Figure 1. Expression of FN with an... | Rockefeller University Press

Figure 1.

$Expression of FN with an internal tag in murine fibroblast lines. (A) Schematic representation of FN polypeptide showing its organization into 12 FN-type I repeats (rectangles), two FN-type II repeats (ovals), 15 FN-type III repeats (circles), three alternatively spliced regions (A, B, and V), and an integrin binding RDG motif in FN-typeIII-10. An internal tag (pHluorin or HaloTag) was inserted between FN-type III 6 and 7 domains. The human FN cDNA expressed here does not contain the variable A and B regions but contains parts of the V region. Domains implicated in FN-FN interactions are shown in red. (B) Immunoblot analysis of FN expressed by KO67 Halo-β1itg fibroblasts (β1itg KO murine fibroblasts reconstituted with Halo-β1itg and expressing paxillin-mCherry), parental and ectopically expressing FN-PH, and comparison with endogenous FN expressed by other cells types, such as primary mouse fibroblasts, HT1080, HeLa, and Ea.hy926 using the pan-FN antibody P1H11. FN-PH was also detected with an anti-GFP antibody, and vinculin and HDAC1 were used as loading controls. (C) Immunoblot analysis of FN-PH and FN-Halo secreted in the medium or present in the cell/ECM fraction of stable lines of KO67 β1itg fibroblasts expressing each tagged FN. (D) Network of FN-PH and FN-Halo (labeled with SiR-Halo ligand) assembled by KO67 β1itg fibroblasts on FN-coated glass and imaged by epifluorescence microscopy. (E) Colocalization of FN-PH and β1itg in a culture of KO67 fibroblasts expressing FN-PH (green) and Halo-β1itg (magenta, labeled with SiR-Halo ligand), imaged in epifluorescence (EpiF) with focus set to the ventral side of the cells. Inset 1 shows the colocalization of FN-PH fibrils with β1itg-rich dorsal adhesions in epifluorescence microscopy with the focus set to the dorsal side of cells. Arrows indicate FN fibers connected to β1itg-rich dorsal adhesions. Inset 2 shows the colocalization of FN-PH fibrils with β1itg-rich focal adhesions at the ventral side of cells using TIRM microscopy. (F) Confocal microscopy images of a KO67 fibroblast (paxillin-mCherry in magenta) expressing Halo-β1itg (gray, labeled with SiR-Halo ligand) and FN-PH (green) cultured overnight on FN-coated glass. FN objects were detected by Imaris surface generation tool (Bitplane) and colored according to their z-position (see multicolor scale bar). The graph shows the z-position of FN-PH positive objects assessed in nine cells plated on FN-coated glass (4,186 objects) and in eight cells plated on uncoated glass (4,469 objects), z position zero corresponding to the middle of the nucleus, negative values indicating objects below the nucleus and positive values indicating objects above the nucleus. Mean position shown as red line. (G) TIRFM images showing the colocalization of fibrils of FN-PH and FN-Halo (labeled with SiR-Halo ligand) with paxillin-mCherry (magenta) and β1itg (Halo-β1itg labeled with SiR-Halo ligand in gray or FN-β1itg in green) in KO67 fibroblasts cultured at low density on an FN-coated glass. The intensity profile of all three fluorescent markers along the orange dotted arrows “a” (across focal adhesions) and “b” (along a focal adhesion) is shown in graphs on the right. The colored arrows on graph “b” indicate the FN fiber extending beyond the paxillin-positive focal adhesion. Scale bar = 10 μm. Source data are available for this figure: SourceData F1.$

Expression of FN with an internal tag in murine fibroblast lines. (A) Schematic representation of FN polypeptide showing its organization into 12 FN-type I repeats (rectangles), two FN-type II repeats (ovals), 15 FN-type III repeats (circles), three alternatively spliced regions (A, B, and V), and an integrin binding RDG motif in FN-typeIII-10. An internal tag (pHluorin or HaloTag) was inserted between FN-type III 6 and 7 domains. The human FN cDNA expressed here does not contain the variable A and B regions but contains parts of the V region. Domains implicated in FN-FN interactions are shown in red. (B) Immunoblot analysis of FN expressed by KO67 Halo-β1itg fibroblasts (β1itg KO murine fibroblasts reconstituted with Halo-β1itg and expressing paxillin-mCherry), parental and ectopically expressing FN-PH, and comparison with endogenous FN expressed by other cells types, such as primary mouse fibroblasts, HT1080, HeLa, and Ea.hy926 using the pan-FN antibody P1H11. FN-PH was also detected with an anti-GFP antibody, and vinculin and HDAC1 were used as loading controls. (C) Immunoblot analysis of FN-PH and FN-Halo secreted in the medium or present in the cell/ECM fraction of stable lines of KO67 β1itg fibroblasts expressing each tagged FN. (D) Network of FN-PH and FN-Halo (labeled with SiR-Halo ligand) assembled by KO67 β1itg fibroblasts on FN-coated glass and imaged by epifluorescence microscopy. (E) Colocalization of FN-PH and β1itg in a culture of KO67 fibroblasts expressing FN-PH (green) and Halo-β1itg (magenta, labeled with SiR-Halo ligand), imaged in epifluorescence (EpiF) with focus set to the ventral side of the cells. Inset 1 shows the colocalization of FN-PH fibrils with β1itg-rich dorsal adhesions in epifluorescence microscopy with the focus set to the dorsal side of cells. Arrows indicate FN fibers connected to β1itg-rich dorsal adhesions. Inset 2 shows the colocalization of FN-PH fibrils with β1itg-rich focal adhesions at the ventral side of cells using TIRM microscopy. (F) Confocal microscopy images of a KO67 fibroblast (paxillin-mCherry in magenta) expressing Halo-β1itg (gray, labeled with SiR-Halo ligand) and FN-PH (green) cultured overnight on FN-coated glass. FN objects were detected by Imaris surface generation tool (Bitplane) and colored according to their z-position (see multicolor scale bar). The graph shows the z-position of FN-PH positive objects assessed in nine cells plated on FN-coated glass (4,186 objects) and in eight cells plated on uncoated glass (4,469 objects), z position zero corresponding to the middle of the nucleus, negative values indicating objects below the nucleus and positive values indicating objects above the nucleus. Mean position shown as red line. (G) TIRFM images showing the colocalization of fibrils of FN-PH and FN-Halo (labeled with SiR-Halo ligand) with paxillin-mCherry (magenta) and β1itg (Halo-β1itg labeled with SiR-Halo ligand in gray or FN-β1itg in green) in KO67 fibroblasts cultured at low density on an FN-coated glass. The intensity profile of all three fluorescent markers along the orange dotted arrows “a” (across focal adhesions) and “b” (along a focal adhesion) is shown in graphs on the right. The colored arrows on graph “b” indicate the FN fiber extending beyond the paxillin-positive focal adhesion. Scale bar = 10 μm. Source data are available for this figure: SourceData F1.