Numb and alpha-Adaptin regulate Sanpodo endocytosis to specify cell fate in Drosophila external sensory organs

Abstract

During asymmetric cell division in Drosophila sensory organ precursors (SOPs), the Numb protein segregates into one of the two daughter cells, in which it inhibits Notch signalling to specify pIIb cell fate. We show here that Numb acts in SOP cells by inducing the endocytosis of Sanpodo, a four-pass transmembrane protein that has previously been shown to regulate Notch signalling in the central nervous system. In sanpodo mutants, SOP cells divide symmetrically into two pIIb cells. We show that Sanpodo is cortical in pIIa, but colocalizes with Notch and Delta in Rab5- and Rab7-positive endocytic vesicles in pIIb. Sanpodo endocytosis requires alpha-Adaptin, a Numb-binding partner involved in clathrin-mediated endocytosis. In numb or alpha-adaptin mutants, Sanpodo is not endocytosed. Surprisingly, this defect is observed already before and during mitosis, which suggests that Numb not only acts in pIIb, but also regulates endocytosis throughout the cell cycle. Numb binds to Sanpodo by means of its phosphotyrosine-binding domain, a region that is essential for Numb function. Our results establish numb- and alpha-adaptin-dependent endocytosis of Sanpodo as the mechanism by which Notch is regulated during external sensory organ development.

Figure 1

Sanpodo is required for asymmetric cell division. (A) 3R6 mutant external sensory (ES) organs on the head show a pronounced loss of microchaetae and interommatidial bristles compared with wild type (high-magnification images). (B) Wild-type ES organs contain one socket (so) cell (Su(H) positive), one neuron (n) cell (Elav positive) and one sheath (sh) cell (Pros positive). In 3R6 mutant ES organs, outer cells are transformed into inner cells (four neurons or two sheath cells and two neurons). (C) In wild-type eye sensory organ precursor (SOP) cells, Numb localizes to the anterior cell cortex throughout mitosis, whereas Numb is mislocalized in a subset of 3R6 mutant mitotic eye SOP cells. (D) The 3R6 mutation changes a glutamine at amino-acid position 91 of the protein Sanpodo into a stop codon. Sanpodo is a 64 kDa protein with four transmembrane domains in the carboxyl terminus and a region in the middle of the protein that is conserved between Drosophila and Anopheles.

Figure 2

Numb and α-Adaptin inhibit Sanpodo localization to the plasma membrane. (A) After cell division, Sanpodo localizes to the membrane in the pIIa cell, where it colocalizes with the membrane marker Gβ13F (open arrowheads), and vesicles in close proximity to the plasma membrane, which can be distinguished from membrane-localized Sanpodo (arrowheads). In the pIIb cell, no colocalization with Gβ13F is observed and Sanpodo localizes to intracellular vesicles instead (arrowheads). (B) In control sensory organ precursor (SOP) cells, Sanpodo localizes to intracellular vesicles in interphase and to the cytoplasm during mitosis. After cell division, Sanpodo is found in endocytic vesicles in the pIIb cell and at the plasma membrane in the pIIa cell. In (C) numb¹⁵ and (D) ada^ear04 mutant SOP cells, Sanpodo localizes predominantly to the plasma membrane throughout the cell cycle.

Figure 3

Sanpodo colocalizes with Notch and Delta in endocytic vesicles in the pIIb cell. (A–E) Sanpodo (Spdo) localizes to the membrane in the pIIa cell but is found in intracellular dots in the anterior pIIb cell. Sanpodo colocalizes strongly with (A) Notch (N) and (B) Delta (Dl) primarily in the pIIb cell. Sanpodo colocalizes with (C) Rab5 (marker for early endosomes) and (D) Rab7 (late endosomes) in the pIIb cell but barely with (E) Rab11 (recycling endosomes). Arrowheads indicate colocalization. Note that these images are projections of several optical slices to better illustrate the difference in the number of vesicles, which are generally found more apical. Therefore, the membrane localization of Sanpodo in the pIIa cell is not as clearly visible as in the single optical slices shown in Fig 2A.

Figure 4

Sanpodo binds to the phosphotyrosine-binding domain of Numb. (A) Numb contains a phosphotyrosine-binding (PTB) domain. Different Numb deletion constructs were used for binding assays. (B) NumbΔPTB alone cannot co-precipitate SanpodoΔTM from S2 cells. IP: immunoprecipitation. (C) Numb04–β-Gal, but not Numb03–β-Gal, can co-precipitate Sanpodo from embryonic lysates. Spdo, Sanpodo.