Rab11 regulates cell-cell communication during collective cell movements

Abstract

Collective cell movements contribute to development and metastasis. The small GTPase Rac is a key regulator of actin dynamics and cell migration but the mechanisms that restrict Rac activation and localization in a group of collectively migrating cells are unknown. Here, we demonstrate that the small GTPases Rab5 and Rab11 regulate Rac activity and polarization during collective cell migration. We use photoactivatable forms of Rac to demonstrate that Rab11 acts on the entire group to ensure that Rac activity is properly restricted to the leading cell through regulation of cell-cell communication. In addition, we show that Rab11 binds to the actin cytoskeleton regulator Moesin and regulates its activation in vivo during migration. Accordingly, reducing the level of Moesin activity also affects cell-cell communication, whereas expressing active Moesin rescues loss of Rab11 function. Our model suggests that Rab11 controls the sensing of the relative levels of Rac activity in a group of cells, leading to the organization of individual cells in a coherent multicellular motile structure.

Figure 1

Rab proteins regulate actin dynamics and Rac activity and polarization. (a) Schematic representation of an egg chamber at stages 9 and 10. pTyr signalling and Rac activity are distinct events. (b) Border cell clusters expressing UAS–Lifeact GFP alone, with UAS–Rab11SN or UAS–Rab5SN at the onset of migration. The expression was driven by slbo–Gal4 at 29 °C. Scale bars, 20 μm. (c) Quantification of protrusion number in the same conditions at the onset and after 25–50% of migration of the indicated genotypes. n = 30, 19, 10 and 10 respectively for control, Rab11SN, Rab5SN and RacN17 at the onset of migration. n = 15, 27, 10 and 10 respectively for control, Rab11SN, Rab5SN and RacN17 at 25–50% of migration. *P < 0.001 (Student's t-test). (d,e) Analysis of protrusion distribution (see Supplementary Fig. S2 for details) in control and Rab11SN-expressing clusters at the onset (d) and after 25–50% (e) of migration. n = 20 clusters. (f–t) Processed FRET signal images of time-lapse series of border cells expressing UAS–Rac Fret alone or together with UAS–Rab11SN or UAS–Rab5SN at the onset of migration. The expression was driven by slbo–Gal4 at 29 °C. Scale bars, 10 μm. (u) Quantification of the total FRET index in the indicated genotypes. n = 30 for each condition. (v) Quantification of the ratio of the FRET index between the front and back of the cluster for the indicated conditions. n = 30 for each condition. *P < 0.001 (Student's t-test). (w) Heat maps representing the FRET index as a function of time at the leading edge of 10 WT and Rab11SN clusters. Error bars show s.e.m.

Figure 2

Local activation of Rac does not rescue the Rab11 loss-of-function phenotype. (a–d) Selected still images from a time-lapse movie of photoactivation of a UAS–PA-RacQ61L-expressing cluster. n = 8. (e–h) Selected still images from time-lapse movies of photoactivation of UAS–PA-RacQ61L in a UAS–Rab11SN background. n = 11. (i–l) Control experiment with UAS–PA-RacQ61L C450M insensitive to light in a UAS–Rab11SN background. n = 10. (m–p) Selected still images from time-lapse movies of photoactivation of UAS–PA-RacQ61L in a UAS–Rab5SN background. n = 8. (q–t) Control experiment with UAS–PA-RacQ61L C450M insensitive to light in a UAS–Rab5SN background. n = 9. The expression was driven by slbo–Gal4 at 29 °C. The blue circles indicate photoactivated regions. The white arrows indicate the direction of migration and the dashed blue arrows the expected direction after photoactivation. Scale bars, 20 μm. (u) Speed measurements for each genetic background presented in a–t. n = 8, 8, 11, 10, 8, 9 respectively for control PA-Rac C450M, PA-Rac control, PA-Rac C450M Rab11SN, PA-Rac Rab11SN, PA-Rac C450M Rab5SN and PA-Rac Rab5SN at the onset of migration. *P < 0.01; NS, not significant (Student's t-test). Error bars show s.e.m.

Figure 3

Local inactivation of Rac reveals a role in cell–cell communication for Rab11. (a–c) Confocal images from an experiment of UAS–PA-RacT17N photoactivation in a control cluster. n =11. (d–f) Confocal images of photoactivation experiments in a UAS–Rab11SN background. n = 13. (g–i) Confocal images of photoactivation experiments in a UAS–Rab5SN background. n = 12. The red circles indicate photoactivated regions. The white arrows indicate the direction of migration. Scale bars, 20 μm. (j) Analysis of protrusion distribution in control and UAS–Rab11SN-expressing clusters. (k) Quantification of protrusions in PA-RacT17N experiments. n 11, 13 and 12 respectively for control, Rab5SN and Rab11SN at the onset=of migration. (l) Quantification of the area of clusters in PA-RacT17N experiments. n = 11, 13 and 12 respectively for control, Rab5SN and Rab11SN at the onset of migration. The expression was driven by slbo–Gal4 at 29 °C. *P < 0.0001 (Student's t-test). Error bars show s.e.m.

Figure 4

Rab11 interacts with and controls Moesin activity. (a) Stably transfected S2 cells expressing GST, GST–Rab11SN, WT or QL and GFP–Moesin or not were lysed and submitted to GST pulldowns. Lysates and immunoprecipitates (IP) were analysed by western blotting using GFP, GST and Moesin antibodies (AB). Western blots are representative of three independent experiments. (b) Representative images showing the intensity and the distribution of pMoesin at the onset of migration in control (CD8–GFP ; n = 11), and in Rab11SN- (n = 16) and Rab5SN- (n = 10) expressing clusters. Scale bars, 10 μm. The expression was driven by the slbo–Gal4 promoter. (c) Representative images showing the intensity and the distribution of Moesin at the onset of migration in control (CD8–GFP ; n = 20), and in Rab11SN- (n = 20) and Rab5SN- (n = 10) expressing clusters. Scale bars, 10 μm. The expression was driven by the slbo–Gal4 promoter. (d) Quantification of the ratio of the mean fluorescence signal of pMoesin to the GFP mean intensity at the interface between border cells and nurse cells. n = 20, 20 and 10 respectively for control, Rab11SN and Rab5SN. (e) Quantification of the ratio of the mean fluorescence signal of Moesin at the interface between border cells (BC) and nurse cells (NC). n = 20, 20 and 10 respectively for control, Rab11SN and Rab5SN. (f) Quantification of the ratio of the mean fluorescence signal of Moesin at the interface between border cells (BC) and nurse cells (NC) on the mean fluorescence signal of Moesin at BC–BC membranes. n = 20, 20 and 10 respectively for control, Rab11SN and Rab5SN. (g) Migration and completion indices after expression of the constructs indicated. n is indicated under each genotype. *P < 0.0001; NS, not significant (Student's t-test). Error bars show s.e.m. Uncropped images of blots are shown in Supplementary Fig. S6.

Figure 5

Moesin regulates protrusion distribution, polarization of Rac activity and cell–cell communication. (a) Migration and completion indices after expression of mcherry RNAi (control) or three different RNAi lines against Moesin. The expression was driven by c306–Gal4 at 32 °C. n is indicated under each genotype *P < 0.0001 (Student's t-test). (b) Analysis of protrusion distribution in RNAi-Moesin-expressing clusters at the onset and after 25–50% of migration. n = 14 clusters. (c) UAS–Rac Fret is expressed in border cells expressing RNAi Moesin and Rac activity is visualized by FRET in living egg chambers at the onset of migration. The expression was driven by c306–Gal4 at 32 °C. Scale bars, 10 μm. (d) Quantification of the total FRET index in control and in RNAi-Moesin-expressing clusters. n = 30 in each condition. (e) Heat maps representing the FRET index as a function of time at the leading edge of 10 RNAi Moesin clusters. (f–h) Confocal images from an experiment of UAS–PA-RacT17N photoactivation in a control cluster The expression was driven by slbo–Gal4 at 32 °C. (i–k) Confocal images of a photoactivation experiment in a UAS–RNAi Moesin background at 32 °C. The red circles indicate photoactivated regions. The white arrows indicate the direction of migration. Scale bars, 20 μm. (l) Quantification of protrusions in PA-RacT17N experiments. n = 11 for each condition. *P < 0.0001 (Student's t-test). Error bars show s.e.m.