Dataset Information

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

ABSTRACT: 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 ?2(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 ?1(I)-collagen and remained intracellular without ascorbate but were assembled into ?1(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.

SUBMITTER: Lu Y

PROVIDER: S-EPMC6425932 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature

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

Lu Yongbo Y Kamel-El Sayed Suzan A SA Wang Kun K Tiede-Lewis LeAnn M LM Grillo Michael A MA Veno Patricia A PA Dusevich Vladimir V Phillips Charlotte L CL Bonewald Lynda F LF Dallas Sarah L SL

Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 20180323 6

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 ...[more]

PMID: 29461659

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Project description:BackgroundImmunohistochemical staining experiments have shown that both hemangiogenesis and lymphangiogenesis occur following severe corneal and conjunctival injury and that the neovascularization of the cornea often has severe visual consequences. To better understand how hemangiogenesis and lymphangiogenesis are induced by different degrees of ocular injury, we investigated patterns of injury-induced corneal neovascularization in live Prox1-GFP/Flk1::myr-mCherry mice, in which blood and lymphatic vessels can be imaged simultaneously in vivo.MethodsThe eyes of Prox1-GFP/Flk1::myr-mCherry mice were injured according to four models based on epithelial debridement of the: A) central cornea (a 1.5-mm-diameter circle of tissue over the corneal apex), B) total cornea, C) bulbar conjunctiva, and D) cornea+bulbar conjunctiva. Corneal blood and lymphatic vessels were imaged on days 0, 3, 7, and 10 post-injury, and the percentages of the cornea containing blood and lymphatic vessels were calculated.ResultsNeither central corneal nor bulbar conjunctival debridement resulted in significant vessel growth in the mouse cornea, whereas total corneal and corneal+bulbar conjunctival debridement did. On day 10 in the central cornea, total cornea, bulbar conjunctiva, and corneal+bulbar conjunctival epithelial debridement models, the percentage of the corneal surface that was occupied by blood vessels (hemangiogenesis) was 1.9±0.8%, 7.14±2.4%, 2.29±1%, and 15.05±2.14%, respectively, and the percentage of the corneal surface that was occupied by lymphatic vessels (lymphangiogenesis) was 2.45±1.51%, 4.85±0.95%, 2.95±1.27%, and 4.15±3.85%, respectively.ConclusionsSubstantial corneal debridement was required to induce corneal neovascularization in the mouse cornea, and the corneal epithelium may therefore be partially responsible for maintaining corneal avascularity.General significanceOur study demonstrates that GFP/Flk1::myr-mCherry mice are a useful model for studying coordinated hemangiogenic and lymphangiogenic responses.

Dataset Information

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

Publications

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

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