Colorful Protein-Based Fluorescent Probes for Collagen Imaging

Abstract

Real-time visualization of collagen is important in studies on tissue formation and remodeling in the research fields of developmental biology and tissue engineering. Our group has previously reported on a fluorescent probe for the specific imaging of collagen in live tissue in situ, consisting of the native collagen binding protein CNA35 labeled with fluorescent dye Oregon Green 488 (CNA35-OG488). The CNA35-OG488 probe has become widely used for collagen imaging. To allow for the use of CNA35-based probes in a broader range of applications, we here present a toolbox of six genetically-encoded collagen probes which are fusions of CNA35 to fluorescent proteins that span the visible spectrum: mTurquoise2, EGFP, mAmetrine, LSSmOrange, tdTomato and mCherry. While CNA35-OG488 requires a chemical conjugation step for labeling with the fluorescent dye, these protein-based probes can be easily produced in high yields by expression in E. coli and purified in one step using Ni2+-affinity chromatography. The probes all bind specifically to collagen, both in vitro and in porcine pericardial tissue. Some first applications of the probes are shown in multicolor imaging of engineered tissue and two-photon imaging of collagen in human skin. The fully-genetic encoding of the new probes makes them easily accessible to all scientists interested in collagen formation and remodeling.

Decoupling the effects of stiffness and fiber density on cellular behaviors via an interpenetrating network of gelatin-methacrylate and collagen

“…To confirm the presence of a fibrillar collagen network within gelMA/collagen scaffolds, gels were stained with EGFP-tagged CNA35, a small protein that preferentially binds to fibrillar collagen [38]. Fluorescent visualization of the EGFP tag demonstrated a homogenous distribution of randomly oriented fibers in all collagen-containing conditions (Figure 2C).…”

“…The gels were incubated in 5 μM collagen-binding adhesion protein 35 fused with enhanced green fluorescent protein (CNA35-EGFP) for 18 hours at 37°C. The CNA35-EGFP, a fluorescently tagged collagen-binding protein that preferentially labels fibrillar collagen, was expressed and purified from E. coli as previously described [38]. The pET28a-EGFP-CNA35 plasmid was a gift from Maarten Merkx (Addgene plasmid # 61603).…”

Discoidin Domain Receptor-1 Regulates Calcific Extracellular Vesicle Release in Vascular Smooth Muscle Cell Fibrocalcific Response via Transforming Growth Factor-β Signaling

“…In DDR-1 −/− vSMCs, lack of collagen feedback from fibrillar collagen in the ECM not only resulted in increased collagen production (p<0.001, Fig.5B), but also led to defined changes in the three-dimensional structure of collagen fibers. The fluorescently labeled collagen binding protein CNA35 28 detected a dense network of amorphous collagen fibers devoid of spatial organization in cultures of DDR-1 −/− vSMCs after 21 days, whereas collagen produced by wild type vSMCs was found to be less dense (p<0.001), more fibrillar and restricted to the immediate vicinity of the vSMCs (Fig.5A). The addition of β-Glycerophosphate in calcifying media increased calcification in DDR −/− vSMCs compared to wild type as detected by a fluorescent calcium tracer 29 (p=0.002).…”

Engineering approaches to study fibrosis in 3-D in vitro systems

“…For instance, collagen-binding fluorescent probes [44,45•] can enable high-resolution imaging of collagen assembly and remodeling in living tissues, producing similar quality of information as SHG [46], but without the need for specialized imaging instrumentation. Molecular sensors have also been designed to non-invasively image the real-time activity of enzymes involved in fibrotic ECM remodeling, such as matrix metalloproteinases (MMPs) [47], as well as lysyl oxidase (LOX) [48], which crosslinks fibers of collagen type I and type III, as well as elastin [49].…”