Fluorescently labeled collagen binding proteins allow specific visualization of collagen in tissues and live cell culture.
Visualization of the formation and orientation of collagen fibers in tissue engineering experiments is crucial for understanding the factors that determine the mechanical properties of tissues. In this study, collagen-specific fluorescent probes were developed using a new approach that takes advantage of the inherent specificity of collagen binding protein domains present in bacterial adhesion proteins (CNA35) and integrins (GST-alpha1I). Both collagen binding domains were obtained as fusion proteins from an Escherichia coli expression system and fluorescently labeled using either amine-reactive succinimide (CNA35) or cysteine-reactive maleimide (GST-alpha1I) dyes. Solid-phase binding assays showed that both protein-based probes are much more specific than dichlorotriazinyl aminofluorescein (DTAF), a fluorescent dye that is currently used to track collagen formation in tissue engineering experiments. The CNA35 probe showed a higher affinity for human collagen type I than did the GST-alpha1I probe (apparent K(d) values of 0.5 and 50 microM, respectively) and showed very little cross-reactivity with noncollagenous extracellular matrix proteins. The CNA35 probe was also superior to both GST-alpha1I and DTAF in visualizing the formation of collagen fibers around live human venous saphena cells. Immunohistological experiments on rat tissue showed colocalization of the CNA35 probe with collagen type I and type III antibodies. The fluorescent probes described here have important advantages over existing methods for visualization of collagen, in particular for monitoring the formation of collagen in live tissue cultures over prolonged time periods.
Laboratory for Cell and Tissue Engineering, Department of Biomedical Engineering, Eindhoven University of Technology, 5600 MB Eindhoven, The Netherlands.
Katy Nash
KN
Krahn
Carlijn V C
CV
Bouten
Sjoerd
S
van Tuijl
Marc A M J
MA
van Zandvoort
Maarten
M
Merkx
D018829:Adhesins, Bacterial / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D003094:Collagen / Q000032:analysis / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism*; D005982:Glutathione Transferase / Q000235:genetics; D006801:Humans; D039423:Integrin alpha1 / Q000235:genetics / Q000378:metabolism*; D007403:Intervertebral Disc / Q000737:chemistry; D008856:Microscopy, Fluorescence; D018482:Muscle, Skeletal / Q000737:chemistry; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D013710:Tendons / Q000737:chemistry
D018829:Adhesins, Bacterial; D005456:Fluorescent Dyes; D039423:Integrin alpha1; D011993:Recombinant Fusion Proteins; C048165:adhesin, Staphylococcus aureus; D003094:Collagen; D005982:Glutathione Transferase