Fluorescein isothiocyanate-labeled human plasma fibronectin in extracellular matrix remodeling.
Fluorescein isothiocyanate (FITC) is a well-known probe for labeling biologically relevant proteins. However, the impact of the labeling procedure on protein structure and biological activities remains unclear. In this work, FITC-labeled human plasma fibronectin (Fn) was developed to gain insight into the dynamic relationship between cells and Fn. The similarities and differences concerning the structure and function between Fn-FITC and standard Fn were evaluated using biochemical as well as cellular approaches. By varying the FITC/Fn ratio, we demonstrated that overlabeling (>10 FITC molecules/Fn molecule) induces probe fluorescence quenching, protein aggregation, and cell growth modifications. A correct balance between reliable fluorescence for detection and no significant modifications to structure and biological function compared with standard Fn was obtained with a final ratio of 3 FITC molecules per Fn molecule (Fn-FITC3). Fn-FITC3, similar to standard Fn, is correctly recruited into the cell matrix network. Also, Fn-FITC3 is proposed to be a powerful molecular tool to investigate Fn organization and cellular behavior concomitantly.
Hoffmann
Celine
C
Equipe de Recherche sur les Relations Matrice Extracellulaire-Cellules (ERRMECe), University of Cergy-Pontoise, 95302 Pontoise Cedex, France.
Leroy-Dudal
Johanne
J
Patel
Salima
S
Gallet
Olivier
O
Pauthe
Emmanuel
E
D000818:Animals; D016466:CHO Cells; D002448:Cell Adhesion; D006224:Cricetinae; D003412:Cricetulus; D005109:Extracellular Matrix / Q000378:metabolism*; D005353:Fibronectins / Q000097:blood*; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry*; D005455:Fluorescent Antibody Technique; D006801:Humans; D006868:Hydrolysis