Controlling Fibronectin Fibrillogenesis Using Visible Light.
AFM
fibrillogenesis
fibronectin
matrix nanomechanics
visible light
Journal
Frontiers in molecular biosciences
ISSN: 2296-889X
Titre abrégé: Front Mol Biosci
Pays: Switzerland
ID NLM: 101653173
Informations de publication
Date de publication:
2020
2020
Historique:
received:
31
01
2020
accepted:
15
06
2020
entrez:
1
8
2020
pubmed:
1
8
2020
medline:
1
8
2020
Statut:
epublish
Résumé
We previously developed a surface-assisted assay to image early steps of cell-induced plasma fibronectin (FN) fibrillogenesis by timelapse atomic force microscopy (AFM). Unexpectedly, complementary attempts to visualize FN fibrillogenesis using fluorescently labeled FN (Alexa Fluor 488 or 568) and live-cell light microscopy initially failed consistently. Further analysis revealed that fibrillar remodeling was inhibited efficiently in the focal area illuminated during fluorescence imaging, but progressed normally elsewhere on the substrate, suggesting photo sensitivity of the FN fibrillogenesis process. In agreement, active cell-driven fibrillar extension of FN could be stopped by transient illumination with visible light during AFM timelapse scanning. Phototoxic effects on the cells could be ruled out, because pre-illuminating the FN layer before cell seeding also blocked subsequent fibrillar formation. Varying the illumination wavelength range between 400 and 640 nm revealed strong inhibition across the visible spectrum up to 560 nm, and a decreasing inhibitory effect at longer wavelengths. The photo effect also affected unlabeled FN, but was enhanced by fluorophore labeling of FN. The inhibitory effect could be reduced when reactive oxygen species (ROS) were removed for the cell imaging medium. Based on these findings, FN fibrillogenesis could be imaged successfully using a labeling dye with a long excitation wavelength (Alexa Fluor 633, excitation at 632 nm) and ROS scavengers, such as oxyrase, in the imaging medium. Fibrillar remodeling of exposed cell-free FN layers by AFM scanning required higher scan forces compared to non-exposed FN, consisting with mechanical stiffing of the FN layer after illumination. In agreement with changes in FN mechanics, cells spreading on pre-exposed FN showed reduced migration speeds, altered focal adhesion arrangement, and changes in mechanosensitive signaling pathways, including reduced FAK (Y397) and paxillin (Y118) phosphorylation. Pre-exposure of FN to visible light prior to cell seeding thus provides a useful tool to delineate mechanosensitive signaling pathway related to FN fibrillogenesis. When using FN-coated cell adhesion substrates, care should be taken when comparing experimental results obtained on non-exposed FN layers in cell culture incubators, or during live-cell fluorescence imaging, as FN fibrillogenesis and mechanosensitive cellular signaling pathways may be affected differently.
Identifiants
pubmed: 32733919
doi: 10.3389/fmolb.2020.00149
pmc: PMC7360794
doi:
Types de publication
Journal Article
Langues
eng
Pagination
149Informations de copyright
Copyright © 2020 Gudzenko and Franz.
Références
Proc Natl Acad Sci U S A. 1994 Apr 26;91(9):3984-8
pubmed: 7513429
J Mol Biol. 1998 Jul 10;280(2):245-58
pubmed: 9654449
Cell. 1981 Jul;25(1):121-32
pubmed: 6791830
Mol Biol Cell. 2015 Sep 15;26(18):3190-204
pubmed: 26371081
J Cell Sci. 1995 Feb;108 ( Pt 2):821-9
pubmed: 7539441
J Cell Biol. 1981 Dec;91(3 Pt 1):673-78
pubmed: 7328116
Methods Mol Biol. 2009;522:261-74
pubmed: 19247612
Am J Clin Oncol. 2003 Aug;26(4):e86-91
pubmed: 12902904
J Biol Chem. 1983 May 25;258(10):6595-601
pubmed: 6133865
Exp Cell Res. 1995 Mar;217(1):109-17
pubmed: 7867709
EMBO J. 1987 Aug;6(8):2343-9
pubmed: 3665879
Proc Natl Acad Sci U S A. 1983 Jan;80(1):137-41
pubmed: 6218503
J Cell Biol. 1995 May;129(3):831-42
pubmed: 7537275
Biol Cell. 2000 Oct;92(7):477-94
pubmed: 11229600
Photochem Photobiol Sci. 2010 Dec;9(12):1589-96
pubmed: 21082123
Photochem Photobiol. 1994 Mar;59(3):284-9
pubmed: 8016206
J Cell Sci. 2002 Oct 15;115(Pt 20):3861-3
pubmed: 12244123
Methods Enzymol. 2010;475:541-64
pubmed: 20627170
Braz J Med Biol Res. 1999 Mar;32(3):255-66
pubmed: 10347781
J Mol Biol. 1982 Jul 25;159(1):109-23
pubmed: 7131558
Fibrogenesis Tissue Repair. 2011 Sep 16;4:21
pubmed: 21923916
Eur Biophys J. 2006 Oct;35(8):695-708
pubmed: 16944124
Biochem J. 1980 Dec 1;191(3):719-27
pubmed: 6456720
J Biol Chem. 1982 May 25;257(10):5831-8
pubmed: 6175633
Int J Dev Biol. 1990 Mar;34(1):139-47
pubmed: 2203453
Biosci Rep. 1984 Dec;4(12):1017-26
pubmed: 6099154
Matrix Biol. 2005 Sep;24(6):389-99
pubmed: 16061370
Biochem J. 1989 Sep 15;262(3):707-12
pubmed: 2556107
J Cell Sci. 2007 Jan 1;120(Pt 1):137-48
pubmed: 17164291
J Pharm Sci. 2009 Sep;98(9):2909-34
pubmed: 18823031
Biochim Biophys Acta. 2002 May 20;1597(1):12-21
pubmed: 12009397
PLoS Biol. 2007 Oct 2;5(10):e268
pubmed: 17914904
Biochem J. 1986 Mar 1;234(2):399-403
pubmed: 3718475
J Biol Chem. 2000 Apr 7;275(14):10673-82
pubmed: 10744764
Annu Rev Cell Dev Biol. 2010;26:397-419
pubmed: 20690820
J Cell Biol. 1992 Nov;119(4):893-903
pubmed: 1385444
J Cell Sci. 2002 Mar 15;115(Pt 6):1221-9
pubmed: 11884521
J Biol Chem. 1995 Nov 3;270(44):26270-7
pubmed: 7592835
Biochem Soc Trans. 2017 Feb 8;45(1):229-236
pubmed: 28202677
Mol Biol Cell. 1997 Dec;8(12):2563-73
pubmed: 9398676
J Biol Chem. 1984 Oct 10;259(19):11901-7
pubmed: 6434532
Exp Cell Res. 2017 Jan 1;350(1):253-266
pubmed: 27919745
Biochim Biophys Acta. 1978 Jun 16;510(1):140-50
pubmed: 667030
Annu Rev Cell Biol. 1985;1:67-90
pubmed: 3916323
Biochim Biophys Acta. 2001 Apr 2;1504(2-3):196-219
pubmed: 11245785
Biochem Biophys Res Commun. 2003 Jun 6;305(3):761-70
pubmed: 12763058
J Cell Biol. 1983 Aug;97(2):466-72
pubmed: 6309861
J Cell Biochem. 1984;26(2):107-16
pubmed: 6084665
Int J Cancer. 2002 Jan 20;97(3):330-5
pubmed: 11774284
J Cell Biol. 2000 Mar 6;148(5):1075-90
pubmed: 10704455
Cell. 1979 Mar;16(3):675-85
pubmed: 222466
Cold Spring Harb Perspect Biol. 2011 Jul 01;3(7):
pubmed: 21576254
J Cell Sci. 1988 Apr;89 ( Pt 4):507-13
pubmed: 3198704
Cell. 1992 Dec 11;71(6):891-4
pubmed: 1458538
Matrix Biol. 2017 Jul;60-61:27-37
pubmed: 27496349
Nat Commun. 2015 Jun 04;6:7275
pubmed: 26041410
Cell. 1983 Dec;35(2 Pt 1):421-31
pubmed: 6317187
J Mol Biol. 2007 Sep 21;372(3):594-607
pubmed: 17686490
J Biol Chem. 1994 Jul 29;269(30):19646-52
pubmed: 7518462
J Biol Chem. 1993 Oct 15;268(29):21883-8
pubmed: 7691819
Photochem Photobiol. 1990 Oct;52(4):761-8
pubmed: 2089424
Cell. 1978 Jun;14(2):377-91
pubmed: 667946
Annu Rev Cell Biol. 1988;4:183-207
pubmed: 2848551
J Cell Sci. 1997 Mar;110 ( Pt 5):569-81
pubmed: 9092939
Cancer Res. 1979 Sep;39(9):3774-9
pubmed: 383288
Photochem Photobiol Sci. 2012 Jan;11(1):38-53
pubmed: 21858349
Biomater Sci. 2017 Jul 25;5(8):1629-1639
pubmed: 28612067
Mol Cell Biol. 1994 Jan;14(1):147-55
pubmed: 7505391
J Biol Chem. 1997 May 16;272(20):13189-95
pubmed: 9148935
PLoS One. 2014 Mar 27;9(3):e93123
pubmed: 24675966
Protein J. 2008 Jun;27(4):223-33
pubmed: 18330684
Adv Protein Chem. 1968;23:121-282
pubmed: 4882248