Spatiotemporal dynamic monitoring of fatty acid–receptor interaction on single living cells by multiplexed Raman imaging Wei Zhanga, Fangjun Linb, Yan Liub, Han Zhanga, Timothy A. Gilbertsonb,1, and Anhong Zhoua,1 aDepartment of Biological Engineering, Utah State University, Logan, UT 84322-4105; and bDepartment of Internal Medicine, University of Central Florida, Orlando, FL 32827-7408 Edited by Sanjiv S. Gambhir, Stanford University, Stanford, CA, and accepted by Editorial Board Member Rakesh K. Jain December 16, 2019 (received for review September 18, 2019) Numerous fatty acid receptors have proven to play critical roles in and 377 residues for the long isoform (GPR120-L), with an ex- normal physiology. Interactions among these receptor types and tracellular N-terminal domain, seven transmembrane domains, their subsequent membrane trafficking has not been fully eluci- and an intracellular C-terminal domain (12). The N-terminal and dated, due in part to the lack of efficient tools to track these transmembrane domains may be responsive to ligands, such as cellular events. In this study, we fabricated the surface-enhanced linoleic acid (LA) and oleic acid (13, 14). Specifically, Arg99 in Raman scattering (SERS)-based molecular sensors for detection of transmembrane domain II has been identified as an essential two putative fatty acid receptors, G protein-coupled receptor 120 residue for GPR120 binding to its ligands (12). The intracellular (GPR120) and cluster of differentiation 36 (CD36), in a spatiotem- region of GPR120 is critical for its interaction with G proteins poral manner in single cells. These SERS probes allowed multiplex and associated scaffold proteins that link with the β-arrestin-2 detection of GPR120 and CD36, as well as a peak that represented system (12). The activation of GPR120 by FA or agonist (e.g., the cell. This multiplexed sensing system enabled the real-time GW9508) can inhibit inflammation, modulate hormone secretion, monitoring of fatty acid-induced receptor activation and dynamic improve glucose disposition, and enhance insulin sensitivity (12, distributions on the cell surface, as well as tracking of the recep- 15). Based on a report that GPR120-deficient mice fed a high-fat tors’ internalization processes on the addition of fatty acid. In- diet developed obesity and glucose intolerance (16), GPR120 is APPLIED BIOLOGICAL SCIENCES creased SERS signals were seen in engineered HEK293 cells with higher fatty acid concentrations, while decreased responses were attracting considerable attention as a potential novel therapeutic found in cell line TBDc1, suggesting that the endocytic process target for the treatment of obesity and diabetes (12, 17). requires innate cellular components. SERS mapping results confirm Cluster of differentiation 36 (CD36) is a scavenger receptor that GPR120 is the primary receptor and may work synergistically that functions in high-affinity tissue uptake of FFAs and contrib- with CD36 in sensing polyunsaturated fatty acids and promoting utes under excessive fat intake to lipid accumulation and metabolic + Ca2 mobilization, further activating the process of fatty acid up- dysfunction (18). As an integral membrane protein, CD36 contains take. The ability to detect receptors’ locations and monitor fatty 472 residues with both C and N termini inside the cell membrane. acid-induced receptor redistribution demonstrates the specificity Ubiquitination sites in the C terminus and palmitoylation sites in and potential of our multiplexed SERS imaging platform in the study of fatty acid–receptor interactions and might provide func- Significance tional information for better understanding their roles in fat in- take and development of fat-induced obesity. We have developed surface-enhanced Raman scattering (SERS) sensing probes that are capable of multiplexed detection of liv- surface-enhanced Raman scattering | GPR120 | CD36 | fatty acid | ing cell membrane receptors in a temporal and spatial manner at taste bud cell the cellular level, providing significant advantages over con- ventional immunostaining. The function of our SERS probes was he storage of excessive energy as fat in the human body is an proven in taste cells based on two receptors involved in fat Tevolutionary advantage allowing protection against starvation signaling: GPR120 and CD36. In situ SERS mapping was applied (1). However, in today’s world, obesity caused by excessive food to visualize and detect the activation of receptors and cellular intake and increased sedentary behavior (2) is predicted to replace processes on fatty acid treatment. The robust and sensitive SERS more traditional health concerns as the most significant cause of probes are useful in studying receptor distribution and function poor health, which can lead to various kinds of diseases, including and are amenable to G protein-coupled receptors, representing heart disease (3) and stroke (4), type II diabetes (5), and cancer the largest category of druggable targets that are linked to a (6). Raising free fatty acids (FAs) in plasma increases insulin re- myriad of diseases, including obesity, diabetes, and cancer. sistance, which further leads to obesity (7). Different types of FAs can either benefit human health (8) or increase the risk of diseases Author contributions: T.A.G. and A.Z. designed research; W.Z., F.L., Y.L., and H.Z. per- formed research; W.Z., F.L., Y.L., T.A.G., and A.Z. contributed new reagents/analytic tools; (9, 10). Understanding the underlying mechanism of dietary fat W.Z., T.A.G., and A.Z. analyzed data; and W.Z., T.A.G., and A.Z. wrote the paper. preference and intake may contribute to decreasing the worldwide The authors declare no competing interest. prevalence of obesity-related diseases. This article is a PNAS Direct Submission. S.S.G. is a guest editor invited by the The free FA receptor (FFAR) acts as an essential component Editorial Board. involving in the initial recognition of dietary fats in a number of Published under the PNAS license. physiological processes. FFARs belong to the G protein-coupled Data Deposition: The raw data generated and analyzed in this study are available at receptor (GPCR) class, the largest superfamily of receptors re- Zenodo (http://dx.doi.org/10.5281/zenodo.3560297). sponsible for signaling between cells and tissues that represent 1To whom correspondence may be addressed. Email: [email protected] or the major druggable targets in pharmacology (11). GPCR 120 [email protected]. (GPR120), expressed mainly in tissues and cells related to energy This article contains supporting information online at https://www.pnas.org/lookup/suppl/ metabolism (lung, gastrointestinal tract, tongue, and adipose tissue), doi:10.1073/pnas.1916238117/-/DCSupplemental. contains 361 amino acid residues for the short isoform (GPR120-S) www.pnas.org/cgi/doi/10.1073/pnas.1916238117 PNAS Latest Articles | 1of10 Downloaded by guest on September 28, 2021 both the C and N termini may regulate the turnover and trafficking on breast cancer cell lines, with results found to be in good of CD36 between the plasma membrane and intracellular organ- agreement with those obtained with conventional sorting by flow elles (19). Lys164 in the hydrophobic pocket of CD36 (18) is the cytometry (32). Our group has successfully applied polyelectrolyte- essential site for binding to ligands, although CD36 may have a coated gold nanorods to visualize the distribution of cancer bio- wide sequence (amino acids 127 to 279) to interact with FAs. A marker EGFR on single living cells (33). deficiency of CD36 (knockout or internalization) results in dys- Here we describe a multiplexed Raman imaging technique functional FA utilization, which may lead to diabetes (19). based on two synthesized SERS probes with the conjugation of FFA transport across cell membrane appears to be a highly specific antibodies to target receptors. This method provides high regulated process involving various membrane proteins, many of sensitivity (up to a million-fold signal enhancement) (34) and is a which are coexpressed in the same tissue. Several levels of regulation powerful tool for detecting and visualizing the distribution of single are coordinated, including translocation from intracellular storage molecules of membrane receptors on single living cell surfaces sites to the cell membrane (20, 21) and posttranslational modifi- (33). The reporter molecule and targeted antibody are adsorbed cation (e.g., phosphorylation, palmitoylation, glycosylation) (21). on gold or silver nanoparticles. The effect of localized surface GPR120 and CD36 are differentially regulated at the tran- plasmons results in significant enhancement of Raman signals scription and translation levels, and the relationship between these when the antibody binds its antigen (receptor or ligand) on the cell two receptors in the regulation of FFAs at molecular recognition membrane, which enables the detection of biomolecules at ultra- and cross-membrane transportation process remains unclear (22). high sensitivity at the single cell level. In addition, alterations of Activated GPR120 interacts with Gαq/11 protein, leading to SERS signals are also very sensitive to changes in the local envi- phospholipase-C (PLC) activation and subsequent elevation in ronment due to molecular events (35, 36). + + intracellular Ca2 concentration. Ca2 mobilization can promote In this study, HEK 293 cells transfected with the GPR120 gene glucagon-like peptide I secretion in taste bud cells and also ac- or