Inhibition of Notch recycling by Numb - François Schweisguth

Apr 8, 2013 - In Drosophila, each bristle sensory organ consists of four distinct cells that are generated via a series of stereotyped asymmetric divisions from ...
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Editorials: Cell Cycle Features

Editorials: Cell Cycle Features

Cell Cycle 12:11, 1–2; June 1, 2013; © 2013 Landes Bioscience

Inhibition of Notch recycling by Numb Relevance and mechanism(s) Lydie Couturier, Khallil Mazouni and François Schweisguth*

The generation of cell fate diversity and the self-renewal of stem cells rely in part on asymmetric cell divisions, whereby a mother cell divides to generate two daughter cells with distinct developmental potential through the unequal segregation of cell fate determinants. In Drosophila, each bristle sensory organ consists of four distinct cells that are generated via a series of stereotyped asymmetric divisions from a single sensory organ precursor cell (SOP).1 SOPs divide asymmetrically to generate an anterior pIIb cell and a posterior pIIa cell. The binary pIIa vs. pIIb decision is regulated by Notch: inhibition of Notch specifies pIIb, whereas activation of Notch leads to pIIa specification. Two cell fate determinants, Numb and Neuralized, localize at the anterior cortex of mitotic SOPs, are specifically inherited by pIIb cells and act in parallel to set up directional Notch signaling (Fig. 1).2,3 The E3 ubiquitin ligase Neuralized regulates the ubiquitin-dependent endocytosis and activity of the ligand Delta, thereby activating Notch in pIIa. By contrast, the mode of action of Numb, a multifunctional membrane-associated protein involved in both endocytosis and proteasome-mediated degradation, has remained a matter of debate. To study the regulation of Notch by Numb in vivo, we have used a functional GFP-tagged Notch to monitor by realtime imaging the localization of Notch. By measuring nuclear activated Notch (i.e., nuclear GFP) in the living organism, we found that Notch was specifically activated in pIIa within 10 min of the ingression of the cytokinetic furrow separating pIIa from pIIb.4 Also during cytokinesis, Notch was rapidly endocytosed from this cytokinetic furrow and accumulated in a Numb-dependent manner in pIIb-sorting

endosomes.5 These observations indicated that Numb regulates the endosomal trafficking of Notch during cytokinesis, and suggested that Numb breaks the symmetry of Notch distribution along the cytokinetic furrow between pIIa and pIIb, hence leading to directional signaling and binary fate decision. Numb is known to directly interact with Sanpodo, a trans-membrane protein that binds Notch and promotes its endocytosis.4,6 Live imaging analysis of a functional Sanpodo-GFP protein combined with antibody uptake assays revealed that Notch and Sanpodo are co-internalized during cytokinesis, suggesting that Notch is endocytosed as Notch-Sanpodo oligomers.4,5 Since Numb regulates the endosomal accumulation of Notch and Sanpodo in pIIb endosomes, it is likely that Numb somehow regulates the accumulation of Notch in sorting endosomes through its interaction with Sanpodo. Since Numb physically interacts with the AP-2 complex, one possibility would be that Numb regulates the AP-2-dependent internalization of Notch-Sanpodo oligomers.7 However, Numb was found to be dispensable for the internalization of both Notch and Sanpodo, indicating that Numb does not inhibit Notch by promoting its internalization from the pIIb plasma membrane.5,8 So, how does Numb regulate the trafficking of Notch-Sanpodo oligomers? Using a functional Numb-GFP protein, we showed that Numb relocalized during cytokinesis from the basal anterior cortex to the apical sorting endosomes that contain internalized Notch and Sanpodo.5 This localization suggested a “recycling inhibition” model, whereby Numb interacts at sorting endosomes

with internalized Notch-Sanpodo oligomers to prevent their recycling. In support of this model, Le Borgne and collaborators8 showed using an elegant recycling assay that Numb inhibits the recycling of Sanpodo in pIIb. How does Numb inhibit the recycling of Notch and Sanpodo? Since Numb interact physically and genetically with AP-1, an adaptor complex involved in sorting and recycling, one possibility would be that Numb somehow inhibits the recycling activity of AP-1 toward Notch and Sanpodo.8 Numb is, however, unlikely to act simply by sequestering AP-1, since Numb and AP-1 do not co-localize. Another non-exclusive possibility is that Numb recruits additional factors, such as the E3 ubiquitin ligase Su(dx)/Itch, to target Notch and Sanpodo toward lysosomal degradation. Also, if Numb acts at sorting endosomes to inhibit the recycling of Notch-Sanpodo oligomers, what might then be the role of AP-2? Several scenarios may be envisaged. First, it may be that the internalization of Notch is modulated by Numb, and that this role was overlooked in our ex vivo antibody uptake assay. Second, Numb may interact with AP-2 in dividing SOPs to increase the level of AP-2 in pIIb,7 thereby contributing to the endocytosis of other Notch regulators. Third, the preformed Numb/AP-2 complexes at the cortex of pIIb may regulate the rapid relocalization of Numb toward sorting endosomes during cytokinesis.5 Future studies are needed to sort out the roles of AP-1 and AP-2 in this context. In our “recycling inhibition” model, Numb does not act as a general inhibitor of Notch, but rather act as a contextdependent inhibitor of Notch, recycling via a linker protein, Sanpodo, connecting

*Correspondence to: François Schweisguth; Email: [email protected] Submitted: 04/08/13; Accepted: 04/13/13 http://dx.doi.org/10.4161/cc.24983 Comment on: Couturier L, et al. Curr Biol 2013; 23:588-93; PMID:23523245; http://dx.doi.org/10.1016/j.cub.2013.03.002 www.landesbioscience.com

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Institut Pasteur; Developmental and Stem Cell Biology Department; Paris, France; CNRS; URA2578; Paris, France

Notch to Numb. Interestingly, Sanpodo is specifically expressed in cells that undergo Numb-dependent asymmetric cell division. According to this model, Numb may inhibit the recycling of various internalized protein cargoes, including integrin and E-Cadherin, in a cell-specific manner depending on the presence of cell-specific linker proteins.

Figure 1. The cell fate determinants Neuralized (A) and Numb (B) are unequally segregated in mitotic SOPs (left panels). During cytokinesis (right panels), Neuralized acts in pIIb by regulating the endocytosis of Delta [green arrow in (A)]. This in turns leads to receptor activation and nuclear translocation of activated Notch in pIIa [orange arrows in (A)]. Sanpodo is equally inherited at mitosis (B) (left panel) and promotes the endocytosis of Notch in pIIa and pIIb [magenta arrows in (B)]. Numb localizes at sorting endosomes in pIIb, interacts with internalized Notch-Sanpodo oligomers and inhibiting their recycling (B).

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