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First published online June 28, 2004
doi: 10.1242/10.1242/jcs.01190

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Fibroblast growth factor 2 endocytosis in endothelial cells proceed via syndecan-4-dependent activation of Rac1 and a Cdc42-dependent macropinocytic pathway

¹ Angiogenesis Research Center and Section of Cardiology, Department of Medicine and Department of Pharmacology and Toxicology, Dartmouth Medical School, One Medical Center Drive, Lebanon, NH 03756, USA
² Department of Pathology, Dartmouth Medical School, One Medical Center Drive, Lebanon, NH 03756, USA

View larger version (73K): [in a new window]   Fig. 1. Internalization of the native and chimera syndecan-4 proteins. (A) RFPEC cells expressing the FcR-S4 construct were decorated with Cy3-labeled (red) human non-immune IgG (niIgG) and then clustered with AlexaFluor 647-labeled (green) FGF2. IgG decorated FcR-S4 chimeras are present on the surface of the cells and in the cytoplasm 20 minutes after FGF2 clustering (red). FGF2 is present both on the cell surface and in the cytoplasm. Note co-localization (yellow) of internalized FGF2 and FcR-S4 chimeras (merged image). This observation is consistent with FGF2-induced oligomerization of FcR-S4 native S4 heterodimers. (B) FcR-S4-expressing cells decorated with biotinylated niIgG followed by clustering with Cy3-labeled clustering antibodies (red). Biotinylated niIgG remaining on the cell surface was visualized by streptavidin-Cy5 (green). Dual stain on the merged image shows cell surface localized FcR-S4 whereas single red stain shows internalized FcR-S4. Right figure shows the merged color image overlaid with a DIC image. Lower figure shows Z-plane projection of the area in the white rectangle.

View larger version (84K): [in a new window]   Fig. 2. Syndecan-4 is internalized from lipid raft regions of the plasma membrane. Confocal microscopy of syndecan-4 endocytosis was carried out in RFPEC expressing the FcR-S4 chimera. The raft portion of the plasma membrane was labeled with BODIPY GM1 (blue) (a) or by transient expression of GPI-GFP construct (f). FcR-S4 chimeras were decorated with biotinylated niIgG and clustering was then initiated by the addition of Cy3-F(ab')2 fragments (red). Five minutes after the initiation of antibody clustering, all internalized FcR-S4 chimeras (b) were seen in close association with GM1-BODIPY (magenta, a,d,c) membrane domains or with GPI-GFP (yellow, h and i). (e,i) Merged color images (d and h, respectively) overlaid with DIC images. The depletion of membrane cholesterol with MßCD fully blocks clustering-induced FcR-S4 internalization (j,k,l,m). Scale bars, 10 µm.

View larger version (111K): [in a new window]   Fig. 3. Syndecan-4 is internalized with endogenous GPI-anchored proteins. (a) FcR-S4 expressing RFPES were labeled with FLAER, decorated by biotinylated niIgG and clustered by Cy-3-F(ab')2. (b) Five minutes after clustering, cells were placed on ice and the cell surface FcR-S4 was detected with streptavidin-Cy5. (c) FLAER-labeled GPI-anchored proteins. (d-f) Cell surface (d,f) and internalized (e,f) FcR-S4 demonstrate high level of colocalization with GPI-anchored proteins. Arrows in f point to examples of FcR-S4- and FLAER-labeled internalized vesicles. Scale bars, 10 µm.

View larger version (76K): [in a new window]   Fig. 4. Syndecan-4 is internalized in the clathrin- and dynamin-independent manner. (a-c) Syndecan-4 endocytosis was studied in FcR-S4 expressing RFPEC transiently transfected with various constructs. Antibody clustering of FcR-S4 chimeras carried out as described in Fig. 1 and 2 leads to internalization of syndecan-4 (a). Note the absence of co-localization of internalized syndecan-4 with clathrin in cells expressing clathrin-eGFP 5 minutes after clustering (b,c). The effect of clathrin dominant negative was studied in cells transiently expressing a c-myc tagged AP180C construct (d). Cell expressing AP180C demonstrates the same FcR-S4 internalization as non-transfected cells (e,f,g,h). The FcR-S4 remaining on the cell surface is shown in green and internalized FcR-S4 is in red. (i-m) The role of dynamin was studied in cells transiently expressing a dominant-negative HA-tagged dyn2K44A construct (i). Note that the cell expressing Dyn2K44A demonstrates high level of FcR-S4 internalization (r,i,l,m). h and m show the color images in g and l, merged with their respective DIC images. In g, h and i internalized FcR-S4 is in red, and in f,g,h,k,i FcR-S4 constructs remaining on the cell surface are in yellow and green. Scale bars, 10 µm.

View larger version (22K): [in a new window]   Fig. 5. Quantitative analysis of syndecan-4 endocytosis was carried out using confocal microscopy as described in the Materials and Methods. The results are presented as a fold change from baseline for each substance. (A) The effects of dominant negative constructs (AP180c) and dynamins 1 and 2 (Dyn1K44A and Dyn2K44A) on clathrin-dependent uptake of syndecan-4 (black bars) and transferrin (gray bars). Note inhibition of transferrin but not syndecan-4 uptake. * P<0.05 versus baseline. (B) The effects of Rac1 and Cdc42 dominant negative and constitutively active constructs and amiloride treatment on FcR-syndecan-4 (black bars), FGF2 (gray bars) and dextran (white bars) uptake was examined in RFPEC. Note a significant inhibition of syndecan-4 and FGF2 uptake in Rac1-DN-expressing cells and increased uptake in Rac1-CA expressing cells. Cdc42-DN and amiloride inhibited uptake of all three substances. *P<0.05 versus baseline endocytosis.

View larger version (29K): [in a new window]   Fig. 6. Rac1 activation by syndecan-4 clustering. Time course of Rac1, RhoA and Cdc42 activation following antibody oligomerization of FcR-S4 chimeras. GST pull down assays with specific substrates were performed as described in the Materials and Methods. Note activation of Rac1 5 minutes after initiation of syndecan-4 oligomerization while neither RhoA nor Cdc42 activity is changed.

View larger version (98K): [in a new window]   Fig. 7. Role of Rho family GTPases in syndecan-4 endocytosis. The effect of pre-treatment with C. difficile toxin B (A) and C3 exotransferase (B) on syndecan-4 endocytosis was examined in antibody-clustered FcR-S4 chimeras. Note that while toxin B treatment completely inhibited FcR-S4 endocytosis, C3 exotransferase had no effect. Scale bars, 10 µm.

View larger version (92K): [in a new window]   Fig. 8. Specific role of Rho GTPases in syndecan-4 endocytosis. The contributions of Rac1, Cdc42 and RhoA GTPases to syndecan-4 endocytosis were studied in FcR-S4-expressing RFPEC transiently transfected with eGFP-tagged dominant negative and constitutively active constructs. (A) A dominant negative Rac1 completely inhibited (left panels) while a constitutively active Rac1 stimulated (right panels) FcR-S4 endocytosis. (B) The expression of a dominant negative Cdc42 construct (left) somewhat inhibited FcR-S4 endocytosis while a constitutively active (right) Cdc42 constructs had no effect. (C) The expression of dominant negative (left) or constitutively active (right) RhoA constructs had no effect on FcR-S4 endocytosis. CA, constitutively active; DN, dominant negative. Scale bars, 10 µm.

View larger version (69K): [in a new window]   Fig. 9. Syndecan-4 and FGF2 uptake proceed via macropinocytosis. FcR-S4-expressing RFPEC cultured with fluorescent dextran (red, a) were decorated by niIgG (green, b) followed by FGF2 (blue, c) clustering. Merged images demonstrate colocalization of dextran and syndecan-4 (d), dextran and FGF2 (e) and all three signals (f). Amiloride pretreatment completely inhibited endocytosis of both dextran (g) and FcR-S4 (h-j). (k) The merged image overlaid with the DIC image. Scale bars, 10 µm.

View larger version (77K): [in a new window]   Fig. 10. Rac1 regulates FGF2 endocytosis. The effect of transient expression of Rac1 dominant negative (Rac1 DN-eGFP, A) and constitutively active (Rac1 CA-eGFP, B) constructs on FGF2 endocytosis was examined in RFPEC treated with fluorescently labeled FGF2 (red). Note decreased FGF2 uptake in cells expressing Rac1 DN-eGFP and increased uptake in cells expressing Rac1 CA-eGFP. Scale bars, 10 µm.

View larger version (14K): [in a new window]   Fig. 11. Flow cytometry analysis of syndecan-4 endocytosis. The effects of membrane cholesterol depletion by treatment with methyl-ß-cyclodextrin (MßCD) and amiloride on uptake of transferrin and FcR-syndecan-4 were analyzed using flow cytometry as described in the Materials and Methods. The data are presented as fold change from baseline. MßCD treatment almost fully blocked syndecan uptake while significantly reducing the level of transferrin internalization. Amiloride treatment inhibited syndecan uptake but increased the rate of transferrin endocytosis. *P<0.05 versus baseline.