Figure 1.
Osteoblasts are able to exert traction force on fibronectin-coated substrate in the absence of β1 integrin and ICAP-1. (A and B) Representative traction forces maps (A) and quantification (B) of the total force applied on fibronectin-coated polyacrylamide gel with a defined rigidity of 5 kPa. β1 integrin KO cells exert less force than the other osteoblasts mutants. The additional deletion of ICAP-1 led to generation of traction forces revealing a novel pathway independent of β1 integrins to generate traction forces on fibronectin. Scale bar, 10 μm. Error bars represent SD. N ≥ 60 cells. ns, adjusted P value >0.05; *, P value ≤ 0.05; **, P value ≤ 0.01; ***, P value ≤ 0.001; ****, P value ≤ 0.0001. (C) Immunofluorescence staining of the ppMyosin (ppMLC antibody, red) and F-actin (phalloidin, green) in the four osteoblasts cell lines showed that deletion of ICAP-1 alone does not change the organization of acto-myosin cytoskeleton but increases slightly the intensity and the thickness of the stress fibers (see quantification of the ppMLC area in E). Deletion of β1 integrin leads to a decrease of the cell area (D) and disorganization and decrease of thickness and number of the ppMLC decorated stress fibers. Scale bar, 20 µm. (D) Quantified cell spreading area from staining of fluorescent F-actin. Error bars represent SD. N ≥ 429 cells. ns, adjusted P value > 0.05; *, P value ≤ 0.05; **, P value ≤ 0.01; ***, P value ≤ 0.001; ****, P value ≤ 0.0001. (E) Quantified ppMLC staining, normalized to cell area. Error bars represent SD. N ≥ 211 cells. ns, adjusted P value > 0.05; *, P value ≤ 0.05; **, P value ≤ 0.01; ***, P value ≤ 0.001; ****, P value ≤ 0.0001. (F) Osteoblasts cells were spread in serum-free medium on uncoated glass for 24 h. Immunofluorescence staining of the extracellular fibronectin (cellular fibronectin antibody, red), F-actin (phalloidin, blue) and β3 integrins (Luc.A5 antibody, green) in the four osteoblasts cell lines were analyzed by fluorescent confocal microscopy. The β1+/+ cell lines orchestrate FN fibrillogenesis events and β1 integrin−/−/icap-1+/+ cells demonstrated very poor organization of synthetized fibronectin. β1 integrin−/−/icap-1−/− on the other side showed significant amount of FN fibrillogenesis. Scale bar, 20 µm. (G) FN fibrils length of thresholded images of deposited and organized FN fibrillogenesis were processed and quantified. 20 images per condition were analyzed. Quantifications reveal that the additional loss of ICAP-1 in β1 KO cell line increases the fibril length compared to the β1 KO cell line, expressing ICAP-1. Error bars represent SD. ns, adjusted P value > 0.05; *, P value ≤ 0.05; **, P value ≤ 0.01; ***, P value ≤ 0.001; ****, P value ≤ 0.0001.

Osteoblasts are able to exert traction force on fibronectin-coated substrate in the absence of β1 integrin and ICAP-1. (A and B) Representative traction forces maps (A) and quantification (B) of the total force applied on fibronectin-coated polyacrylamide gel with a defined rigidity of 5 kPa. β1 integrin KO cells exert less force than the other osteoblasts mutants. The additional deletion of ICAP-1 led to generation of traction forces revealing a novel pathway independent of β1 integrins to generate traction forces on fibronectin. Scale bar, 10 μm. Error bars represent SD. N ≥ 60 cells. ns, adjusted P value >0.05; *, P value ≤ 0.05; **, P value ≤ 0.01; ***, P value ≤ 0.001; ****, P value ≤ 0.0001. (C) Immunofluorescence staining of the ppMyosin (ppMLC antibody, red) and F-actin (phalloidin, green) in the four osteoblasts cell lines showed that deletion of ICAP-1 alone does not change the organization of acto-myosin cytoskeleton but increases slightly the intensity and the thickness of the stress fibers (see quantification of the ppMLC area in E). Deletion of β1 integrin leads to a decrease of the cell area (D) and disorganization and decrease of thickness and number of the ppMLC decorated stress fibers. Scale bar, 20 µm. (D) Quantified cell spreading area from staining of fluorescent F-actin. Error bars represent SD. N ≥ 429 cells. ns, adjusted P value > 0.05; *, P value ≤ 0.05; **, P value ≤ 0.01; ***, P value ≤ 0.001; ****, P value ≤ 0.0001. (E) Quantified ppMLC staining, normalized to cell area. Error bars represent SD. N ≥ 211 cells. ns, adjusted P value > 0.05; *, P value ≤ 0.05; **, P value ≤ 0.01; ***, P value ≤ 0.001; ****, P value ≤ 0.0001. (F) Osteoblasts cells were spread in serum-free medium on uncoated glass for 24 h. Immunofluorescence staining of the extracellular fibronectin (cellular fibronectin antibody, red), F-actin (phalloidin, blue) and β3 integrins (Luc.A5 antibody, green) in the four osteoblasts cell lines were analyzed by fluorescent confocal microscopy. The β1+/+ cell lines orchestrate FN fibrillogenesis events and β1 integrin−/−/icap-1+/+ cells demonstrated very poor organization of synthetized fibronectin. β1 integrin−/−/icap-1−/− on the other side showed significant amount of FN fibrillogenesis. Scale bar, 20 µm. (G) FN fibrils length of thresholded images of deposited and organized FN fibrillogenesis were processed and quantified. 20 images per condition were analyzed. Quantifications reveal that the additional loss of ICAP-1 in β1 KO cell line increases the fibril length compared to the β1 KO cell line, expressing ICAP-1. Error bars represent SD. ns, adjusted P value > 0.05; *, P value ≤ 0.05; **, P value ≤ 0.01; ***, P value ≤ 0.001; ****, P value ≤ 0.0001.

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