Col‐F, a fluorescent probe for ex vivo confocal imaging of collagen and elastin in animal tissues

Biela, Eva; Galas, Jerzy; Lee, Brian; Johnson, Gary L.; Darzynkiewicz, Zbigniew; Dobrucki, Jurek

doi:10.1002/cyto.a.22264

Cited by 32 publications

(30 citation statements)

References 26 publications

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“…A few investigators have used fluorescently tagged bacterial proteins that bind collagen or have used other collagen-binding proteins as probes for imaging collagen in live and fixed cells and tissues. (16)(17)(18) However, to our knowledge, these approaches have not yet been used for long-term time-lapse imaging studies to visualize the dynamic mechanisms for extracellular assembly of collagen fibril networks. Additional drawbacks are that these probes are somewhat non-specific and bind to many types of fibrillar collagens and they are less useful for observing intracellular steps in the assembly process.…”

Section: Introductionmentioning

confidence: 99%

Live Imaging of Type I Collagen Assembly Dynamics in Osteoblasts Stably Expressing GFP and mCherry-Tagged Collagen Constructs

Sayed

Wang

et al. 2018

Journal of Bone and Mineral Research

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Type I collagen is the most abundant extracellular matrix protein in bone and other connective tissues and plays key roles in normal and pathological bone formation as well as in connective tissue disorders and fibrosis. Although much is known about the collagen biosynthetic pathway and its regulatory steps, the mechanisms by which it is assembled extracellularly are less clear. We have generated GFPtpz and mCherry-tagged collagen fusion constructs for live imaging of type I collagen assembly by replacing the α2(I)-procollagen N-terminal propeptide with GFPtpz or mCherry. These novel imaging probes were stably transfected into MLO-A5 osteoblast-like cells and fibronectin-null mouse embryonic fibroblasts (FN-null-MEFs) and used for imaging type I collagen assembly dynamics and its dependence on fibronectin. Both fusion proteins co-precipitated with α1(I)-collagen and remained intracellular without ascorbate but were assembled into α1(I) collagen-containing extracellular fibrils in the presence of ascorbate. Immunogold-EM confirmed their ultrastuctural localization in banded collagen fibrils. Live cell imaging in stably transfected MLO-A5 cells revealed the highly dynamic nature of collagen assembly and showed that during assembly the fibril networks are continually stretched and contracted due to the underlying cell motion. We also observed that cell-generated forces can physically reshape the collagen fibrils. Using co-cultures of mCherry- and GFPtpz-collagen expressing cells, we show that multiple cells contribute collagen to form collagen fiber bundles. Immuno-EM further showed that individual collagen fibrils can receive contributions of collagen from more than one cell. Live cell imaging in FN-null-MEFs expressing GFPtpz-collagen showed that collagen assembly was both dependent upon and dynamically integrated with fibronectin assembly. These GFP-collagen fusion constructs provide a powerful tool for imaging collagen in living cells and have revealed novel and fundamental insights into the dynamic mechanisms for the extracellular assembly of collagen. © 2018 American Society for Bone and Mineral Research.

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Section: Introductionmentioning

confidence: 99%

Live Imaging of Type I Collagen Assembly Dynamics in Osteoblasts Stably Expressing GFP and mCherry-Tagged Collagen Constructs

Sayed

Wang

et al. 2018

Journal of Bone and Mineral Research

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“…To visualize production of ECM over time after the click cell assembly, the cells were stained with fluorescent probe markers specific for collagen and elastin (Fig. 7)35. Upon tissue assembly, the cells were treated with the dye marker and fixed with 4% formalin for various durations.…”

Section: Resultsmentioning

confidence: 99%

Scaffold Free Bio-orthogonal Assembly of 3-Dimensional Cardiac Tissue via Cell Surface Engineering

Rogozhnikov

O’Brien

Elahipanah

et al. 2016

Sci Rep

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There has been tremendous interest in constructing in vitro cardiac tissue for a range of fundamental studies of cardiac development and disease and as a commercial system to evaluate therapeutic drug discovery prioritization and toxicity. Although there has been progress towards studying 2-dimensional cardiac function in vitro, there remain challenging obstacles to generate rapid and efficient scaffold-free 3-dimensional multiple cell type co-culture cardiac tissue models. Herein, we develop a programmed rapid self-assembly strategy to induce specific and stable cell-cell contacts among multiple cell types found in heart tissue to generate 3D tissues through cell-surface engineering based on liposome delivery and fusion to display bio-orthogonal functional groups from cell membranes. We generate, for the first time, a scaffold free and stable self assembled 3 cell line co-culture 3D cardiac tissue model by assembling cardiomyocytes, endothelial cells and cardiac fibroblast cells via a rapid inter-cell click ligation process. We compare and analyze the function of the 3D cardiac tissue chips with 2D co-culture monolayers by assessing cardiac specific markers, electromechanical cell coupling, beating rates and evaluating drug toxicity.

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“…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].…”

Section: New Directions For the Evaluation Of In Vitro Fibrosis Platfmentioning

confidence: 99%

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

Porras

Hutson

Berger

et al. 2016

Current Opinion in Biotechnology

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Fibrotic diseases occur in virtually every tissue of the body and are a major cause of mortality, yet they remain largely untreatable and poorly understood on a mechanistic level. The development of anti-fibrotic agents has been hampered, in part, by the insufficient fibrosis biomimicry provided by traditional in vitro platforms. This review focuses on recent advancements toward creating 3-D platforms that mimic key features of fibrosis, as well as the application of novel imaging and sensor techniques to analyze dynamic extracellular matrix remodeling. Several opportunities are highlighted to apply new tools from the fields of biomaterials, imaging, and systems biology to yield pathophysiologically-relevant in vitro platforms that improve our understanding of fibrosis and may enable identification of potential treatment targets.

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Col‐F, a fluorescent probe for ex vivo confocal imaging of collagen and elastin in animal tissues

Cited by 32 publications

References 26 publications

Live Imaging of Type I Collagen Assembly Dynamics in Osteoblasts Stably Expressing GFP and mCherry-Tagged Collagen Constructs

Live Imaging of Type I Collagen Assembly Dynamics in Osteoblasts Stably Expressing GFP and mCherry-Tagged Collagen Constructs

Scaffold Free Bio-orthogonal Assembly of 3-Dimensional Cardiac Tissue via Cell Surface Engineering

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

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