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Biomedical Imaging and Instrumentation

Biomedical Imaging and Instrumentation - Poster Session E

Poster G2 - Using Noncanonical Amino Acids to Label the Extracellular Matrix in Intact Organs

Saturday, October 26, 2024

10:00 AM – 11:00 AM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Kasey M. Shao (she/her/hers)

Student researcher
Princeton University
Cincinnati, Ohio, United States

Co-Author(s)

EF

Eric Fowler, PhD

Postdoctoral associate
Princeton University
Lawrence Township, New Jersey, United States

Primary Investigator(s)

Celeste M. Nelson, PhD (she/her/hers)

Wilke Family Professor in Bioengineering
Princeton University
Princeton, New Jersey, United States

Introduction: Fluorescent noncanonical amino acid tagging (FUNCAT) uses click chemistry to introduce fluorescent tags into proteins that have been synthesized by cells in the presence of noncanonical amino acids. Because it is a bioorthogonal metabolic labeling technique, FUNCAT is ideally suited to provide spatial information about proteins that are otherwise difficult to visualize, including the extracellular matrix (ECM) of non-genetic model systems. Here, we optimized FUNCAT for high-fidelity visualization of the ECM of the intact lungs of chicken embryos.

Materials and

Methods: Lungs were dissected from chicken embryos at days 4 and 5 after fertilization (E4 and E5). Lung explants were cultured in methionine-free culture media and were supplemented with the synthetic methionine replacement, L-azidohomoalanine (AHA). A click reaction was then performed between AHA and dibenzocyclooctyne (DBCO)-Alexa Fluor conjugated dye in order to label the AHA that had incorporated into proteins. Lung explants were then cleared and whole mounts were imaged to analyze ECM localization.

Results, Conclusions, and Discussions: Our data show that FUNCAT is effective for visualizing ECM proteins in embryonic chicken lungs. We find an increase in ECM production around the epithelium in E4 lungs, in regions distal to sites of branch initiation. We also find an increase in ECM production at the tips of branches in E5 lungs, consistent with the need for ECM remodeling during branch initiation and extension. Our data suggest that the ECM may play a role in defining the sites of early branch initiation and in reinforcing branch outgrowth. Further studies will continue to develop and optimize FUNCAT protocols and define spatiotemporal patterns of ECM production.

Acknowledgements (Optional):