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 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.

Macrophages directly contribute collagen to scar formation during zebrafish heart regeneration and mouse heart repair

“…Macrophages directly contribute to collagen deposition and scar formation. To address whether macrophages contribute directly to collagen deposition in the P7 and adult mouse heart post-MI, we utilised a transgenic mouse line that expresses a collagen-GFP fusion protein to fluorescently label type-1 collagen fibrils 36 . We initially characterised GFPtpz-collagen deposition in adult mice post-MI ( Supplementary Fig.…”

The Connexin 43 Carboxyl Terminal Mimetic Peptide αCT1 Prompts Differentiation of a Collagen Scar Matrix Resembling Unwounded Skin

“…A tool that might assist with work of this type is GFP-tagged collagen and mouse lines expressing this protein. 74 In vitro experiments are presently underway using live-cell imaging of fibroblasts derived from GFP-collagen expressing mice to observe patterns of collagen laid down by motile cells and determine whether αCT1 increases the disorganization of newly secreted ECM. A further limitation is that we do not understand the molecular bases of the effect of the peptide on dermal collagen order in vivo.…”

Three‐dimensional intravital imaging in bone research

“…Hence, other methodologies should be adopted when dynamic events involved in bone formation, such as extracellular collagen assembly, are the imaging target. Lu et al revealed the dynamics of in vitro extracellular collagen assembly using time‐lapse confocal imaging. Imaging in cultured osteoblasts expressing fluorescent collagen and fibronectin‐null cells showed that collagen assembly is highly dynamic and influenced by constantly stretching and contracting from motile cells, and that collagen assembly is dependent on fibronectin deposition as a dynamically integrative event.…”