Journal: bioRxiv

Article Title: Basement membrane regulates fibronectin organization using sliding focal adhesions driven by a contractile winch

doi: 10.1101/618686

Figure Lengend Snippet: (A) Maximum-intensity projections of immunofluorescence confocal images of SCC-9 cells seeded on glass or basement membrane overnight. (B) Quantification of results in (A) showing FN fibrillar matrix deposition per cell normalized to data from cells on glass. n =20. (C) Total internal reflection fluorescence (TIRF) images of SCC-9 cells sparsely seeded on glass or various extracellular matrix protein coatings at 20μg/ml for 8hrs. (D-F) Quantification of results from C showing normalized (to glass) total amounts of FN fibrillar matrix deposition, number of FN matrix fibers per cell, and average area of each FN matrix fiber on different substrates. n ≥13. p ≤ 0.001 was achieved in all comparisons between each of the conditions of Matrigel, laminin and collagen IV with each of the conditions of collagen I, vitronectin and glass, except in the comparison of FN matrix fiber size between collagen IV and vitronectin (**p ≤ 0.01) and glass (*p ≤ 0.05). (G) Quantification of normalized average FN matrix deposition by each cell growing on various concentrations and combinations of Matrigel, laminin and collagen IV. n ≥30. Error bars: SEM. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. Scale bars: 10 μm. See also and .

Article Snippet: Matrigel, laminin, collagen IV, collagen I, perlecan and vitronectin were diluted to 20 μg/ml or other indicated concentrations and aliquoted into glass-bottom MatTek dishes or chambers (ibidi).

Techniques: Immunofluorescence, Membrane, Fluorescence, Comparison

Journal: bioRxiv

Article Title: Basement membrane regulates fibronectin organization using sliding focal adhesions driven by a contractile winch

doi: 10.1101/618686

Figure Lengend Snippet: (A) Maximum-intensity projections of immunofluorescence confocal images of MDA-MB-231 cells seeded on glass or Matrigel with or without exogenous FN (20μg/ml) in the medium. (B) Quantification of results from A showing normalized FN fibrillar matrix deposition per cell. n ≥15. (C) Quantification of normalized FN matrix deposition per SCC-9 cell cultured in FN-containing medium (20μg/ml) containing DMSO vehicle, FN-containing medium containing cycloheximide (20μg/ml) or FN-free medium containing cycloheximide. n =20. (D) Maximum-intensity projections of immunofluorescence confocal images comparing assembly of FN matrix fibers at the bottom of SCC-9 cells seeded on collagen I or Matrigel for 1hr. (E) Quantification of the amount of FN matrix from images shown in D. n =12 and 22 for number of cells on collagen I and Matrigel, respectively. (F) Deoxycholate-insoluble FN matrix fibers. Cells growing on Matrigel or collagen I for 1hr were incubated with 10% deoxycholic acid for 10mins before washing and staining with anti-FN. (G) Schematic diagram of the Matrigel-vitronectin patterned coating experiment. (H) Maximum-intensity projections of immunofluorescence confocal images showing FN fibrillogenesis by SCC-9 cells growing at the boundary between Matrigel and vitronectin. Error bars: SEM. **p ≤ 0.01, ***p ≤ 0.001. Scale bars: 10 μm (A, D, F, H); 40 μm (G).

Article Snippet: Matrigel, laminin, collagen IV, collagen I, perlecan and vitronectin were diluted to 20 μg/ml or other indicated concentrations and aliquoted into glass-bottom MatTek dishes or chambers (ibidi).

Techniques: Immunofluorescence, Cell Culture, Incubation, Staining

Journal: bioRxiv

Article Title: Basement membrane regulates fibronectin organization using sliding focal adhesions driven by a contractile winch

doi: 10.1101/618686

Figure Lengend Snippet: (A) TIRF images of SCC-9 cells seeded on collagen I, vitronectin, collagen IV and Matrigel. Note the patterns of focal adhesions and their spatial relationship with FN fibers highlighted in the insets. (B) Time-lapse image series showing two focal adhesions sliding medially toward the ventral cell center (but shown directed upward for clarity) that are assembling FN matrix fibers. Scale bars: 20 μm (A); 5 μm (insets, B).

Article Snippet: Matrigel, laminin, collagen IV, collagen I, perlecan and vitronectin were diluted to 20 μg/ml or other indicated concentrations and aliquoted into glass-bottom MatTek dishes or chambers (ibidi).

Techniques:

Journal: bioRxiv

Article Title: Basement membrane regulates fibronectin organization using sliding focal adhesions driven by a contractile winch

doi: 10.1101/618686

Figure Lengend Snippet: (A) Effects of blebbistatin (50μM), Y27632 (20μm), and C3 transferase (4μg/ml) treatment on FN matrix assembly by SCC9 cells growing on Matrigel. n ≥30. (B) TIRF image of an SCC-9 cell growing on Matrigel. Magnified insets show myosin IIA is associated with actin stress fibers between two sets of parallel focal adhesions. (C) Effects of myosin IIA and myosin IIB knock-out on FN matrix assembly by HFFs on Matrigel. n =15. (D) Time-lapse montage of focal adhesions (green) sliding toward each other, showing shortening actin stress fibers (blue) and FN (magenta) matrix fiber elongation. (E) Trailing edge of a cell on Matrigel; white arrow indicates direction of cell movement. Time-lapse montage of the inset shows FN fibrillogenesis (magenta) by retracting focal adhesions (green). (F) Traction force maps of SCC-9 cells growing on vitronectin, Matrigel or collagen IV. (G) Quantification of average traction force magnitude (A.U.). n =20. Schematic representation of the classical fibronectin matrix assembly model (H) and the current winch model (I). Error bars: SEM. ***p ≤ 0.001. Scale bars: 10 μm (B, D left, F); 5 μm (D right, E). See also .

Article Snippet: Matrigel, laminin, collagen IV, collagen I, perlecan and vitronectin were diluted to 20 μg/ml or other indicated concentrations and aliquoted into glass-bottom MatTek dishes or chambers (ibidi).

Techniques: Knock-Out

Journal: bioRxiv

Article Title: Basement membrane regulates fibronectin organization using sliding focal adhesions driven by a contractile winch

doi: 10.1101/618686

Figure Lengend Snippet: (A) Confocal images of an SCC-9 cell seeded on Matrigel for 6 hrs and immunostained for paxillin, integrin α5β1, and fibronectin. Remaining fibronectin matrix fibers form primarily near the cell periphery after integrin α5β1 blocking (B) or knock-out (C). (D) Quantification of fibronectin matrix of results from B and C. n =15 for blocking, n =20 for knock-out. (E) Merged TIRF live imaging of assembled fibronectin matrix fibers immediately before integrin α5β1 blocking and 3hrs after blocking; note the new fibronectin matrix fibers (magenta) assembled on the cell edge after blocking. (F) Integrin αVβ3 aggregation in focal adhesions of cells on vitronectin or collagen I was lost on Matrigel and could not be rescued by mixing Matrigel with vitronectin for coating the substrate. (G) Cells growing at the boundary between Matrigel and vitronectin. Note that within a single cell, only the region of the cell body on vitronectin shows integrin αVβ3 clustering, while the regions on Matrigel are more effective at assembling fibronectin matrix than on vitronectin. Error bars: SEM. ***p ≤ 0.001. Scale bars: 10 μm (A, E, F); 20 μm (B, C, G). See also .

Article Snippet: Matrigel, laminin, collagen IV, collagen I, perlecan and vitronectin were diluted to 20 μg/ml or other indicated concentrations and aliquoted into glass-bottom MatTek dishes or chambers (ibidi).

Techniques: Blocking Assay, Knock-Out, Imaging