additive (46 < n < 188). (I) Percentage of BCs presented in H having completed 0, 25, 50, 75, and 100% (standard errors of the mean ≤ 4.3). Error bars are standard error of the mean.

Figure 4.

Evi5 regulates Rab11 during BC migration. (A–F) Representative images showing the distribution of GFP-Sec15 at the onset of migration (stage 9) for the indicated conditions. The dotted lines outline BC clusters as determined by the GFP signal. The insets show a higher magnification of the regions marked by dashed line squares. Bar, 5 µm. (G and H) Computational analysis of the conditions (A–F) as in Fig. S1 F (504 < n vesicles < 1,682; ***, P < 0.05; KS test and rank sum test; Fig. S1 G). Green crosses indicate the medians of the thresholded distribution.

Figure 5.

Evi5 is necessary to properly localize active RTKs at the leading edge of BCs. (A–D) Representative images showing the distribution of pTyr at the onset of the migration process in stage 9 egg chambers in the indicated conditions. Nuclei are stained with DAPI (blue). A grayscale image of the red channel is shown for every image. Dashed lines outline the BC cluster in the colored image. The arrows point to regions with a high pTyr signal. Bar, 5 µm. (E) Quantification of pTyr fluorescence ratio in the posterior half of the cluster (F(P)) to the anterior half (F(A)) at the onset of migration for the indicated conditions. (10 < n < 17; ***, P < 0.005; t test). Error bars are standard deviations. (F) Schematic representation of the endocytic cycle regulating the polarization of RTKs during BC migration. Vesicle trafficking steps illustrated in green and in blue are regulated by Rab5 and Rab11, respectively. Moreover, Evi5, the Rab11-GAP protein identified in this study as a novel regulator of BC migration, is shown in red. RN-tre is shown as the potential Rab5-GAP involved in BC, as it was found in RNAi screen as necessary for BC migration, and its mammalian orthologue is known to act on Rab5. E.E., early endosome; R.E., recycling endosome.