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.
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DOI：
10.1002/jbmr.3409
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发表时间：
2018-06
期刊：
影响因子：
--
通讯作者：
Dallas SL
中科院分区：
文献类型：
--
作者：
Lu Y;Kamel-El Sayed SA;Wang K;Tiede-Lewis LM;Grillo MA;Veno PA;Dusevich V;Phillips CL;Bonewald LF;Dallas SL
I型胶原蛋白是骨和其他结缔组织中最丰富的细胞外基质蛋白，在正常和病理骨形成以及结缔组织疾病和纤维化中起着关键作用。尽管人们对胶原蛋白的生物合成途径及其调控步骤了解甚多，但其在细胞外组装的机制尚不清楚。我们用GFPtpz或mCherry替代α2(I)-前胶原n端前肽，生成了GFPtpz和mCherry标记的胶原融合构建体，用于I型胶原组装的实时成像。这些新型成像探针被稳定地转染到MLO-A5成骨细胞样细胞和无纤维连接蛋白的小鼠胚胎成纤维细胞（FN-null-MEFs）中，用于成像I型胶原组装动力学及其对纤维连接蛋白的依赖。两种融合蛋白都与α1(I)-胶原共沉淀，在没有抗坏血酸的情况下留在细胞内，但在抗坏血酸存在的情况下组装成含有α1(I)-胶原的细胞外原纤维。免疫金电镜证实了它们在带状胶原原纤维中的超微结构定位。在稳定转染的MLO-A5细胞中，活细胞成像揭示了胶原组装的高度动态性质，并显示在组装过程中，原纤维网络由于潜在的细胞运动而不断拉伸和收缩。我们还观察到，细胞产生的力量可以物理地重塑胶原原纤维。通过mCherry-和gfptpz -胶原表达细胞的共培养，我们发现多个细胞贡献胶原形成胶原纤维束。免疫电镜进一步显示，单个胶原原纤维可以接受来自多个细胞的胶原蛋白的贡献。表达gfptpz -胶原的FN-null-MEFs的活细胞成像显示，胶原组装既依赖于纤维连接蛋白组装，又与纤维连接蛋白组装动态整合。这些gfp -胶原融合结构为活细胞中的胶原成像提供了强大的工具，并揭示了胶原细胞外组装动态机制的新颖和基本见解。
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. ImmunoEM 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.
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影响因子：
2.7
作者：
Aleksandrova A;Czirók A;Szabó A;Filla MB;Hossain MJ;Whelan PF;Lansford R;Rongish BJ
通讯作者：
Rongish BJ
影响因子：
2.1
作者：
Banos, Charles C.;Thomas, Amelia H.;Kuo, Catherine K.
通讯作者：
Kuo, Catherine K.
影响因子：
5.6
作者：
HULMES, DJS;KADLER, KE;PROCKOP, DJ
通讯作者：
PROCKOP, DJ
DOI：
10.1007/978-1-61779-415-5_26
发表时间：
2012-01-01
期刊：
BONE RESEARCH PROTOCOLS, SECOND EDITION
影响因子：
--
作者：
Dallas, Sarah L.;Veno, Patricia A.
通讯作者：
Veno, Patricia A.
影响因子：
7.5
作者：
Kadler KE;Hill A;Canty-Laird EG
通讯作者：
Canty-Laird EG