1. Volume 21, Issue 1, Pages 87-95 (January 2011)
AP-1 Controls the Trafficking of Notch and Sanpodo toward E-Cadherin Junctions in Sensory Organ Precursors 
Najate Benhra, Sylvie Lallet, Mathieu Cotton, Stéphanie Le Bras, Aurore Dussert, Roland Le Borgne 
Current Biology 
Volume 21, Issue 1, Pages (January 2011)
DOI: /j.cub
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2. Current Biology 2011 21, 87-95DOI: (10.1016/j.cub.2010.12.010)
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3. Figure 1
AP-1 Loss of Function Causes a Notch Gain-of-Function Phenotype in SOP Lineage
(A) Control adult sensory organ showing one socket and one shaft.
(B and C) Tissue-specific gene silencing of the AP-47 (B) or AP-1γ (C) subunits of AP-1 using the ap-GAL4 driver leads to an excess of socket cells (white arrows).
(D and D′) Homozygous clones of AP-47SHE11 on the adult thorax (dashed lines) are identified by the loss of a bristle marker Stubble (Sb, shorter and thicker bristles). AP-47SHE11 sensory organ shows an excess of socket cells (D) and AP-47SHE11 sensory organ shows no phenotype (D′).
(E–G) Projection of confocal sections of a wild-type notum (E), a notum expressing AP-47dsRNA (F), and a homozygous clone for AP-47SHE11 (G) at 24–26 hr after puparium formation (APF) stained with Cut (sensory cell marker, green) and Su(H) (socket cell marker, red). AP-47SHE11 mutant cells are delimited by the dotted lines and are visualized by the absence of nls-GFP (blue in G). In contrast to wild-type organs containing one Su(H)-positive cell (E and asterisk in G), the mutant organ contains two or three Su(H)-positive cells (F and G).
(H) Quantification of the different lineages observed at 24–26 hr APF in AP-47 SHE11 clones, in ap>AP-1γdsRNA and ap>AP-47dsRNA flies. The table indicates the number of different organs observed per genotype. For AP-47SHE11 clones, all mutant organs per notum were quantified. For ap>AP-1γdsRNA and ap>AP-47dsRNA flies, quantification was done in a square area delimited by the four dorsocentral machrochaetae. ElaV was used to identify the neuron in SO lineage.
(I–N) AP-1-dependent Notch gain-of-function phenotype is ligand dependent and requires spdo activity.
(I) Somatic clones of DeltaRev10, SerRX106 on an adult thorax show loss of external structures, socket, and shaft cells of the microchaete.
(J) A similar balding phenotype is observed in the triple AP-47SHE11, DeltaRev10, SerRX106 mutant clones.
(K) Clones of WASp3 show some bald cuticle.
(L) AP-47SHE11, WASp3 homozygous clones show an excess of external cells with double shaft.
(M) spdoG104 clones show some bald cuticle.
(N) A similar phenotype is observed in the double AP-47SHE11, spdoG104 homozygous clones. Somatic clones (dashed lines) were induced with Ubx-FLP and were identified by the loss of a bristle marker Sb.
Arrows show positions lacking the sensory external cell shaft and socket (K, M, and N). Asterisks indicate mutant organs with an excess of external cells (L). Note that AP-47 mutation also causes a depigmentation of the adult cuticle. Anterior is up and scale bar is 10 μm in (E)–(G).
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4. Figure 2
AP-1 Controls the Localization of Spdo and Localizes to LqfR and Rab11 Positive Compartments
(A–E″) Localization of Spdo (red) in wild-type (A–A″ and C–C″), AP-47SHE11 (B–B″ and D–D″), or AP-2α40-31 (E–E″) mutant cells. Mutant cells are identified by the absence of nls-GFP (green). (A)–(E) show apical and (A′)–(E′) show basolateral confocal slices, respectively. (A″)–(E″) show orthogonal sections of cells from (A)–(E′), respectively. In AP-47SHE11 SOP mutant cells (B–B″), Spdo accumulates apically at the level of DE-Cad (green; together with nlsGFP in A–E″). In wild-type pIIa-pIIb cells, Spdo is mostly intracellular in pIIb (white arrowheads) and localizes at the cell cortex of pIIa (C′). Low levels of Spdo are also found at the apical plasma membrane of both SOP daughter cells (C). In AP-47SHE11 SOP daughter cells, Spdo accumulates at the apical level, as well as at the junction of both cells (D–D″). On average, AP-47SHE11 SOP daughter cells contain five times more apical Spdo than wild-type SOP daughter cells (4.8 ± 2.37 standard deviation; p < ; n = 14; one-sample t test). Spdo localization at the basolateral level (D′ and D″) remains similar to that of wild-type cells (C′ and C″).
(F–F″) AP-1 localizes with Spdo at the TGN and in RE. Localization of AP-1γ (green) in pIIa/pIIb cells relative to Spdo (red) and LqfR (blue in F, red in F″) is shown. White arrowheads indicate dotted structures positive for the three markers, whereas blue arrowheads label structures positive for AP-1γ and LqfR.
(G and H) Localization of AP-1 (green) relative to Rab11 (red) (G) and localization of Spdo (red) relative to Rab11 (green) (H). White arrowheads indicate dotted structures positive for the two markers.
(I–L) Colocalization of Spdo with AP-1 relies on Spdo's first N-terminal 18 amino acids.
(I–J′) Localization of AP-47-VFP (green in I–J′) with Spdo-mChFP (red in I and I′) or with SpdoΔ18-mChFP (red in J and J′) expressed under the control of the neurP72-GAL4 driver. (I′) and (J′) are orthogonal confocal slices of cells shown in (I) and (J), respectively.
(K and L) Quantification of the number of vesicles (size > 0.03 μm2) positive for AP-47-VFP (green), Spdo-mChFP (red in K), or SpdoΔ18-mChFP (red in L) and for both (yellow) in the two SOP daughter cells (no distinction between anterior and posterior cell; one-sample t test; ∗∗∗p < ; n = 20 SOP daughter cells).
In all panels, anterior is left; scale bar is 5 μm in (A)–(J′). See also Figure S2.
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5. Figure 3 Apical Recycling of Spdo in AP-1-Depleted Cells
(A) Schematic representation of the SpdoL2:mChFP construct.
(B) Schematic representation of the endocytosis-recycling assay. Pupal nota were dissected in culture medium and incubated for 15 min on ice with a rabbit anti-RFP recognizing the mChFP tag present in the second extracellular loop of Spdo. After washing, either nota were fixed (cell surface staining) or Spdo2::mChFP-anti-RFP complexes were allowed to be internalized and recycled back to the cell surface for 45 min (45 min chase) prior to fixation and immunodetection of internalized antibodies. Because of the presence of the apically secreted cuticle, anti-RFP antibodies only have access to the basolateral pool of SpdoL2::mChFP, enabling us to test for apical recycling of Spdo [40].
(C–H″′) In the control, anti-RFP present at the cell surface (C–C″) is efficiently internalized and reaches apical endosomes (D–D″′) but is poorly recycled back to the apical plasma membrane (D″′). In cells depleted of AP-47, although anti-RFP initially present at the basolateral plasma membrane (E–E″) is efficiently internalized, a substantial pool of anti-RFP appears at the apical plasma membrane and at adherens junctions after the chase (F–F″′), indicating that SpdoL2::mChFP is efficiently recycled back to the apical plasma membrane in AP-1-depleted cells. In contrast, in cells depleted of AP-2 (AP-50dsRNA), endocytosis from the basolateral plasma membrane is drastically reduced (G–H″′). SpdoL2::mChFP, AP-47dsRNA, and AP-50dsRNA are expressed under the control of the sca-GAL4 driver. In all panels, anterior is left and scale bar is 10 μm. See also Figure S3.
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6. Figure 4
Notch Is Stabilized with Spdo at the Junction between SOP Daughter Cells Mutant for AP-47
Apical localization of NECD (red in A, B, E, and F; grey in A′, B′, E′, and F′) or NICD (red in C and D; grey in C′ and D′) and DE-Cad (green) in AP-47SHE11 (B, B′, D, and D′), spdoG104 (E and E′), and AP-47SHE11, spdoG104 (F and F′) mutant clones versus control (A, A′, C, and C′) SOP daughter cells. Sensory organs are identified with Senseless (A–B′ and E–F′; not shown) or Spdo (blue in C and D; grey in C′ and D′). NECD and NICD are stabilized at the apical junction between SOP daughter cells mutant for AP-47SHE11 (B, B′, D, and D′) compared to wild-type SOP daughter cells (A, A′, C, and C′). NECD is also stabilized at the junction of SOP daughter cells mutant for AP-47SHE11, spdoG104 (F and F′). Although SOP daughter cells mutant for spdoG104 (E and E′) or AP-47SHE11, spdoG104 (F and F′) show higher levels of NECD apically compared to wild-type cells (A and A′), NECD is not stabilized at the junction between spdoG104 SOP daughter cells (E and E′). (A′)–(F′) are higher magnification of (A)–(F), respectively. Mutant cells were identified by the loss of nuclear GFP (not shown). Anterior is left; scale bar is 1 μm in (A′)–(F′) and 5 μm in (A)–(F). See also Figure S4.
Current Biology  , 87-95DOI: ( /j.cub )
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