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Directional Delta and Notch trafficking in Sara endosomes during asymmetric cell division

Abstract

Endocytosis has a crucial role during Notch signalling after the asymmetric division of fly sensory organ precursors (SOPs): directional signalling is mediated by differential endocytosis of the ligand Delta and the Notch effector Sanpodo in one of the SOP daughters, pIIb1,2,3. Here we show a new mechanism of directional signalling on the basis of the trafficking of Delta and Notch molecules already internalized in the SOP and subsequently targeted to the other daughter cell, pIIa. Internalized Delta and Notch traffic to an endosome marked by the protein Sara4,5. During SOP mitosis, Sara endosomes containing Notch and Delta move to the central spindle and then to pIIa. Subsequently, in pIIa (but not in pIIb) Notch appears cleaved in Sara endosomes in a γ-secretase- and Delta internalization-dependent manner, indicating that the release of the intracellular Notch tail to activate Notch target genes has occurred. We thus uncover a new mechanism to bias signalling even before asymmetric endocytosis of Sanpodo and Delta takes place in the daughter cells: already during SOP mitosis, asymmetric targeting of Delta and Notch-containing Sara endosomes will increase Notch signalling in pIIa and decrease it in pIIb.

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Figure 1: Directional transport of internalized Delta/Notch during asymmetric cell division.
Figure 2: Endosome dynamics during asymmetric cell division.
Figure 3: Mistargeting of Sara endosomes causes cell fate transformation.
Figure 4: Cleaved Notch in Sara endosomes.

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Acknowledgements

We thank S. Bray, P. Bryant, D. Glover, C. González, E. Lai, C. Micchelli, M. Muskavitch, M. O’Connor, J. Posakony, F. Schweisguth, J. Skeath and A. Wodarz for providing reagents, and the M.G.-G. laboratory members, A. Martinez-Arias and F. Schweisguth for critically reading the manuscript. We thank S. Sigrist and W. Fouquet for providing us the opportunity to perform stimulated emission depletion (STED) microscopy. We thank A. Schwabedissen, D. Backash, C. Alliod and A. Beguin for technical assistance. M.F. thanks M. P. Euzenot for support. M.F. has benefited from EMBO and Human Frontier Science Program (HFSP) long-term postdoctoral fellowships and F.C. from a Fondation pour la recherche médicale (FRM) postdoctoral fellowship. This work was supported by the Max Planck Society, Volkswagen, an FP6 Strep (ONCASYM), the Swiss National Science Foundation (SNF), SystemsX (LipidX) and HFSP.

Author Contributions F.C. conducted the experiments depicted in Figs 1e, f, 2a–i, Supplementary Figs 1c, d, 6, 7, 12, 13a, b and 21, and Supplementary Movies 1, 2, 4–7 and 11. M.F. developed the live antibody uptake assay, conducted the experiments depicted in Figs 1a–d, g, h, 2j–l, 3 and 4, Supplementary Figs 1a, b, e–h, 2–5, 8–11, 13c, d and 14–20, and Supplementary Movies 3 and 8–10, and contributed to the writing of the manuscript. J.A.K. provided reagents for the study before their publication. M.G.-G. planned the project, analysed the experiments together with F.C. and M.F. and wrote the manuscript.

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Correspondence to M. Fürthauer or M. González-Gaitán.

Supplementary information

Supplementary Information

This file contains Supplementary Notes, Supplementary Methods, Supplementary References, Supplementary Figures 1-21 with Legends and Supplementary Movie Legends 1-11. (PDF 5983 kb)

Supplementary Movie 1

This movie shows Segregation of internalized Delta during SOP division (see file s1 for full legend). (MOV 4976 kb)

Supplementary Movie 2

This movie shows Segregation of internalized Notch during SOP division (see file s1 for full legend). (MOV 7154 kb)

Supplementary Movie 3

This movie shows that Sara endosomes transport internalized delta to the posterior daughter cell (see file S1 for full legend). (MOV 7177 kb)

Supplementary Movie 4

This movie shows Segregation of SaraGFP-labeled endosomes (green) during asymmetric SOP division (see file s1 for full legend). (MOV 203 kb)

Supplementary Movie 5

This movie shoes Segregation of SaraGFP endosomes (green) in symmetrically dividing epidermal cells (see file s1 for full legend). (MOV 2478 kb)

Supplementary Movie 6

This movie shows Segregation of 2xFYVE-GFP endosomes (green) during asymmetric SOP division (see file s1 for full legend). (MOV 2571 kb)

Supplementary Movie 7

This movie shows Segregation of Rab5-GFP endosomes (green) during asymmetric SOP division (see file s1 for full legend). (MOV 2597 kb)

Supplementary Movie 8

This movie shows Symmetric Segregation of SaraGFP endosomes (green) upon Lgl3A misexpression (see file s1 for full legend). (MOV 9878 kb)

Supplementary Movie 9

This movie sows Symmetric segregation of internalized Delta (green) upon Lgl3A misexpression (see file s1 for full legend). (MOV 7885 kb)

Supplementary Movie 10

This movie shows Symmetric segregation of internalized Notch (green) upon Lgl3A misexpression (see file s1 for full legend). (MOV 5908 kb)

Supplementary Movie 11

This movie shows Asymmetric segregation of SaraGFP endosomes (green) in larval brain neuroblasts (see file s1 for full legend). (MOV 3588 kb)

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Coumailleau, F., Fürthauer, M., Knoblich, J. et al. Directional Delta and Notch trafficking in Sara endosomes during asymmetric cell division. Nature 458, 1051–1055 (2009). https://doi.org/10.1038/nature07854

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