Extended Data Fig. 1: Photoconversion assay controls and two extreme regimes of Dpp transport.
From: Morphogen gradient scaling by recycling of intracellular Dpp
a–d, Photoconversion assay. Test of efficiency of the acid wash in the photoconversion experiment using GBP-Dendra2: GFP-Dpp expressing discs have been incubated in GBP-Dendra2 for 50 min at 4 °C (the nanobody is only bound to the extracellular pool) and subsequently acid-washed to remove the label of the extracellular pool. Confocal image of eGFP-DppLOP expressing disc (a) and corresponding images of Dendra2* (b) before photoconversion (left) and 40 min following photoconversion (right; see Materials and Methods). Note that no detectable Dendra2* signal is observed 40 min after the acid wash, indicating that the extracellular pool of nanobodies has been efficiently removed and that the potential extracellular leftover (below the detection limit) cannot lead to an observable recovery in intracellular compartments. c, Comparison between eGFP-DppGal4 gradient profiles and gradient profiles formed by photoconverted Dendra2* propagated into the posterior compartment of the discs (photoconversion experiments as in Fig. 1a). Bar plot showing ϕ=λ/l of eGFP-DppGal4 gradient profiles and photoconverted Dendra2* gradient profiles for large discs. Bars, standard deviations. Two-tailed two sample t-test, p-value = 0.2353. d, Fluorescence intensity of Dendra2* in a ROI of 6x35 µm at the source boundary in the photoconversion experiment in Fig. 1a. Measured Dendra2* fluorescence (blue dots) is plotted as a function of time after the photoconversion event. The red line represents the theoretical dynamics of Dendra2* fluorescence signal considering the parameterized values for large discs. n = 4 biologically independent samples. Data represented as mean values ± s.e.m. e–h, Acid wash efficiently removes the extracellular pool. Confocal images of eGFP-DppLOP gradient (green in e, f), and extracellular eGFP-DppLOP pools monitored by means of an extracellular immunostaining (see Materials and Methods, Supplementary Information section 2.3.2) by using a GBP-Alexa555 nanobody against GFP (g, h; red in e, f) before (e, g) and after (f, h) acid wash. Acid wash in these conditions largely reduces the extracellular staining down to 9% of the signal. Scale bar: 10 µm. i, Acid wash does not affect internalized GBP-Alexa555. Confocal images of eGFP-DppLOP (top, green) and GBP-Alexa555 internalized for 40 min (bottom, red) before (left) and after acid wash (right). The GBP-Alexa555 signal decreases by 2.3 ± 0.6% after acid wash. j, Acid wash: effect of pH on GBP binding to GFP from larval extracts. Immunoblot of GFP which was bound to GFP-Trap beads (Chromotek, GFP-Trap beads, lanes 3-7) and GFP dissociated from GFP-Trap beads (supernatant, lanes 8-12) following treatment at different pH. FT, flowthrough (lane 1), PD, pulldown (lane 2). For gel source data, see Supplementary Fig. 1a. k, Stacked bar chart showing the relative contribution of the different modules to Dpp transport in the two theoretical extreme regimes of morphogen transport: extracellular diffusion (ExD20) and transcytosis (Tr) regimes. The relative contribution of different modules is expressed as the ratio λi2/λ2 with the index i corresponding to each of the four modules (i = u,b,r,t). Note that the unbound module contributes almost exclusively to λ2 in ExD and the transcytosis module, in Tr. l, Theoretical values of the 8 transport rates characteristic for ExD (rate values as in reference20) and Tr regimes of morphogen transport. m, n, FRAP recovery with respect to the two extreme theoretical regimes. Red lines, calculated recovery curves in a FRAP experiment for a set of parameter values corresponding to the extreme Tr (m) and ExD17 regimes (n). Blue dots, average of the experimental recovery curves in discs of l = 144 µm average posterior length. n = 9 biologically independent samples. Data represented as mean values ± s.e.m. The coefficient of determination R2 characterizes how well the calculated curves fit the experimental FRAP data. λ, decay length of the Dpp gradient profile calculated using equation (1) and the set of parameter values corresponding to Tr and ExD (see Supplementary Information section 4.2). Bars, s.e.m. Scale bar, 10 µm (a, h, i).
