High fidelity visualization of cell-to-cell variation and temporal dynamics in nascent extracellular matrix formation.
High fidelity visualization of cell-to-cell variation and temporal dynamics in nascent extracellular matrix formation.
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DOI：
10.1038/srep38852
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发表时间：
2016-12-12
影响因子：
4.6
通讯作者：
Mauck RL
中科院分区：
文献类型：
--
作者：
McLeod CM;Mauck RL
细胞外基质动力学是组织形态发生、内稳态、损伤和修复的关键。这种基质的时空组织具有深远的生物学意义，但使用标准技术进行监测具有挑战性。在此，我们通过使用非经典氨基酸标记来对在生物正交甲硫氨酸类似物存在下合成的细胞外基质进行荧光标记，从而应对这些挑战。该策略以高分辨率标记基质蛋白，且不影响其分布或力学功能。我们证明蛋白质基质的组织和时间动态取决于微环境的生物物理特征，包括生物材料支架以及细胞自身构建的微环境。脉冲标记实验表明，在未成熟的构建体中，新生基质呈高度纤维状，并与预先存在的基质相互交错，而在更成熟的构建体中，新生基质缺乏纤维状组织，并保留在紧邻细胞的空间内。抑制胶原蛋白交联会增加基质合成，但会损害基质组织。最后，这些数据表明在经历软骨形成的软骨细胞和间充质干细胞之间存在显著的细胞间异质性。总之，这些结果引入了荧光非经典氨基酸标记作为一种研究时空基质组织的策略，并证明了其在单细胞水平上识别表型、微环境和基质组装差异的能力。
Extracellular matrix dynamics are key to tissue morphogenesis, homeostasis, injury, and repair. The spatiotemporal organization of this matrix has profound biological implications, but is challenging to monitor using standard techniques. Here, we address these challenges by using noncanonical amino acid tagging to fluorescently label extracellular matrix synthesized in the presence of bio-orthogonal methionine analogs. This strategy labels matrix proteins with high resolution, without compromising their distribution or mechanical function. We demonstrate that the organization and temporal dynamics of the proteinaceous matrix depend on the biophysical features of the microenvironment, including the biomaterial scaffold and the niche constructed by cells themselves. Pulse labeling experiments reveal that, in immature constructs, nascent matrix is highly fibrous and interdigitates with pre-existing matrix, while in more developed constructs, nascent matrix lacks fibrous organization and is retained in the immediate pericellular space. Inhibition of collagen crosslinking increases matrix synthesis, but compromises matrix organization. Finally, these data demonstrate marked cell-to-cell heterogeneity amongst both chondrocytes and mesenchymal stem cells undergoing chondrogenesis. Collectively, these results introduce fluorescent noncanonical amino acid tagging as a strategy to investigate spatiotemporal matrix organization, and demonstrate its ability to identify differences in phenotype, microenvironment, and matrix assembly at the single cell level.
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影响因子：
46.2
作者：
Jewett JC;Bertozzi CR
通讯作者：
Bertozzi CR
影响因子：
3.1
作者：
Farrell, Megan J.;Comeau, Eric S.;Mauck, Robert L.
通讯作者：
Mauck, Robert L.
影响因子：
4.7
作者：
Hong, Vu;Steinmetz, Nicole F.;Manchester, Marianne;Finn, M. G.
通讯作者：
Finn, M. G.
影响因子：
16.6
作者：
Beahm, Brendan J.;Dehnert, Karen W.;Derr, Nicolas L.;Kuhn, Joachim;Eberhart, Johann K.;Spillmann, Dorothe;Amacher, Sharon L.;Bertozzi, Carolyn R.
通讯作者：
Bertozzi, Carolyn R.
影响因子：
14.9
作者：
UniProt Consortium
通讯作者：
UniProt Consortium