Interaction of Notch signaling modulator Numb with α-Adaptin regulates endocytosis of Notch pathway components and cell fate determination of neural stem cells

Abstract

The ability to balance self-renewal and differentiation is a hallmark of stem cells. In Drosophila neural stem cells (NSCs), Numb/Notch (N) signaling plays a key role in this process. However, the molecular and cellular mechanisms underlying Numb function in a stem cell setting remain poorly defined. Here we show that α-Adaptin (α-Ada), a subunit of the endocytic AP-2 complex, interacts with Numb through a new mode of interaction to regulate NSC homeostasis. In α-ada mutants, N pathway component Sanpodo and the N receptor itself exhibited altered trafficking, and N signaling was up-regulated in the intermediate progenitors of type II NSC lineages, leading to their transformation into ectopic NSCs. Surprisingly, although the Ear domain of α-Ada interacts with the C terminus of Numb and is important for α-Ada function in the sensory organ precursor lineage, it was dispensable in the NSCs. Instead, α-Ada could regulate Sanpodo, N trafficking, and NSC homeostasis by interacting with Numb through new domains in both proteins previously not known to mediate their interaction. This interaction could be bypassed when α-Ada was directly fused to the phospho-tyrosine binding domain of Numb. Our results identify a critical role for the AP-2-mediated endocytosis in regulating NSC behavior and reveal a new mechanism by which Numb regulates NSC behavior through N. These findings are likely to have important implications for cancer biology.

FIGURE 1.

The AP-2 complex acts to prevent NB overproliferation. A, schematic view of a type II NB lineage consists of NB (pink), immature IP (brown), mature IP (light pink), GMC (light blue), and postmitotic neurons (blue). B, during each cycle of NB mitosis, Numb (orange) is asymmetrically segregated into the immature IP. As a consequence, Notch signaling is activated in the NB (green) but inhibited in immature IPs (gray). C, α-ada¹ or α-ada⁵ mutant larval brains stained with the NB marker Deadpan (Dpn) and GMC and neuronal marker Prospero (Pros), containing supernumerary NBs but few neurons in the CB (boxed) and ventral nerve cord (VNC, bracket) areas. D, α-Ada protein levels were greatly reduced in α-ada¹ or α-ada⁵ mutant larval brains, indicating that both are strong hypomorphic alleles. Note that the UAS-EGFP-α-Ada transgene specifically expressed in NBs by the 1407-GAL4 driver showed similar protein level as endogenous α-Ada. E, WT or α-ada¹ mutant MARCM clones induced from type II NBs that are marked by CD8-GFP. Neurons are marked with Elav. F, quantification of data from E. G, schematic drawing of the AP-2 complex. H, AP-2σ^KG02457 mutant MARCM clones contained ectopic NB (Dpn⁺Pros⁻, yellow arrowhead). Scale bars: 20 μm (E) and 10 μm (H).

FIGURE 2.

α-Ada and Numb promote Spdo endocytosis. A, WT (G-G”, I-I”) or α-ada¹ (H-H”, J-J”) mutant NBs at metaphase or telophase stages triple-labeled with Mira, Spdo, and DNA. B, cortical localization of Spdo in numb¹⁵ mutant NBs and adjacent IPs (pink arrowhead). C, MARCM analysis of type II NB lineages of WT control, α-ada¹ mutant, spdo^G104 mutant, and α-ada¹; spdo^G104 double mutant genotypes. NBs were marked with stars. The number of type II NBs (> 10 μm) per clone in these various genotypes are quantified in D. Scale bars = 10 μm (A and B) and 20 μm (C).

FIGURE 3.

Forced cortical localization of Spdo is not sufficient to promote the formation of ectopic NBs from IPs. A, schematic representation of Numb and Spdo domain structures. These various Numb or Spdo deletion or mutant versions were used for coimmunoprecipitation experiments or in vivo functional assays. B, interaction between Spdo and Numb-PTB. FLAG-tagged PTB or the M domains of Numb and a Myc-tagged Spdo with the C-terminal transmembrane domain deleted (SpdoΔTM) were expressed in HEK-293T cells, and coimmunoprecipitation (IP) experiments were performed as indicated. C, evidence that the Spdo YTNPAF motif is essential for Spdo-Numb interaction. FLAG-tagged full-length or truncated versions of SpdoΔTM and Myc-tagged Numb were expressed in HEK-293T cells, and coimmunoprecipitation experiments were performed as indicated. D, telophase NBs expressing either the Spdo-GFP or ΔNm-Spdo-GFP transgene were triple-labeled with GFP, Mira, and DNA. The arrowheads point to vesicular (upper) versus cortical (lower) GFP localization in future IP daughter cells. Note that ΔNm-Spdo-GFP also localized to some intracellular membrane structures. E, type II lineage NB clones of various genotypes marked with CD8-GFP (encircled by the dashed line). The Mira⁺ primary NB in each clone is marked with a star. F, the effects of NB-specific overexpression of Nb or ΔNm-Spdo or Nb and ΔNm-Spdo, driven by 1407-GAL4, on type II NB maintenance. Quantification of data from E is shown in F. Note that ΔNm-Spdo overexpression driven by 1407-GAL4 does not change the total NB number per brain lobe. NS, not significant. *, p < 0.0001, Student's t test. Scale bars = 10 μm (D), 20 μm (E), and 100 μm (F).

FIGURE 4.

The functional relationships among α-Ada, Numb, Spdo, and N. A, distribution of N as detected with the anti-N extracellular domain (N^ECD) antibody in WT and α-ada⁵ NBs at the metaphase stage. B, N distribution in a numb¹⁵ mutant NB MARCM clone marked with CD8-GFP (red). NBs are labeled with stars. White open or closed arrowheads point to N distribution at the cell cortex of control or numb¹⁵ mutant NBs, whereas pink open or closed arrowheads indicate N localization at the cell cortex of control or numb¹⁵ mutant IPs, respectively. The dashed line indicates the boundary between control (±, left) and numb¹⁵ mutant (right) cells. C and D, quantification of the relative distribution of N immunofluorescence at the cell cortex or cytoplasm of α-ada⁵ versus control (C) or numb¹⁵ mutant versus control (D) NBs or IPs. E, NBs in the signal larval brain lobe of various genotypes were marked with Mira. Data from E were quantified in F. *, p < 0.0001. Scale bars = 10 μm (A and B) and 100 μm (E).

FIGURE 5.

The Ada-Ear domain, important for α-Ada function in the SOPs, is dispensable in the NBs. The α-ada^ear5 mutant showed cell fate transformation in external sensory (ES) organs (A) but no phenotype in the type II NB lineages (B). Bracket, NB; arrowhead, immature IP. C, Spdo was localized to the cell cortex of α-ada¹ NBs but remained at the cytosol of α-ada^ear5 NBs. D, schematic representation of the α-Ada domain structures and various α-Ada deletion constructs used for generating transgenic lines and for coimmunoprecipitation experiments. E, interaction between Numb and α-Ada Trunk. FLAG-tagged full-length or truncated forms of α-Ada and Myc-tagged full-length Numb were expressed in HEK293T cells. Cell extracts were immunoprecipitated (IP) with anti-FLAG antibody, followed by Western blotting with anti-FLAG or anti-Myc antibodies. F, rescue of the α-ada⁵ mutant phenotype (H, H') by NB-specific expression (driven by 1407-GAL4) of full-length (Ada-FL) or truncated versions (Ada-ΔEar or Ada-Trunk) of the α-Ada transgenes. G, quantification of data from F. *, p < 0.0001. Scale bars = 10 μm (C) and 100 μm (F).

FIGURE 6.

An Nb-PTB-α-Ada chimera recapitulates the functional AP-2/Numb complex. A, schematic diagrams of Nb-FL, Nb-PTB, and Nb-PTB-α-Ada chimeric constructs. C, effects of overexpressing Nb-PTB, α-Ada, or Nb-PTB-α-Ada on type II NB number in WT or α-ada¹ mutants. Data quantification is shown in B. *, p < 0.0001. D, N and Nb-PTB-GFP-α-Ada vesicles (arrowhead) colocalize (solid arrowheads) in the differentiating daughter cells but not NBs (star), in α-ada¹; 1407>Nb-PTB-GFP-α-Ada background. E, in α-ada¹; 1407>GFP-α-Ada background, N (open arrowheads) and GFP-α-Ada positive vesicles (solid arrowheads) rarely colocalize in either the NBs (star) or differentiating daughter cells. Scale bars = 100 μm (C) and 5 μm (D and E).

FIGURE 7.

The function of N ligand Delta in regulating stem cell homeostasis in the type II NB lineages. A, WT NBs at metaphase, anaphase, or telophase stages were triple-labeled with Neur, Mira, and DNA. B–E, NB lineage-specific overexpression of the Tom or Dl RNAi transgenes led to a complete depletion of type II NBs (B) and complete suppression of NB overproliferation in α-ada¹ mutants (D). Arrowheads in B indicate type II NB lineages. Quantification of NB number is shown in C and E. *, p < 0.0001. F, NB lineage-specific overexpression of a full-length Dl transgene had no effect on NB number. Quantification of NB number is shown in G. Scale bars = 10 μm (A) and 100 μm (B and D).