title: High fidelity visualization of cell-to-cell variation and temporal dynamics in nascent extracellular matrix formation.
abstract: 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.
authors.affiliation: Department of Bioengineering, University of Pennsylvania, Philadelphia, PA 19104, USA.|McKay Orthopaedic Research Laboratory, Department of Orthopaedic Surgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.|Translational Musculoskeletal Research Center, Philadelphia VA Medical Center, Philadelphia, PA 19104, USA.
authors.forename: Claire M
authors.initials: CM
authors.lastname: McLeod
authors.affiliation: Department of Bioengineering, University of Pennsylvania, Philadelphia, PA 19104, USA.|McKay Orthopaedic Research Laboratory, Department of Orthopaedic Surgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.|Translational Musculoskeletal Research Center, Philadelphia VA Medical Center, Philadelphia, PA 19104, USA.
authors.forename: Robert L
authors.initials: RL
authors.lastname: Mauck
mesh_terms: D000409:Alanine / Q000031:analogs & derivatives / Q000378:metabolism; D000480:Alkynes / Q000378:metabolism; D000818:Animals; D002417:Cattle; D060833:Cellular Microenvironment; D019902:Chondrocytes / Q000378:metabolism* / Q000648:ultrastructure; D020219:Chondrogenesis; D005109:Extracellular Matrix / Q000378:metabolism* / Q000648:ultrastructure; D016326:Extracellular Matrix Proteins / Q000378:metabolism; D019085:Fluorescent Antibody Technique, Direct; D005456:Fluorescent Dyes / Q000032:analysis; D005998:Glycine / Q000031:analogs & derivatives / Q000378:metabolism; D059630:Mesenchymal Stem Cells / Q000378:metabolism* / Q000648:ultrastructure; D018613:Microscopy, Confocal / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*; D011806:Quinolinium Compounds / Q000032:analysis; D059010:Single-Cell Analysis
chemical_list: C481052:Alexa fluor 546; D000480:Alkynes; D016326:Extracellular Matrix Proteins; D005456:Fluorescent Dyes; D011806:Quinolinium Compounds; C506098:azidohomoalanine; C551041:homopropargylglycine; D000409:Alanine; D005998:Glycine