Journal: Nature cell biology

Article Title: Godzilla-dependent transcytosis promotes Wingless signalling in Drosophila wing imaginal discs

doi: 10.1038/ncb3325

Figure Lengend Snippet: a, Diagram depicting the tag switching construct inserted at the wingless locus. b, Steady state distribution of endogenous Wingless relative to the Golgi marker GMAP in a 3 rd instar wing imaginal disc. 82% of Wingless-positive punctae are in the apical half (n= 175). c , Absence of HA immunoreactivity in an imaginal disc from wg KO [F-Wg OLLAS -F-Wg HA ]/UAS-Flp ; hedgehog-Gal4 tub-Gal80 ts larva cultured at 18°C. d, HA immunoreactivity can be seen 2 hrs after transfer to 29°C and mostly colocalises with GMAP (white arrows). e, From 3 hrs onwards, anti-HA is found in both GMAP-negative (blue arrows) as well as GMAP-positive (white arrows) vesicles. f, Steady state distribution of Wingless OLLAS in a Wg OLLAS -F-Wg HA ]/UAS-Flp ; hedgehog-Gal4 tub-Gal80 ts imaginal disc. Note the absence of Wingless HA . g, h, HA immunoreactive vesicles first appear in the apical region 2 hrs the after temperature shift (g), and increase in number during the following 2 hrs (h). i, Wingless HA is seen at ever more basolateral positions 8 hrs after transfer to 29°C. j, Another 8 hrs later, Wingless HA has completely replaced Wingless OLLAS and HA immunoreactivity can also be detected in nearby non expressing cells (white arrows). k, This and all subsequent panels show hemizygous shibire ts wing imaginal discs at 3 rd instar. When endocytosis is allowed to proceed normally (18°C ), steady state extracellular Wingless is mainly found at the basolateral surface (approximate plane of focus is shown as a grey line across a diagrammatic cell on the left). l-n, Preventing Dynamin-dependent endocytosis leads to Wingless accumulation at the apical surface and concomitant depletion from the basolateral surface (15, 30, and 60 min at 34°C). o, Steady state extracellular Wingless with normal endocytosis (18°C). p, Extracellular Wingless after endocytosis block (60 min at 34°C). q, r, Restoration of Dynamin activity by transfer to 18°C for 30 min (q) and 90 min (r). This leads to removal of accumulated apical extracellular Wingless and replenishment of the basolateral pool. Data from panels b-j, i.e. the number of punctae, as provided in the text, were pooled from at least 3 discs per experimental condition. Each time point in panels k-n is representative of >10 discs from 3 independent experiments. For panels o-r, >5 discs from three independent experiments were analysed. Scale bars represent 20 μm in b-j and 24 μm in k-r.

Article Snippet: The following primary antibodies were used: mouse anti-Wingless (1:1000 for standard protocol and 1:300 for extracellular staining; DSHB 4D4), rabbit anti-HA (1:1000, Cell Signalling), rat anti-OLLAS (1:10, Abnova), chicken anti-GFP (1:1000, Abcam), mouse anti-Integrin (1:100, DSHB CF.6G11), mouse anti-Dlp (1:50, DSHB 13G8), guinea pig anti-Senseless (1:1000, gift from H. Bellen), mouse anti-V5 (1:500, Invitrogen), goat anti-GMAP (1:100, gift from S. Munro, MRC-LMB Cambridge) guinea pig anti-Godzilla (1:1500; ).

Techniques: Construct, Marker, Cell Culture, Expressing, Blocking Assay, Activity Assay