TMED10 mediates the loading of neosynthesised Sonic Hedgehog in COPII vesicles for efficient secretion and signalling

Abstract

The morphogen Sonic Hedgehog (SHH) plays an important role in coordinating embryonic development. Short- and long-range SHH signalling occurs through a variety of membrane-associated and membrane-free forms. However, the molecular mechanisms that govern the early events of the trafficking of neosynthesised SHH in mammalian cells are still poorly understood. Here, we employed the retention using selective hooks (RUSH) system to show that newly-synthesised SHH is trafficked through the classical biosynthetic secretory pathway, using TMED10 as an endoplasmic reticulum (ER) cargo receptor for efficient ER-to-Golgi transport and Rab6 vesicles for Golgi-to-cell surface trafficking. TMED10 and SHH colocalized at ER exit sites (ERES), and TMED10 depletion significantly delays SHH loading onto ERES and subsequent exit leading to significant SHH release defects. Finally, we utilised the Drosophila wing imaginal disc model to demonstrate that the homologue of TMED10, Baiser (Bai), participates in Hedgehog (Hh) secretion and signalling in vivo. In conclusion, our work highlights the role of TMED10 in cargo-specific egress from the ER and sheds light on novel important partners of neosynthesised SHH secretion with potential impact on embryonic development.

Keywords: Biosynthetic pathway; ERES; Endoplasmic reticulum; Membrane trafficking; p24 proteins.

Fig. 1

Characterisation of SHH-RUSH system. A Schematic illustrating the SHH-RUSH reporter system. The Streptavidin-KDEL hook (grey) was encoded on the same plasmid as SHH-RUSH (green) with an internal ribosome entry site (IRES, black) to enable expression of both genes. B Cartoon illustrating the SHH-RUSH retention and release upon biotin addition. C Representative western blot of SVG-A cell lysates either non-transfected (NT), transfected with untagged SHH (SHH) or with SHH-RUSH probed with antibodies against SHH. GAPDH was used as a loading control. D SHH-ELISA of supernatant taken from cells either non-transfected (NT) or transfected with SHH-RUSH and cultured in the absence or presence of biotin for 24 h. Data is represented as mean ± SD from two individual experiments. Unpaired t-test p value < 0.05 (*). E RT-qPCR analysis of Gli1 mRNA expression (using primer set a) in RNA extracted from 3T3 fibroblasts treated with supernatants taken from cells either non-transfected (NT), mock-transfected (Mock), or transfected with SHH-RUSH and cultured in the absence or presence of biotin for 24 h. Data is represented as mean ± SD from two individual experiments. Unpaired t-test p value < 0.01 (**). F RT-qPCR analysis of Gli1 mRNA expression (using primer set b) from NIH 3T3 fibroblasts treated with supernatants taken from cells either non-transfected (NT), or transfected with untagged SHH (SHH WT) or SHH-RUSH. Data is represented as mean ± SD from two independent experiments. Unpaired t-test p value < 0.05 (*). ns non-significant. G Representative confocal microscopy images of SVG-A cells expressing SHH-RUSH and fixed at indicated timepoints after biotin addition. Scale bar = 10 µm. H Representative confocal images of SVG-A cells co-transfected with SHH-RUSH (purple) and a medial Golgi marker, Mannosidase-II-EGFP (Mann-II-EGFP; green). The left micrograph shows a snapshot at 5 min post biotin addition. Scale bar = 10 µm. The right panel highlights zoomed insets of the Golgi area at indicated timepoints post biotin addition from the white dotted square on the left image. I Quantification of the mean fluorescence intensity of SHH-RUSH in the Golgi area (black line) and colocalisation (blue line) of the two channels over time. Data is represented as mean ± SD from three individual experiments.

Fig. 2

SHH-RUSH follows the classical biosynthetic pathway. A Representative confocal images from a single plane in a z-stack of images from cells transfected with SHH-RUSH and stained with antibodies against Rab6 or Rab7, and fixed at 45 min post biotin addition. B Quantification of colocalisation between SHH-RUSH and endogenous Rab5, Rab6 or Rab7 from whole cells in which the Golgi was masked, at different timepoints post biotin addition. Data is represented as mean ± SD, n = 30 cells. Unpaired t-test p value < 0.0001 (****) or < 0.001 (***) or < 0.01 (**). C Representative TIRF images of cells co-transfected with SHH-RUSH and GFP-Rab6a under the weak L30 promoter associated to Video S3. The left panel shows a still image taken at 1898s post biotin addition. Scale bar = 10 µm. The right panel highlights zoomed insets showing a double-positive SHH-RUSH/Rab6a vesicle arriving into the focal plane then fusing with the plasma membrane. D Schematic illustrating the SHH-RUSH cell surface staining assay. E Quantification by flow cytometry of the ratio of surface staining (anti-SHH + AF-647) over the total mCherry signal mean fluorescence intensities is represented at different timepoints post biotin addition in cells treated with siRNA against Rab6a (siRab6a), or Rab7a (siRab7a), or a non-targeting control (siCTRL). Data was normalised to t = 0 and plotted as mean ± SD from four independent experiments. Unpaired t-test p value < 0.05 (*).

Fig. 3

TMED10 regulates SHH transport through the biosynthetic pathway. A Representative western blot of lysates taken from SVG-A cells treated with siCTRL or siTMED10. Blots were then probed with antibodies against TMED10. GAPDH was used as a loading control. B Quantification of western blot in (A), data plotted as mean ± SD, n = 3 independent experiments, *p < 0.05. C Quantification of SHH-NLuc released into cell culture media from cells treated with siCTRL or siTMED10 over 24 h. Data plotted as mean ± SD, n = 6 independent experiments, **p < 0.01. D Quantification by flow cytometry of the ratio of surface staining (anti-SHH + AF-647) over the total mCherry signal mean fluorescence intensities is represented at different timepoints post biotin addition in cells treated with siRNA against TMED10 (siTMED10) or siCTRL. Data was normalised to t = 0 and plotted as mean ± SD from 3 independent experiments. Unpaired t-test p value < 0.0001 (****). E Flow cytometry analysis of TfR surface staining at different timepoints post biotin addition from SVG-A cells transfected with TfR-RUSH and treated with siTMED10 or siCTRL. Data was normalised to t = 0 and plotted as mean ± SD from 3 independent experiments. No significant differences were measured between the siTMED10 and siCTRL. F Colocalisation analysis of SHH-RUSH with cis-Golgi marker GM130. The left panel shows representative confocal images of cells treated with siCTRL or siTMED10 and transfected with SHH-RUSH (magenta), fixed at 15 min post biotin addition, permeabilized and stained for GM130 (green). Scale bar = 10 µm. The right panel shows the quantification of colocalisation between SHH-RUSH and GM130 at 0 min and 15 min post-biotin addition. Data is plotted as mean ± SD (n = 20 cells). Unpaired t-test p value < 0.0001 (****). G Colocalisation analysis of SHH-RUSH with trans-Golgi marker TGN46. The left panel shows representative confocal images of cells treated with siCTRL or siTMED10 and transfected with SHH-RUSH (purple), fixed at 45 min post biotin addition, permeabilized and stained for TGN46 (green). Scale bar = 10 µm. The right panel shows the quantification of colocalisation between SHH-RUSH and TGN46 at 0 min and 45 min post-biotin addition. Data is plotted as mean ± SD (n = 20 cells). Unpaired t-test p value < 0.05 (*). H TMED10 dynamics during SHH intracellular dynamics associated to Video S5. The left panel shows a still image at 0 s post biotin addition of SVG-A cells co-transfected with GFP-TMED10 and SHH-RUSH. Scale bar = 10 µm. The right panel highlights zoomed images of a double-positive TMED10:SHH-RUSH vesicle trafficking together towards a perinuclear region at indicated timepoints. Scale bar = 2 µm.

Fig. 4

TMED10 associates with SHH-RUSH at ERES and aids in its exit from ER. A Live confocal imaging of ER exit site marker SEC24D (green) and SHH-RUSH (purple) associated to Video S7. The left panel shows a still image of a cell co-transfected with SEC24D-EGFP and SHH-RUSH at 0 s post biotin addition. Scale bar = 10 µm. The right panel highlights zoomed images of a SHH-RUSH vesicle leaving a SEC24D-positive ERES. Scale bar = 2 µm. B Quantification of SHH-RUSH at SEC24D-positive puncta over time post-biotin addition. Data is plotted as mean fluorescence intensity of SHH-RUSH (magenta) and SEC24D-EGFP (green) and is representative of 5 individual cells. C Representative confocal images from a single panel in a z-stack of cells treated with siCTRL or siTMED10 and transfected with SHH-RUSH, fixed at either 0 min or 15 min post biotin addition, permeabilized and stained with antibodies against SEC31. Scale bar = 10 µm. D Quantification of SHH-RUSH colocalisation with SEC31 over time in siCTRL and siTMED10 treated cells. Data is plotted as mean ± SD (n = 30 cells). Unpaired t-test p value < 0.01 (**) or < 0.0001 (****). E Quantification of the ratio of SHH-RUSH intensity at SEC31-positive puncta at 15 min versus 0 min post biotin addition in siCTRL or siTMED10 treated cells. Dotted line (red) represents relative SHH-RUSH intensity at 0 min post biotin addition. Data is plotted as mean ± SD (n = 30 cells). Unpaired t-test p value < 0.001 (***).

Fig. 5

Loss of baiser function affects Hh secretion and signalling in vivo. A Left panel: cartoon illustrating pupal lethality rescue assay and larval wing imaginal disc (WID) outgrowth assay. In the “tester line”, expression of Hh is increased in the WID posterior domain (in pink) leading to distal anterior tissue outgrowth. This can be rescued upon decreasing Hh level or Hh extracellular secretion. Middle panel: lethality rescue assay—percentage of escaper animals in”tester line” control, and overexpressing bai RNAi-s in the posterior domain of the “tester line”. Right panel: WID outgrowth assay—percentage of outgrown, moderately and fully rescued discs with respect to their genotypes. Sample numbers (n) are indicated above the graphs. B–D Hh target gene expression in wild-type and bai RNAi wing discs. B Engrailed, C Patched (Ptc) and D anti-β-gal (dpp-lacZ). The anterior/posterior (A/P) compartment boundaries are marked with yellow dotted lines. Placement of insets are indicated by white rectangles. White arrows point to the areas in the anterior compartment, where proximal Hh target gene expression is different between wild-type and bai RNAi conditions. Scale bar – 50 µm. E–G Quantification of Engrailed (E, n = 8), Patched (F, n = 10) and dpp-lacZ (G, n = 10) in the anterior compartment of wild-type control (black) and bai RNAi (magenta) discs. The A/P boundary is indicated by black arrows. H Left panel: Cartoon illustrating the dorsal depletion of bai using RNAi, with ventral area serving as an internal control. Middle and right panels: Extracellular staining of endogenous Hh (middle) and filamentous Actin (right) in discs with bai RNAi. Dotted yellow line labels boundary between dorsal and ventral compartments. Scale bar – 50 µm. Quantification of extracellular Hh and Actin intensity levels between the dorsal and ventral compartments are shown next to the corresponding immunostainings. Data plotted as mean ± SD, n = 10. I, J Distal ectopic dpp-lacZ gene expression measured in discs overexpressing Hh in producing cells for 1 day (I) or 4 days (J). Cartoons next to the fluorescent panels illustrate dpp-lacZ expression pattern (green). The pink rectangles indicate the domain of expression of UAS-Hh together with GFP RNAi (control), disp RNAi (positive control) and bai RNAi. White arrows point to ectopic dpp-lacZ expression (or in case of disp and bai RNAi the lack of it) in cuboidal cells of the WID. Note, that 1-day induction of Hh overexpression does not lead to changes in WID morphology, and that bai RNAi in Hh producing cells is capable of suppressing ectopic distal dpp-lacZ expression. Additional examples can be found on Supplementary Fig. S6. Scale bar – 100 µm.