Notch does not traffic to late endosomes in awd mutant cells It has been shown that Notch signaling can also be en hanced by blocking Oligomycin A FDA MVB formation with mutations in the endosomal sorting complex required for transport genes tsg101, vps25 and vps20, or by promoting early endosome maturation with over expression of con stitutively active Rab5. Since the awd mutant is defective in Notch signaling, it is unlikely that the Notch containing vesicles in awd mutant cells have passed into late endosomes. This notion is supported by the lack of significant co localization of Notch containing vesicles in MARCM awd mutant clones with Rab7, the late endosomal marker. As well, transition from early endosomes to late endosomes is accompanied by acidification of the luminal contents, which can be detected by Lysotracker staining.

Inhibitors,Modulators,Libraries Consistent with the notion that Notch containing vesicles in awd mu tant cells cannot Inhibitors,Modulators,Libraries enter MVB and late endosomes, we ob served no difference in Lysotracker positive vesicles in awd and awd mutant cells. In addition, the Notch containing vesicles in MARCM awd mutant clones are not Rab11 positive recycling endosomes, either. We next sought to follow the time course of Notch localization in live cells. Wing discs are an ideal and standardized system for this purpose since they can be cultured ex vivo for a prolonged period of time. Note that in this established ex vivo system, internalization of Notch is detected by binding to NECD antibody, without binding to spatially expressed ligands. Therefore, the system strictly measures the kinetics of vesicular trans port, not the endogenous signaling process.

We first established that at the steady state, Notch accumulated on the awd cell surface. In wild type cells, internalized Inhibitors,Modulators,Libraries Notch follows a typical time course, at 20 minutes after initiation of endocytosis, Notch is mostly in Avl positive early endosomes while some has passed into Rab7 positive late endosomes. At one hour after endocytosis, the Notch signal is barely detectable, consistent with the degrad ation time course. Also, in wild type cells, Avl staining is Inhibitors,Modulators,Libraries much more pronounced at 20 minutes than at one hour. This is likely because in this label and chase experiment, a large number of Avl positive vesicles were formed syn chronously after initiation of endocytosis. Concentrated Avl was then lost after early endosomes matured and were incorporated into late endosomes.

In awd mutant, on the other hand, accumulated Notch is mostly on cell surface or in Avl positive early endo somes at 20 minutes Inhibitors,Modulators,Libraries and remains in these early endo somes even one hour after internalization. The Notch signal shows no localization to the late endosomes. inhibitor Abiraterone Note that some of the Rab7 positive vesicles shown in Figure 7C are very close to or surrounded by the Notch signal but are not overlapping.