Spatial restriction of receptor tyrosine kinase activity through a polarized endocytic cycle controls border cell migration

Abstract

Border cell migration is a stereotyped migration occurring during the development of the Drosophila egg chamber. During this process, a cluster composed of six to eight follicle cells migrates between nurse cells toward the oocyte. Receptor tyrosine kinases (RTKs) are enriched at the leading edge of the follicle cells and establish the directionality of their migration. Endocytosis has been shown to play a role in the maintenance of this polarization; however, the mechanisms involved are largely unknown. In this study, we show that border cell migration requires the function of the small GTPases Rab5 and Rab11 that regulate trafficking through the early and the recycling endosome, respectively. Expression of a dominant negative form of rab11 induces a loss of the polarization of RTK activity, which correlates with a severe migration phenotype. In addition, we demonstrate that the exocyst component Sec15 is distributed in structures that are polarized during the migration process in a Rab11-dependent manner and that the down-regulation of different subunits of the exocyst also affects migration. Together, our data demonstrate a fundamental role for a plasma membrane-endosome trafficking cycle in the maintenance of active RTK at the leading edge of border cells during their migration.

Fig. 1.

Expression of dominant negative forms of Rab5 and Rab11 blocks border cell migration. (A) Representative examples of border cell migration in a control stage 10 egg chamber (Top) and when border cells express rab11^SN (Middle and Bottom). Border cells are additionally expressing CD8::GFP (green) and are stained with phalloidin (red) and DAPI (blue). The border cell clusters are indicated by arrowheads. Anterior is left and posterior is right. The percentage of migration compared with the total migration distance is indicated. (B and C) Normalized migration and completion indexes after expression of different dominant negative Rab proteins in border cells or after down-regulation of rab11 by dsRNA (26 < n < 105). Indexes were normalized to c306-Gal4 [dsRNA(rab11)]; slbo-Gal4 (YFP::rabX4^TN); or slbo-Gal4, UAS CD8::GFP.

Fig. 2.

Rab11 regulates the spatial distribution of active RTKs and genetically interacts with their downstream target Rac1. (A) Representative images showing the distribution of β-Integrin, E-Cadherin, and pTyr (red) at the onset of migration in stage 9 control egg chambers and when rab11^SN is expressed in border cells. Border cells are additionally expressing CD8::GFP (green) and are stained with DAPI (blue). A grayscale image of the red channel is shown for every image. A dashed line delimits the border cell cluster in the grayscale image. Anterior is left and posterior is right. (B) Quantification of the ratio of the mean fluorescent signal of CD8::GFP, E-Cadherin, β-Integrin, and pTyr determined in an area of the leading edge [F(P)] divided by the signal at the trailing edge [F(A), SI Materials and Methods] in control clusters (blue bars) and when rab11^SN is expressed (red bars). Error bars are SEM (13 < n < 50; ***P < 0.005, Student's t test). (C) Normalized migration indexes for the indicated conditions. Hatched bars represent the difference between the expected indexes if the phenotypes were only additive and the experimental indexes. Indexes were normalized to slbo-gal4. Error bars are SEM (three individual experiments, total number of egg chambers: 92 < n < 247).

Fig. 3.

The exocyst component Sec15 is polarized in a Rab11-dependent manner during border cell migration. (A) Representative images showing the distribution of GFP::Sec15 at the onset, in the middle, and at the end of the migration process in control egg chambers and when rab11^SN is expressed. Egg chambers were additionally stained with phalloidin (red) and DAPI (blue). A grayscale image of the green channel is shown for every image. A dashed line delimits the border cell cluster in the grayscale image. (B) Quantification of the ratio of Sec15 punctae that are distributed in the posterior half of the cluster [V#(P)] to the anterior half [V#(A)] at the onset, in the middle, and at the end of the migration process in control egg chambers (blue bars) and when rab11^SN is expressed (red bars). (6 < n < 12, error bars are SEM; ***P < 0.005, **P < 0.01, Student's t test).

Fig. 4.

The exocyst component Sec15 is necessary for border cell migration. (A) Normalized migration and completion indexes (normalized to c306-Gal4) for clusters expressing dsRNA against the indicated gene (36 < n < 146). (B) Representative images showing the distribution of pTyr (red) at the onset of the migration process in stage 9 control egg chambers and when sec15 expression is down-regulated in border cells by dsRNA. Nuclei are stained with DAPI (blue). A grayscale image of the red channel is shown for every image. A dashed line delimits the border cell cluster in the grayscale image. Anterior is left and posterior is right. (C) Quantification of pTyr fluorescence ratio (Fig. 3) in control (blue bar) and when sec15 is down-regulated in border cells (red bar). (16 < n < 18, error bars are SEM; ***P < 0.005, Student's t test).