Fluorescent imaging of protein myristoylation during cellular differentiation and development

Abstract

Protein post-translational modifications (PTMs) serve to give proteins new cellular functions and can influence spatial distribution and enzymatic activity, greatly enriching the complexity of the proteome. Lipidation is a PTM that regulates protein stability, function, and subcellular localization. To complement advances in proteomic identification of lipidated proteins, we have developed a method to image the spatial distribution of proteins that have been co- and post-translationally modified via the addition of myristic acid (Myr) to the N terminus. In this work, we use a Myr analog, 12-azidododecanoic acid (12-ADA), to facilitate fluorescent detection of myristoylated proteins in vitro and in vivo. The azide moiety of 12-ADA does not react to natural biological chemistries, but is selectively reactive with alkyne functionalized fluorescent dyes. We find that the spatial distribution of myristoylated proteins varies dramatically between undifferentiated and differentiated muscle cells in vitro. Further, we demonstrate that our methodology can visualize the distribution of myristoylated proteins in zebrafish muscle in vivo. Selective protein labeling with noncanonical fatty acids, such as 12-ADA, can be used to determine the biological function of myristoylation and other lipid-based PTMs and can be extended to study deregulated protein lipidation in disease states.

Keywords

• fatty acid/transferase
• muscle
• fluorescence microscopy
• cells and tissues
• lipidation
• click chemistry