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First published online 28 February 2007
doi: 10.1242/dev.02811

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The O-fucosyltransferase O-fut1 is an extracellular component that is essential for the constitutive endocytic trafficking of Notch in Drosophila

¹ Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency, 4-1-8 Honcho Kawaguchi, Saitama, Japan.
² Department of Biological Science and Technology, Tokyo University of Science, Chiba 278-8510, Japan.
³ Genome and Drug Research Center, Tokyo University of Science, Chiba 278-8510, Japan.
⁴ Department of Biological Sciences, Tokyo Metropolitan University, Tokyo 192-0397, Japan.
⁵ Department of Biochemistry, Osaka University, Graduate School of Medicine, Osaka 565-0871, Japan.

View larger version (64K): [in this window] [in a new window]   Fig. 1. Notch accumulated in O-fut1- cells. (A-D) O-fut1 mutant clones, indicated by the expression of GFP (green), generated by the MARCM method in late third-instar wing discs, and stained with an anti-Notch antibody (C17.9C6, magenta). Apical (A), subapical (B, 1.2 µm beneath the apical level), basal (C, 6.6 µm) and vertical (D) sections of wing disc epithelium are shown. Strong Notch staining was detected in all O-fut1- cells. Arrows indicate a small clone surrounded by wild-type cells (B,C). (E) Notch failed to localize to the adherens junctions in O-fut1- clones. Late third-instar wing discs that contained O-fut1- somatic clones were stained with anti-DE-Cadherin (green), and anti-Notch antibodies (C17.9C6, magenta). The clone boundary is indicated by a white dashed line.

View larger version (87K): [in this window] [in a new window]   Fig. 2. Notch was endocytosed but not transported to early endosomes in O-fut1- cells. (A-K) Live wing discs containing Notch- (A,B) or O-fut1- (C-K) clones were incubated with an anti-Notch extracellular antibody (rat1; magenta) to detect Notch on the plasma membrane, and allowed to endocytose Notch for 20 minutes (A-J) or 10 hours (K). Mutant cells were distinguished by the lack of GFP (green, A-F,K) or are labeled O-fut1- (G-J). Wing discs were also stained with an anti-Hrs antibody (G-J, shown in green). Optical sections corresponding to the apical region (A,C,G) or to 2 µm (D,H) or 8 µm (E,I) beneath the apical level, and optical vertical sections (B,F,J) are shown. (L) Living wing discs with O-fut1- clones (indicated by O-fut1-) incubated with fluorescent dextran (green) for 10 minutes, chased for 20 minutes at 25°C, and stained with an anti-Notch antibody (C17.9C6; magenta). Higher magnification of one or two dextran-positive vesicles in wild-type (arrowhead) and O-fut1- (open arrowhead) cells are shown as insets in the upper left and lower right, respectively. The clone boundaries are demarcated by a white dashed line. All wing discs were isolated from late third-instar larvae.

View larger version (106K): [in this window] [in a new window]   Fig. 3. Most Notch was not co-localized with ER markers in O-fut1- cells. (A-E) O-fut1- somatic clones (labeled O-fut1-) generated in wing discs expressing ER marker PDI-GFP (A-C) or ER-CFP (driven by sd-GAL4; D,E), and stained with anti-Notch (C17.9C6, magenta) and anti-GFP (green) antibodies. (A) Normal confocal image showing the subapical region of the wing disc epithelium, where Notch accumulated in O-fut1- cells. (B) The resolution of A was improved using deconvolution. (C) Optical vertical section of B. (D) Deconvolution was used to improve the resolution of this image. (E) Optical vertical section of D. (F) A positive control for deconvolution analysis. Wing discs expressing PDI-GFP were incubated with a rat anti-GFP antibody, and stained simultaneously with two anti-rat secondary antibodies, conjugated to Alexa 488 or Cy3. All wing discs were isolated from late third-instar larvae. Right panels show merged images of the left and middle panels. Higher magnifications of O-fut1- cells are shown in the small panels at right (B-F). Clone boundaries are indicated by white dashed lines (A-E).

View larger version (53K): [in this window] [in a new window]   Fig. 4. O-fut1 was required for the endocytic transportation of Notch in a manner independent of its enzymatic activity. (A) Genomic organization of the Gmd locus. Exons are shown as boxes, and the predicted coding region is in black. The regions 3' to the Pelement insertion site of GS13045 are deleted in GmdH43 (0.4 kb) and GmdH78 (0.8 kb). (B) Concentration of GDP-fucose in Gmd mutants. GDP-fucose in wild-type and heterozygous and homozygous Gmd mutant larvae was measured in duplicate. ND, not detected. (C-F) Wild-type (C,E) and GmdH78 homozygous (D,F) wing discs stained with anti-Wg (C,D) and anti-En antibodies (E,F). (G) Uptake of fluorescent dextran by live GmdH78 homozygous mutant wing discs after a 10 minute incubation and a 20 minute chase at 25°C. Dextran and Notch are shown in green and magenta, respectively. Some dextran-positive vesicles are indicated by arrowheads. (H,I) Notch staining in wild-type (H) and GmdH78 (I) wing discs. (J) O-fut1- clones generated in GmdH78 wing discs, then stained with anti-Notch (magenta) and anti-myc (clone marker, green) antibodies. The clone boundary is indicated by a white dashed line. All wing discs were isolated from late third-instar larvae.

View larger version (73K): [in this window] [in a new window]   Fig. 5. Notch turnover is modulated by O-fut1. (A-D) The expression of N+-GV3 (magenta) was induced by a 1 hour heat shock at 37°C (B-D) or was not induced (A), and the wing discs were subsequently incubated at room temperature for another 30 minutes (B), 6 hours (C) or 12 hours (D). O-fut1- clones are denoted by the absence of GFP (green). The clone boundaries are indicated by white dashed lines in (B). (E) Expression of a mutant form of N+-GV3 (NEGF-GV3, magenta), which lacks the fifth to 23rd EGF-like repeats, was induced by a 1 hour heat shock followed by a 6 hour incubation at room temperature. O-fut1- clones lack GFP (green). (F-I) O-fut1 (F-H) or O-fut1-G3 (I) driven by en-GAL4 were overexpressed (green) in the posterior compartment of wing discs, and N+-GV3 expression (magenta) was induced by a 3 hour heat shock at 37°C (G-I) or was not induced (F). Wing discs were dissected 30 minutes (G) or 24 hours (H,I) after heat shock. (J-L) Adult wings. (J) Wild-type. (K,L) Wing phenotype induced by the overexpression of wild-type O-fut1 (K) or O-fut1-G3 (L) driven by sd-GAL4. Higher magnification images of the wing margin are shown at right. (M,N) wg expression (magenta) was detected in the wild-type wing disc (M) or the wing disc overexpressing O-fut1 (green) under the control of sd-GAL4 (N). The right panel of N shows a merged image of Wg and O-fut1 expression. All wing discs were isolated from late third-instar larvae.

View larger version (50K): [in this window] [in a new window]   Fig. 6. O-fut1 is secreted and internalized by cells. (A,B) Intracellular localization of O-fut1 was examined in Garland cells overexpressing either ER-CFP (A, green) or N+-GV3 (B, green) and Ofut1-myc (A,B, magenta). ER-CFP and O-fut1-myc were driven by da-GAL4, and N+-GV3 was induced by a 2 hour heat shock that ended 30 minutes before fixation. (A',B') Higher magnifications of A and B, respectively. Arrows indicate O-fut1 and Notch double-positive vesicles, which were distant from the peri-nuclear ER (B'). (C) S2 cells were transfected with the expression constructs for O-fut1-myc and Notch (lanes 1,2), or NotchEC (lanes 3,4). Lysates prepared from these cells were immunoprecipitated using anti-Myc (lanes 1,3) or anti-Notch (lanes 2,4) antibodies, and the blots were probed with the anti-Notch antibody (C17.9C6) or anti-Myc antibody (9E10). The expression of Notch and O-fut1 in the lysates was confirmed by western blot (Lysate, lower panels). (D) O-fut1 was incorporated into cells in a Notch-dependent manner. S2 cells expressing Notch (green) were incubated with the O-fut1-ER- protein (magenta). The brightfield image is superimposed in the right panel. (E-H) The endocytosis defect in the Ofut1 knockdown cells was rescued by the addition of extracellular Ofut1. S2 cells expressing Notch (E) or Notch and O-fut1-IR (F,G) were incubated with anti-Notch antibody (rat1, magenta), fixed, and costained with anti-Hrs antibody (green). (G) Live cells expressing Notch and O-fut1-IR were incubated with O-fut1-ER- for 20 minutes before the addition of anti-Notch antibody. (E'-G') Corresponding higher magnification images of E-G. Arrows indicate Notch and Hrs double-positive vesicles. (H) Average ratio of Notch and Hrs double-positive vesicles shown as the percentage of Notch-positive vesicles. Mean ± s.d. from triplicate assays (more than 20 cells each) are shown. (I,J) The N+-GV3 protein (magenta) 8 hours after heat shock for 1 hour in thirdinstar wing discs overexpressing O-fut1 (I) or O-fut1-ER- (J) (green) driven by en-GAL4. (K,L) Adult wings. O-fut1 (K) and O-fut1-ER- (L) were overexpressed under the control of en-GAL4, and the resulting wing phenotypes were examined. (M) Fluorescent dextran uptake by Delta and Serrate double-mutant cells in live wing discs after a 10 minute incubation and a 20 minute chase at 25°C. Clone boundary and dextran-positive vesicles are indicated by dotted lines and white arrowheads, respectively. All wing discs were isolated from late thirdinstar larvae.