“Fix and Click” for Assay of Sphingolipid Signaling in Single Primary Human Intestinal Epithelial Cells

Abstract

Capillary electrophoresis with fluorescence detection (CE-F) is a powerful method to measure enzyme activation in single cells. However, cellular enzymatic assays used in CE-F routinely utilize reporter substrates that possess a bulky fluorophore that may impact enzyme kinetics. To address these challenges, we describe a “fix and click” method utilizing an alkyne-terminated enzyme activation reporter, aldehyde-based fixation, and a click chemistry reaction to attach a fluorophore prior to analysis by single-cell CE-F. The “fix and click” strategy was utilized to investigate sphingolipid signaling in both immortalized cell lines and primary human colonic epithelial cells. When the sphingosine alkyne reporter was loaded into cells, this reporter was metabolized to ceramide (31.6 ± 3.3% peak area) without the production of sphingosine-1-phosphate. In contrast, when the reporter sphingosine fluorescein was introduced into cells, sphingosine fluorescein was converted to sphingosine-1-phosphate and downstream products (32.8 ± 5.7% peak area) without the formation of ceramide. Sphingolipid metabolism was measured in single cells from both differentiated and stem/proliferative human colonic epithelium using “fix and click” paired with CE-F to highlight the diversity of sphingosine metabolism in single cells from primary human colonic epithelium. This novel method will find widespread utility for the performance of single-cell enzyme assays by virtue of its ability to temporally and spatially separate cellular reactions with alkyne-terminated reporters, followed by the assay of enzyme activation at a later time and place.

Analytical Techniques for Single-Cell Biochemical Assays of Lipids

“…Many separation techniques have been applied to assay single cells, including TLC, capillary electrophoresis (CE), and even HPLC (4,(185)(186)(187)(188). To date, CE is the most prevalent method used for lipid assays in single cells, with TLC as an emerging technique.…”

“…Use of multiple fluorophores with different spectral properties further increases the number of analytes that can be simultaneously measured in CE. As long as the analytes remain fluorescent, CE can be used to track downstream metabolites as they form from a reporter molecule (4,187). Single-cell lipid assays by CE can also be fully automated and integrated with microscopy methods (185).…”

“…However, some members of these lipid groups such as GLs and free fatty acids (FFAs) act as signaling molecules to regulate energy homeostasis, insulin secretion, gene expression, cell survival, and cell proliferation (3). Members of the SL family such as ceramide, sphingosine, and their metabolites play a role in signal transduction pathways, while other SL members direct protein sorting, mediate cell-to-cell interactions, or form signaling hubs (lipid microdomains and rafts) (4). Members of the remaining four lipid categories (saccharolipids, polyketides, sterol lipids, and prenol lipids) play diverse roles including participation in lipid bilayers, biosynthetic pathways, and signaling pathways, as well as antioxidant functions (1).…”

Capillary Electrophoresis: A Three-Year Literature Review

“…In a recent study, a CE separation coupled with fluorescence detection was conducted to investigate sphingolipid signaling in both immortalized cells from a cell line and primary human colonic epithelial cells . The authors developed a fix and click approach to address the fluorescence signal issue.…”

“…In a recent study, a CE separation coupled with fluorescence detection was conducted to investigate sphingolipid signaling in both immortalized cells from a cell line and primary human colonic epithelial cells. 202 The authors developed a fix and click approach to address the fluorescence signal issue. Through quantitative or qualitative measurements of genetic, proteomic, or metabolic products, the CE-fluorescence-based single-cell analysis demonstrated its ability to investigate molecular heterogeneity in morphologically similar cell populations.…”