Glypican-1 drives unconventional secretion of fibroblast growth factor 2
- Heidelberg University, Biochemistry Center, Germany
- Institut für Chemie und Biochemie, Freie Universität Berlin, Germany
Figures
Figure 1
Glypican-1 (GPC1) and fibroblast growth factor 2 (FGF2) are in spatial proximity to a cellular context.
(A) HeLa S3 cells stably expressing either FGF2-BirA or myc-tagged BirA (control) in a doxycycline-dependent manner were cultured as detailed in the Materials and methods section. Whole cell lysates …
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Figure 1—source data 1
Raw data of the BioID screening procedure identifying proteins in proximity to fibroblast growth factor 2 (FGF2) in intact cells.
- https://cdn.elifesciences.org/articles/75545/elife-75545-fig1-data1-v2.xlsx
Figure 2 with 2 supplements
Efficient secretion of fibroblast growth factor 2 (FGF2) to cell surfaces depends on Glypican-1 (GPC1).
(A) Quantitative analysis of FGF2 secretion under the experimental conditions indicated measured by cell surface biotinylation. Standard deviations are shown (n = 4). (B) Quantitative analysis of …
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Figure 2—source data 1
Raw data of the cell surface biotinylation and flow cytometry experiments quantifying fibroblast growth factor 2 (FGF2) secretion under the conditions indicated.
- https://cdn.elifesciences.org/articles/75545/elife-75545-fig2-data1-v2.xlsx
Figure 2—figure supplement 1
Characterization of engineered HeLa S3 cells used to quantify fibroblast growth factor 2 (FGF2) secretion efficiencies.
(A) HeLa S3 cell lines with different knockout backgrounds along with overexpression of Glypican-1 (GPC1) and GPC5, respectively, as indicated. (B) HeLa S3 cell lines with a GPC1 knockout background …
Figure 2—figure supplement 2
Visualization of endocytosis of fluorescent transferrin versus fibroblast growth factor 2 (FGF2)-GFP in HeLa S3 cells comparing wild-type, Glypican-1 (GPC1) knockout and GPC1 knockout plus GPC1 overexpression conditions.
Endocytosis of recombinant FGF2-GFP (5 µg/ml, in green) and Transferrin-Alexa Fluor546 (25 µg/ml, in red) was visualized for the times indicated in HeLa S3 wild-type, GPC1 knockout, and GPC1 …
Figure 3
Fibroblast growth factor 2 (FGF2)-GFP binding to cell surfaces is increased in Glypican-1 (GPC1)-overexpressing cells.
(A) Quantitative analysis of the FGF2-GFP binding capacity of cell surfaces under the experimental conditions indicated using flow cytometry. Standard deviations are shown (n = 5). Statistical …
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Figure 3—source data 1
Raw data of experiments quantifying fibroblast growth factor 2 (FGF2) binding to cell surfaces as well as glycosaminoglycan and heparan sulfate contents of cells under the conditions indicated.
- https://cdn.elifesciences.org/articles/75545/elife-75545-fig3-data1-v2.xlsx
Figure 4
Glypican-1 (GPC1) is the principal heparan sulfate proteoglycan involved in unconventional secretion of fibroblast growth factor 2 (FGF2).
(A) Representative example for the raw data of cell surface biotinylation experiments used to quantify and to statistically evaluate unconventional secretion of FGF2 under the conditions indicated …
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Figure 4—source data 1
Raw data of the cell surface biotinylation and flow cytometry experiments quantifying fibroblast growth factor 2 (FGF2) secretion and FGF2 cell surface binding under the conditions indicated.
- https://cdn.elifesciences.org/articles/75545/elife-75545-fig4-data1-v2.xlsx
Figure 5
Glypican-1 (GPC1) overexpression results in increased fibroblast growth factor 2 (FGF2) secretion efficiencies.
FGF2 secretion efficiencies in wild-type and GPC1-overexpressing cells were assessed by TIRF microscopy using anti-GFP nanobodies to detect single FGF2-GFP molecules on cell surfaces as described …
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Figure 5—source data 1
Raw data of the cell-based TIRF experiments quantifying fibroblast growth factor 2 (FGF2) recruitment at the inner plasma membrane leaflet and FGF2 translocation to the outer plasma membrane leaflet under the conditions indicated.
- https://cdn.elifesciences.org/articles/75545/elife-75545-fig5-data1-v2.xlsx
Figure 6 with 2 supplements
Glypican-1 (GPC1) and fibroblast growth factor 2 (FGF2) form a strong pair of interaction partners.
Recombinant constructs encoding soluble ectodomains of GPC1 (panel A), GPC1 ΔHS (panel B; a mutant form to which heparan sulfate chains cannot be added), GPC5 (panel C), GPC6 (panel D), and SDC4 …
Figure 6—figure supplement 1
Expression and purification of soluble recombinant forms of fibroblast growth factor 2 (FGF2) and the various heparan sulfate proteoglycans.
FGF2 was expressed and purified from Escherichia coli cells. All heparan sulfate proteoglycans indicated were expressed and purified from HEK293 cells. For details, see Materials and methods. Upper …
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Figure 6—figure supplement 1—source data 1
Unprocessed and uncropped image file of a SDS-PAGE analysis of the recombinant forms of GPC family members and fibroblast growth factor 2 (FGF2) used in the BLI experiments shown in Fig.
- https://cdn.elifesciences.org/articles/75545/elife-75545-fig6-figsupp1-data1-v2.zip
Figure 6—figure supplement 2
Interaction studies between different kinds of heparan sulfate proteoglycans and growth factors or cytokines using biolayer interferometry.
Heparan sulfate proteoglycans were loaded onto BLI sensors as indicated by the color code. In some cases, they were treated with heparinase III or chondroitinase ABC as indicated. The binding …
Figure 7 with 1 supplement
Quantitative characterization of the disaccharide contents of heparan sulfate chains derived from Glypican-1 (GPC1), GPC5, and SDC4.
The recombinant forms of GPC1, GPC5, and SDC4 described in Figure 6 and Figure 6—figure supplement 1 were treated with a mixture of the heparinases I + II and III to release the disaccharide units …
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Figure 7—source data 1
Raw data of the analytical HPLC experiments quantifying disaccharide units of the heparan sulfate chains of the proteoglycans indicated.
- https://cdn.elifesciences.org/articles/75545/elife-75545-fig7-data1-v2.xlsx
Figure 7—figure supplement 1
Characterization of synthetic dissacharide standards corresponding to the building blocks of heparan sulfate chains using an analytical HPLC analysis.
The standards used were commercial products (Iduron, UK). They were characterized for their retention times on a strong anion exchange column operated by a HPLC system. For experimental details, see …
Figure 8
Glypican-1 (GPC1) is dispensable for fibroblast growth factor 2 (FGF2) signaling.
Various forms of HeLa cells including wild-type and GPC1 knockout cells as well as cells overexpressing GPC1 in either a wild-type or a GPC1 knockout background were treated with recombinant FGF2 at …
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Figure 8—source data 1
Raw data of the cell-based signaling experiments quantifying fibroblast growth factor 2 (FGF2)-induced signaling cascades under the conditions indicated.
- https://cdn.elifesciences.org/articles/75545/elife-75545-fig8-data1-v2.xlsx
Videos
Video 1
Endocytosis of recombinant fibroblast growth factor 2 (FGF2)-GFP (5 µg/ml, in green) and Transferrin-Alexa Fluor546 (25 µg/ml, in red) was visualized in HeLa S3 wild-type cells.
One frame corresponds to 1 min. The data shown are representative for three independent experiments. Scale bar = 20 µm.
Video 2
Endocytosis of recombinant fibroblast growth factor 2 (FGF2)-GFP (5 µg/ml, in green) and Transferrin-Alexa Fluor546 (25 µg/ml, in red) was visualized in HeLa S3 Glypican-1 (GPC1) knockout cells.
One frame corresponds to 1 min. The data shown are representative for three independent experiments. Scale bar = 20 µm.
Video 3
Endocytosis of recombinant fibroblast growth factor 2 (FGF2)-GFP (5 µg/ml, in green) and Transferrin-Alexa Fluor546 (25 µg/ml, in red) was visualized in HeLa S3 knockout cells overexpressing Glypican-1 (GPC1).
One frame corresponds to 1 min. The data shown are representative for three independent experiments. Scale bar = 20 µm.
Additional files
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Transparent reporting form
- https://cdn.elifesciences.org/articles/75545/elife-75545-transrepform1-v2.docx
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Source data 1
- https://cdn.elifesciences.org/articles/75545/elife-75545-data1-v2.zip
