A nanobody-based toolset to investigate the role of protein localization and dispersal in Drosophila
- University of Basel, Switzerland
- University of Zurich, Switzerland
Figures
Figure 1 with 3 supplements
The GrabFP constructs localize to distinct regions along the apical-basal axis.
(A) Linear representation of the six different versions of the GrabFP system; the constructs exist in two topologies with the GFP-nanobody (vhhGFP4) either facing extracellular (Ext) or …
Figure 1—figure supplement 1
Localization of the GrabFPIntra tools.
(A–C) Optical cross-sections of wing discs expressing the intracellular versions of morphotrapInt (A), GrabFP-AInt (B) and GrabFP-BInt (C) in the disc proper (ptc::Gal4). The GrabFP constructs are …
Figure 1—figure supplement 2
Expression of the GrabFP system allows normal wing development.
(A–E) Male wings of indicated genotypes. Expression of the GrabFPExt tools (using hh::Gal4 (A–D) or r4::Gal4 (E)) does not interfere with wing development and yields viable and fertile flies. Solely …
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Figure 1—figure supplement 2—source data 1
Source data for wing area quantification.
Only male wings were used. Intervein area IV4-5 in µm2 as indicated in pannel A.
- https://doi.org/10.7554/eLife.22549.005
Figure 1—figure supplement 3
Quantification and analysis of protein distribution along the A-B axis.
Procedure for obtaining relative concentration profiles along the A-B axis of DP cells for the basolateral marked Nrv1-YFP (A–D) and the apical marked Crb-GFP (E–H). (A) Optical cross-section of a …
Figure 2 with 2 supplements
Mislocalization of transmembrane proteins using the GrabFPExt system.
(A) In the GrabFPExt system, the GFP-nanobody (vhhGFP4) faces the extracellular space and can interact with extracellular-tagged transmembrane proteins. (B–C) Optical cross-section of wing disc …
Figure 2—figure supplement 1
Examples of target protein mislocalization using the GrabFPExtra system.
(A–B) Effects of GrabFP-AExt expression on the localization of Dally-YFP (A) and PMCA-YFP (B). Optical cross-sections of target proteins alone (Ctrl., left) or co-expressed with GrabFP-AExt …
Figure 2—figure supplement 2
Modulation of EGFP fluorescent properties by vhhGFP4 binding in vitro.
(A) Coomassie staining of SDS-PAGE loaded with purified eGFP (25 kDa) and vhhGFP4 (14 kDa). See Materials and methods for details. (B) Fluorescence in vitro essay to estimate changes in eGFP …
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Figure 2—figure supplement 2—source data 1
Source data pannels C-E.
- https://doi.org/10.7554/eLife.22549.010
Figure 3 with 1 supplement
Mislocalization of GFP/YFP-tagged proteins using the GrabFPInt system.
(A) With the GFP-nanobody facing the cytosol, the GrabFPInt system can interact with cytosolic proteins and transmembrane proteins tagged along their cytosolic portion. (B–C) Optical cross-sections …
Figure 3—figure supplement 1
Examples of GFP/YFP-target protein mislocalization using the GrabFPIntra system.
(A) Optical cross-section of DP cells expressing Arm-YFP (Ctrl., left) and Arm-YFP together with GrabFP-BInt (middle). Average Arm-YFP protein distribution along the A-B axis in the absence (black) …
Figure 4
GrabFP-BInt-mediated Sqh-GFP mislocalization results in changes of DP cell shape.
(A) Optical cross-section of a wing disc expressing Sqh-GFP (green), stained for Dlg (blue). In the magnifications, the junctional level is marked by a dashed line. (B–D) Optical cross-sections of …
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Figure 4—source data 1
Source data for apical surface area.
- https://doi.org/10.7554/eLife.22549.014
Figure 5
The Dpp morphogen spreads in the apical and basolateral compartment.
(A) Wing disc expressing eGFP-Dpp in the central Dpp stripe and eGFP fluorescence profile (bottom). (B) Optical cross-section of a wing disc as shown in (A) additionally stained for Dlg (blue). …
Figure 6 with 1 supplement
The GrabFPExt system can interfere with specific sub-fractions of the Dpp morphogen gradient.
Optical cross sections of wing discs either expressing eGFP-Dpp (green) in the stripe source (A) or eGFP-Dpp in the stripe and the different versions of the GrabFP system (red, B–D) in the posterior …
Figure 6—figure supplement 1
Quantification of differential eGFP-Dpp accumulation by morphotrap, GrabFP-BExt and GrabFP-AExt.
eGFP-Dpp immobilization pattern along the A-B axis in the posterior compartment when either morphotrap (A), GrabFP-BExt (B) or GrabFP-AExt (C) are expressed in posterior cells (hh::Gal4). Left …
Figure 7 with 1 supplement
Basolateral Dpp spreading is required for patterning and size control.
(A–B) p-Mad staining in representative dppd8/d12 mutant wing disc rescued by eGFP-Dpp (A) and in dppd8/d12 wing disc rescued by eGFP-Dpp expressing morphotrap in the posterior compartment (hh::Gal4, …
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Figure 7—source data 1
Wing pouch area and IV4-5 area.
Only male wing discs and wings were included in the quantification.
- https://doi.org/10.7554/eLife.22549.019
Figure 7—figure supplement 1
morphotrap expression in PPE cells interferes with luminal Dpp spreading.
(A) Expression of UAS-GFPNLS under control of the AGir::Gal4 driver line in a third instar wing imaginal disc. The activity of AGir::Gal4 is restricted to PPE cells and a small group of cells in the …
Figure 8
Basal Dpp is required to control wing size.
(A) Schematic representation of GrabFP-ECM localization when expressed in the larval fat body. (B) Wing disc optical cross-section of an animal expressing GrabFP-ECM in the fat body, stained for …
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Figure 8—source data 1
Wing pouch and blade areas.
Only male wing discs and wings were included in the quantification.
- https://doi.org/10.7554/eLife.22549.022
Author response image 1
N-YFP mislocalization causes the induction of an ectopic Wg stripe.
(A) Representative wing disc of a N-YFP (green) hemizygous male (control) stained for Wg (upper panel, blue; lower panel, black). Wg is expressed along the D/V boundary. (B) Basolateral …
Author response image 2
Mislocalization of Crb-GFP affects the localization of apical Crb interaction partners.
(A-D) Optical cross-sections of Crb-GFP (green) homozygous GFP-tagged wing discs showing the anterior control compartment (Ctrl., left) and the posterior compartment expressing GrabFP-BExt (right). …
Author response image 3
Detection of a secreted VHH-mCherry fusion in the wing disc lumen.
(A) Schematic representation of the experimental setup. We expressed a fusion of the mouse CD8 signal peptide, the vhhGFP4 GFP-nanobody and mCherry (secretedVHH-mCherry) under the control of hh::Gal4…
