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1.
FIGURE 6:

FIGURE 6:. From: Physiological type I collagen organization induces the formation of a novel class of linear invadosomes.

Linear invadosome kinetics and dynamics. (A) Linear invadosomes (white arrow) appear in the early stage of adhesion (from 15 min) on fibrillar collagen I. F-actin: red; Tks5: green; nuclei: blue. Scale bar: 7 μm. (B and C) Images from time-lapse video microscopy of BAECs transfected with Tks5-GFP (green) on fibrillar collagen I (red) showing static (B) and motile (C) linear invadosomes. Scale bar: 2 μm.

Amélie Juin, et al. Mol Biol Cell. 2012 January 15;23(2):297-309.
2.
FIGURE 7:

FIGURE 7:. From: Physiological type I collagen organization induces the formation of a novel class of linear invadosomes.

Linear invadosomes, a new class of β1 and β3 integrin–independent invadosomes. (A and B) MEFs were seeded on a mixed gelatin-FITC/collagen I matrix and stained respectively for β1 and β3 integrins. Linear invadosomes (white arrows) do not colocalize with integrins. β1 and β3: red; Tks5: green. Scale bar: 10 μm. Right panels, zoom (7×) of the white square. (C and D) MEFs were seeded on gelatin-FITC (C) or on mixed gelatin-FITC/collagen I matrix (D) and stained for Tks5 (green) and F-actin (red). Linear invadosomes and associated degradation are observed only in the presence of collagen I fibrils Gelatin-FITC: gray. Scale bar: 10 μm. (E) β1/ MEFs were seeded on the mixed matrix and exhibited functional linear invadosomes. Tks5: green; F-actin: red; gelatin-FITC: gray. Scale bar: 7 μm. (F) The same result was obtained with β3/MEFs. Tks5: green; F-actin: red; gelatin-FITC: gray. Scale bar: 5 μm. (G and H) Quantification of the number of linear invadosomes per micrometer squared and the fractional linear invadosomes area between WT, β3/, and β1/MEFs. NS: No significant difference.

Amélie Juin, et al. Mol Biol Cell. 2012 January 15;23(2):297-309.
3.
FIGURE 5:

FIGURE 5:. From: Physiological type I collagen organization induces the formation of a novel class of linear invadosomes.

Degradation activity of collagen I–induced linear F-actin structures in Src-3T3 cells. (A) Src-3T3 cells were seeded on a mixed matrix of fluorescent gelatin/fluorescent collagen I. Linear structures (white arrows) in Src-3T3 cells are associated with fibrils and degradation area of gelatin. Collagen I: red; Tks5: green; gelatin-FITC: gray. (B) Four hours after seeding, cells were fixed and analyzed for gelatin degradation (in situ zymography). Src-3T3cells are able to degrade gelatin. Gelatin-FITC: gray; degradation area: black. However, on the mixed matrix, the presence of collagen I fibrils is seen to enhance the degradation activity compared with gelatin alone (mean ± SD; n = 30; three experiments; *** p < 0.001 compared with control). Scale bar: 20 μm. (C) Quantification of cells exhibiting linear structures and rosettes on mixed matrix after 4 and 24 h (n = 300; three experiments; *** p < 0.001 compared with control). (D and E) Src-3T3 cells were seeded on fluorescent collagen I. Four hours after seeding, the majority of cells exhibits linear structures, and collagen I fibrils are maintained (D). Twenty-four hours after seeding, most collagen I fibrils have been degraded. This is concomitant with reformation of classical rosettes in most cells (E).

Amélie Juin, et al. Mol Biol Cell. 2012 January 15;23(2):297-309.
4.
FIGURE 2:

FIGURE 2:. From: Physiological type I collagen organization induces the formation of a novel class of linear invadosomes.

Molecular characterization of linear F-actin structures induced in the presence of collagen I fibrils. (A) BAECs were seeded on 0.4 mg/ml fibrillar collagen I and processed for fluorescent staining: F-actin (red), nuclei (blue), and invadosome markers (green), such as phosphotyrosine (4G10), N-WASP, paxillin, and Tks5. Merged image indicates colocalization of linear F-actin structures with phosphotyrosine, N-WASP, and Tks5, but not with paxillin. In zoom of white square (3.5×) colocalization of phosphotyrosine and paxillin can be noted with focal adhesions at the extremity of stress fibers. N-WASP and Tks5 colocalize with linear F-actin structures but not with focal adhesions. Scale bar: 5 μm. (B) Top panel, Src-3T3 cells seeded on glass coverslips exhibited classical “rosettes.” F-actin: red; Tks5: green. Scale bar: 10 μm. Right, zoom (3.5×) of the white square shows the presence of Tks5 in a “rosette.” Bottom panel, Src-3T3 cells were seeded on collagen I fibrils and exhibited linear F-actin structures (white arrows) F-actin: red; Tks5: green. Scale bar: 10 μm. Right, zoom (3×) of the white square shows a localization of Tks5 in collagen I–induced structures (white arrows). (C) MDA-MB-231 were seeded on glass coverslips, where invadopodia were observed (top panel), and on collagen I fibrils, where invadopodia were linearized (bottom panel). F-actin: red; Tks5: green. Scale bars: 10 μm. Zoom: 4.4×. (D) RAW 264.7-derived macrophages form rosettes of podosomes on glass coverslips (top panel, scale bar: 5 μm) and F-actin structures on collagen I fibrils (bottom panel, scale bar: 6 μm). F-actin: red; cortactin: green. Zoom; 4.4×.

Amélie Juin, et al. Mol Biol Cell. 2012 January 15;23(2):297-309.
5.
FIGURE 3:

FIGURE 3:. From: Physiological type I collagen organization induces the formation of a novel class of linear invadosomes.

Tks5 is required for collagen I–induced linear F-actin structure formation. (A) Protein extracts were analyzed by Western blotting. All cell types tested express Tks5, with the exception of PAECs. Tubulin content is shown as a loading control. (B) Immunoblot showing Tks5 expression level in PAECs transiently transfected with Tks5-GFP or GFP alone. (C) PAECs transiently transfected with a Tks5-GFP present linear F-actin structures on collagen I fibril matrix. F-actin (red) colocalizes with Tks5-GFP construct in these structures (white arrows). Insets are zoomed images of white squares. Scale bar: 10 μm. (D) PAECs were transfected with a GFP control construct unable to promote formation of these structures. Insets are zoomed images of white squares. Scale bar: 10 μm. (E) BAECs were transfected with a control siRNA (CT) or an siRNA targeting Tks5 (siRNA1 or siRNA2). Cells with a down-regulated expression of Tks5 present a decrease in linear F-actin structure formation. Shown is the percentage of BAECs presenting collagen I–induced actin structures ± SD (n = 1200; three experiments; ** p < 0.01). Protein extracts of BAECs transfected with indicated siRNA were analyzed by Western blotting.

Amélie Juin, et al. Mol Biol Cell. 2012 January 15;23(2):297-309.
6.
FIGURE 8:

FIGURE 8:. From: Physiological type I collagen organization induces the formation of a novel class of linear invadosomes.

Linear invadosome formation in a three-dimensional environment. (A) Schematic representation of BAECs showing linear invadosomes (green lines) in a collagen I environment. The red and blue dotted lines correspond to focal planes of 0 and 1.6 μm, respectively. (B and C) Confocal and IRM images of BAECs reveal that linear invadosomes are localized at the bottom of the cell (Z: 0 μm) in contact with collagen I fibrils. F-actin: red; Tks5: green. Note that focal adhesions stained for β1 (gray levels) can be detected at the ventral part of BAECs, although they never colocalize with linear invadosomes. Linear invadosomes (white square) are present at the apical part of BAECs, as shown by the focal plane and selected IRM images (Z: 1.6 μm). Insets are zoomed images of the white squares. Scale bar: 5 μm. (D) Representative confocal images of BAECs embedded in a three-dimensional fibrillar collagen I matrix. F-actin (red) colocalizes with Tks5 (green) at linear invadosome level (white arrows), as depicted on merged image. Z-cut section reveals that linear invadosomes do exist in three dimensions. Nuclei: blue.

Amélie Juin, et al. Mol Biol Cell. 2012 January 15;23(2):297-309.
7.
FIGURE 4:

FIGURE 4:. From: Physiological type I collagen organization induces the formation of a novel class of linear invadosomes.

Degradation activity of collagen I–induced linear F-actin structures in BAECs. (A and B) BAECs were seeded on fluorescent gelatin-FITC or on a mixed matrix (gelatin-FITC + collagen I fibrils) and stained for Tks5 (green) and nuclei (blue). After 24 h, BAECs show degradation activity of gelatin only on the mixed matrix containing collagen I fibrils. Scale bar: 10 μm. (C) To confirm the association of linear F-actin structures with the degradation activity, BAECs were seeded on a mixed gelatin/collagen matrix with collagen I fibrils labeled with succinimidyl-ester-568. Linear F-actin structures were formed along fibrils and a degradation area were observed underneath. Scale bar: 5 μm. (D) Quantification of the degradation activity that was observed only in the collagen I fibril condition (mean ± SD; n = 100; three experiments; *** p < 0.001 compared with control gelatin/collagen I fibrils). (E) Tks5 (green) and MT1-MMP (red) colocalize in the linear F-actin structures formed in BAECs on the mixed matrix. Scale bar: 5 μm. (F) In the same condition (gelatin/collagen I fibrils), BAECs were treated overnight with the MMP inhibitor GM6001 at 5 μM. Scale bar: 5 μm. (G) For analysis of collagen I fibril degradation, BAECs were seeded on collagen I fibrils labeled with succinimidyl-ester-568. No degradation of collagen I fibrils can be observed after 24 h. After 3 d, the density of collagen I fibrils decreased, and after 6 d, the degradation was complete. Scale bar: 35 μm.

Amélie Juin, et al. Mol Biol Cell. 2012 January 15;23(2):297-309.
8.
FIGURE 1:

FIGURE 1:. From: Physiological type I collagen organization induces the formation of a novel class of linear invadosomes.

Fibrillar collagen I induces linear F-actin structures. (A) Representative confocal microscopy images of linear F-actin structures (white arrows) associated with a reorganization of actin cytoskeleton in LSECs on collagen I matrix. Control cells (CT) were seeded on glass. F-actin (red) and nuclei (blue) were stained respectively with phalloidin and Hoechst. Right panels show zoom of the white squares. Scale bar: 7 μm. (B) Linear F-actin structures are observed only on collagen I. Shown is the percentage of LSECs presenting linear F-actin structures. Results are expressed as mean ± SD. (n = 200; three experiments; ***p < 0.001). (C) IRM images showing control (CT = glass) and acetic acid (AA) at room temperature (Collagen I RT) or 37°C (Collagen I 37°C) conditions of collagen I coating. Neutral pH and warmer conditions increased fibrillogenesis. Scale bar: 5 μm. (D) Collagen I fibrillogenesis induces the formation of linear F-actin microdomains. Shown is the percentage of LSECs presenting linear F-actin structures (mean ± SD; n = 200; three experiments; *** p < 0.001 as compared with control). (E) Confocal images of LSECs on fibrillar collagen-FITC (green). Actin structures (white arrows) are formed exclusively along collagen I fibrils. Scale bars: 5 μm. A three-dimensional reconstruction shows the association between linear F-actin structures (red) and collagen I fibers (green).

Amélie Juin, et al. Mol Biol Cell. 2012 January 15;23(2):297-309.

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