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 -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 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 , modulate hormone secretion, monitoring of fatty acid-induced receptor activation and dynamic improve glucose disposition, and enhance 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 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 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 glucagon-like peptide I secretion in taste bud cells and also ac- or CD36 gene and immortalized or primary taste bud cells were tivates phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K), used as cell models (Fig. 1). The taste bud-derived (TBD) cell which further induces glucose uptake (12). Interaction of ligands line TBDc1 expresses both CD36 and GPR120 (SI Appendix, Fig. with CD36 has been shown to induce CD36-mediated intracellular S1). Calcium imaging was performed to measure the intracellular signaling in many cases, often initiated by Src tyrosine kinases and responses to LA treatment, while Raman spectroscopy was used involving pathways linked to angiogenesis, inflammation, or ath- to investigate the process of LA recognition by the receptors in erosclerosis. FA interactions with CD36 can also induce intracel- real time by single point measurement (0 to 6 min) and single cell lular calcium release from the endoplasmic reticulum (ER) via a mapping (0 to 21 min). The potential internalization of receptors phospholipase Cβ (PLCβ)andIP3 pathway (18). The rise in in- in three cell lines on LA or agonist activation was analyzed along tracellular calcium levels can be monitored in real time in living with our Raman spectroscopy results. cells via the use of Ca-sensitive dyes, such as Fura-2 and Fluo-4 + (23, 24). Currently, in vitro Ca2 signaling in taste bud cells is the Results primary approach to studying the role of GPR120 and CD36 in Calcium Imaging. To validate our choice of cell line for study- sensing of dietary fat (25); however, the extracellular process of ing LA signaling in these experiments, we initially performed + GPR120 and/or CD36 binding with FFAs to trigger FFA uptake, ratiometric Ca2 imaging using HEK cell lines expressing GPR120 subsequent signaling, and receptor endocytosis and recycling or CD36 and the TBDc1. Four concentrations of LA (5, 20, 30, remains incompletely understood. and 60 μM) known to stimulate calcium changes in native taste As for other cell membrane receptors, the distribution of cells were tested. As shown in Fig. 2, there was a concentration- GPR120 and CD36 receptors on cells and tissues is visualized by dependent rise in intracellular calcium in all three cell lines, immunohistochemistry (IHC) or immunofluorescence staining pointing to their suitability for use in the current study. In addition, + (13, 26). Various measurement techniques have been developed the logistic regression curve between LA concentration and Ca2 and applied to detect receptors and ligand–receptor interactions. intensity shows that the FFARs on each cell surface appear satu- Radiolabeled labeled reporters are widely used to detect plasma rated over 60 μMLA(Fig.2D–F, Inset). It is well documented membrane receptors and the internalization of receptors (27). that FAs interact with GPR120 and CD36 to induce intracellular The main disadvantage of this assay is the safety concerns as- calcium release from the ER by PLCβ, which in turn triggers sociated with radiolabeling. Alternatively, fluorescence-labeled calcium flux from membrane store-operated calcium channels receptor internalization into intracellular compartments can be (Fig. 1) (17, 18). In our experiments, HEK293 and TBDc1 cells + visualized under fluorescence microscopy (28). However, in fluo- showed rapidly increasing intracellular Ca2 levels (within ∼3min), rescence labeling-based imaging, the cells are fixed, and fluores- indicating that a minimum of 3 min is necessary for complete cence images may bias observations; for example, the receptor activation of the transduction machinery in these cells. We con- integrity on fixed cells may be destroyed. The dynamic events sidered this time course when designing experiments aimed at of receptor activation or deactivation, even conformational monitoring the activity of the two putative FFARs, GPR120 and change, can be accessed by receptor fluorescence resonance CD36, using specifically designed SERS probes for the purpose of energy transfer (FRET) and bioluminescence resonance energy monitoring the activity and distribution of these receptors on living transfer (BRET) in a near-native system (29); however, FRET cell membranes before, during, and after LA treatment. may suffer from time-dependent decreases in signal intensity due to exposure to incident laser light (photobleaching), and geneti- Real-Time Monitoring of SERS Peaks in Single Point Measurement. cally encoded specific fluorescence proteins are sensitive to the The incubation time required for SERS probes with cells to bind local environment (such as pH and temperature) (30), and BRET with inactivated receptors varies from a few hours to longer might not be suitable for image acquisition due to lower temporal depending on the incubation conditions (37, 38). In this study, it and spatial resolution (29). was found that at least 16 h of incubation was essential to observe Surface-enhanced Raman scattering (SERS) imaging is a non- a significant SERS signal increase (SI Appendix,Fig.S2), which is invasive optical imaging tool that can visualize targeted receptors much slower than FA binding with receptors and translocation in biological or chemical samples in a temporal and spatial man- (within minutes) (17, 21, 39). Thus, 24 h was used in all the sub- ner. The SERS technique has been used as a 2D/3D tracing sequent experiments. Based on manufacturer’s information, the method for precise localization of folate and luteinizing hormone- immunogenic sequence of GPR120 antibody (H-10; Santa Cruz releasing hormone on the membrane (31). SERS mapping has also Biotechnology) is amino acids 78 to 232, which cover the first been applied to detect cancer stem cell markers CD44 and CD24 (99 to 112) and second (178 to 204) extracellular domains. While

2of10 | www.pnas.org/cgi/doi/10.1073/pnas.1916238117 Zhang et al. Downloaded by guest on September 28, 2021 GPR120 CD36 GPR120 CD36

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469 472 Y YY Y YY Y Y Y Y Y UU Y N C C 3 7 464 466 palmitoylation site ß ß arrestin 2 Ca2+ ion APPLIED BIOLOGICAL SCIENCES U ubiquitination site fatty acid

Fig. 1. Application of the SERS and calcium imaging system in sensing FA on a single living cell.

CD36 antibody (sc-7309; Santa Cruz Biotechnology) has been indicated that the peak intensities of GPR120, CD36, and phe- shown to interact with the domain spanning amino acids 155 to nylalanine were consistent across the 6-min data collection period, 183 (40, 41). In general, the concentration for 50% of maximal suggesting that receptors on the cell surface, antibody on the effect (EC50) of FFA to receptors (e.g., 1.288 μMforLAto SERS probe, and addition of LA are all essential components in GPR120) (42, 43) is much higher than the affinity constants of such a sensing system. Furthermore, as expected, the cell peak antibody to receptors (which vary from 10 to 200 pM) (44). (i.e., phenylalanine peak) was independent of LA stimulation and The structures of both receptors imply that the LA and antibody served as an important internal control. would bind or interact with receptors in their different extracel- In the LA treatment groups of the HEK 293 cell line, the lular regions, making it possible to apply our antibody-based SERS GPR120 or CD36 SERS peak responded rapidly (∼2 min) and was nanoparticles to detect the receptors and probe the LA–receptor significantly elevated at 6 min (Fig. 3 A and B). In simultaneous interaction. Three control groups (SI Appendix, Figs. S3–S5) application of two SERS probes in TBDc1 cells, the GPR120

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Fig. 2. Representative calcium images (A–C) and intracellular calcium level variation (D–F) of three cell lines on application of different concentrations of LA. Warmer colors are indicative of increasing intercellular Ca2+ concentration. (A and D) HEK293 GPR120. (B and E) HEK293 CD36. (C and F) TBDc1. (Insets) Logistic calibration curves of each cell line. (Scale bar: 20 μm.)

Zhang et al. PNAS Latest Articles | 3of10 Downloaded by guest on September 28, 2021 Fig. 3. Contour maps of Raman peaks (cell, GPR120 and CD36 peaks) in the first 6 min of three cell lines. (A) HEK293-GPR120. (B) HEK293-CD36. (C) TBDc1. HEK293 cells were incubated with DOX for 24 h before addition of the SERS probe. With accumulation of LA in the cell system, Raman spectra of the same − − location in one cell were recorded every 30 s in the range of 994 to 1,345 cm 1. Peaks of 1,002, 1,078, and 1,328 cm 1 were assigned to the cell, GPR120, and CD36, respectively. The peak intensity during the first 6 min (in 30-s increments) is illustrated in the right column of each contour map (n = 10∼13). One representative Raman spectrum (4 min) is shown in the upper part of each contour map. One representative light image was also included in each contour map. The results represent fold activity over the basal level of the SERS peak at the beginning of the experiment. (Scale bar: 10 μm.)

4of10 | www.pnas.org/cgi/doi/10.1073/pnas.1916238117 Zhang et al. Downloaded by guest on September 28, 2021 SERS peak began to rise dramatically after 1 min, plateaued from To further validate the FA specificity of the system, a medium- 3 to 5 min, and then increased again. While CD36 SERS peak chain saturated fatty acid myristic acid (MA; C14) and a long- changed more slowly but consistently until 4.5 min, and then chain polyunsaturated fatty acid docosahexaenoic acid (DHA; continued to increase after 5.5 min. The cell peak remained con- C22:6 [n-3]) were also tested to verify the feasibility of real-time, sistent for the 6-min period in all cell lines (Fig. 3C). in situ SERS mapping in monitoring interaction between other fatty acids and these two receptors. In MA experiments, the Localization of Receptors and Monitoring of FA–Receptor Interaction SERS mapping results showed little effect in activating GPR120 (LA Treatment). In our study, SERS mapping images over the first (as observed in the first 3 min) but altered the distribution of 21 min after introduction of LA show the dynamic distribution of CD36 in HEK293 cell lines (SI Appendix, Fig. S6). The addition GPR120 and CD36 on the cell surface (Fig. 4). The Raman of MA seemed to interact with CD36 on TBDc1 cells, as scanning area (black dashed rectangle in white image) covered reflected by the relative redistribution of CD36. These results the entire single cell. The sequential mapping images showed suggest that some medium-chain saturated fatty acids, like MA, that the receptor expression sites on both HEK293-GPR120 can alter the location of CD36 on the cell membrane via their binding with this protein (45). However, MA cannot influence (Fig. 4A) and HEK293-CD36 cells (Fig. 4B) increased gradu- the location of GPR120, since it apparently does not serve as an ally with stronger SERS signals (the spectra noted by triangles effective ligand for this receptor (46). In addition, treatment with for GPR120 and circles for CD36), indicating that more SERS another polyunsaturated fatty acid, DHA, SERS mapping results probes are bound with receptors on HEK293 cells. Moreover, indicate that binding sites of both receptors in HEK293 cell lines C the SERS-binding sites on TBDc1 cells (Fig. 4 ) changed no- are dynamically increased over the 21-min period (SI Appendix, ticeably, especially for the CD36 sites (green). In addition, SERS Fig. S7), confirming that DHA has activity at both GPR120 and intensity in TBDc1 cells was decreased in general during the 21- CD36 (47, 48). The expression and distribution of CD36 in TBDc1 min period but recovered slightly approximately every 10 min are altered after DHA is gradually introduced to the TBDc1 cells, (see the spectra at 9 min and 21 min), implying the time frame of implying the potential internalization of DHA mainly by CD36 receptor recycling in TBDc1 cells, which is close to what has may also be occurring (SI Appendix,Fig.S7), as was the case for been reported previously (21). LA (48). All these findings are consistent with the existence of APPLIED BIOLOGICAL SCIENCES

Fig. 4. Real-time SERS mapping to monitor the distribution of two receptors on the cell surface after the cells were incubated with SERS probes for 24 h, then infused by LA from 0 min (0 μM) to 6 min (150 μM). (A) HEK293-GPR120. (B) HEK293-CD36. (C) TBDc1. Each column includes a representative white image, a sequential Raman mapping image with virtual color, and corresponding Raman spectrum indicated by a cross, circle, and triangle in mapping images. The − − spectra in each column have the same vertical ranges. A 1,078 cm 1 peak was used to create the mapping of the GPR120 distribution (red), and 1,328 cm 1 was used to create the mapping of the CD36 distribution (green). Each Raman mapping image covers a single cell, as denoted by the black dashed line in the white image. LA was introduced during the first 6-min period. (Scale bar: 10 μm.)

Zhang et al. PNAS Latest Articles | 5of10 Downloaded by guest on September 28, 2021 a functional connection between the effects of ligand–receptor peak was reduced dramatically with GW9508 treatment in TBDc1 binding on localization of receptors in cells that can be detected cells, while CD36 was maintained at a relatively high level (SI and monitored in real time by the SERS probes. Consistent with Appendix,Fig.S9C and D). previous studies, those ligands that interact with CD36 (e.g., LA, DHA, MA) alter its cellular distribution, while those that do not Evaluation of Primary Taste Bud Cells. To test freshly isolated taste interact with CD36 (e.g., capric acid [CA]) have little to no effect bud cells, the cells were incubated with SERS probes for a (SI Appendix,Fig.S8). Known ligands for GPR120 (e.g., LA, shorter period (4 h) in the presence or absence of LA. The SERS DHA) have a similar specific effect on its distribution changes mapping images of the distribution of two receptors in primary (Fig. 4 and SI Appendix,Fig.S7). cells showed overall stronger signals in the control group and weaker signals in the LA (60 μM) treatment group (Fig. 6 A and Internalization of GPR120 and CD36. Membrane uptake of FAs is B). Furthermore, point mapping on multiple cells demonstrated the first step in cellular FA utilization and metabolic regulation a limited decrease in the GPR120 SERS signal and a significant (19). To verify our speculation that cells would be activated after decline in the CD36 SERS signal in primary cells from circum- receptor binding with FAs and begin to transport FAs across the vallate (CV) papillae (Fig. 6C), while the primary cell from cell membrane, SERS intensities on three cell lines were mea- fungiform (FF) papillae showed significant inhibition of both sured after SERS probe incubation with cells for 24 h. Fig. 5 A SERS signals after LA treatment (Fig. 6D). and B show enhanced SERS signals with increasing concentra- tions of LA in HEK293 cells expressing either GPR120 or CD36. Discussion However, in TBDc1 cells expressing both receptors in a more 4-Mercaptobenzoic acid (MBA) is often used as a Raman re- native system, GPR120 SERS and CD36 SERS signals show a porter, because the Au-S linkage can form a stable and well- concentration-dependent decrease in intensity during activation defined monolayer on a Au surface, and its two relatively large − with LA. SERS peaks (at 1,078 and 1,580 cm 1) have been well character- To determine the specificity of these responses to ligands of ized (50). 5, 5-Dithio-bis-(2-nitrobenzoic acid) (DTNB) is able to these two receptors, parallel experiments were conducted with decompose into two monomers that can form an Au-S linkage as − another FA, CA, which does not activate either of these two well, giving one dominant SERS peak (1,328 cm 1) that apparently receptor types (49). When various concentrations of CA were does not overlap with either of the two MBA SERS peaks (51). In induced into these cell lines, both SERS peaks were consistent this study, MBA and DTNB were selected as Raman reporters with the control group (0 μM) (SI Appendix, Fig. S8). We next conjugated, along with antibodies, to gold nanorod surfaces to used a compound, GW9508, that acts as an agonist to GPR120 form the MBA anti-GPR120 SERS probe and DTNB anti-CD36 but is ineffective for activating CD36. As expected, CD36 SERS probe, respectively. SERS signals were not affected by GW9508 in HEK293 cells Most mammalian cells have a characteristic Raman peak − expressing CD36. Similar to previous results with LA, GW9508 around 1,002 cm 1 assigning the presence of phenylalanine (50, showed a concentration-dependent increase in GPR120 SERS 52), which has been selected as a sensitive marker for moni- signals in HEK-GPR120 cells up to 30 μM, where it dropped off at toring cellular protein structure (53). With a small spectral scan − higher concentrations (SI Appendix, Fig. S9 A and B). Further window (994 to 1,345 cm 1), it covers all three well-defined peaks − demonstrating the specificity of the system, the GPR120 SERS of interest, including the cell characteristic peak (1,002 cm 1),

Fig. 5. SERS spectra after cells were pretreated by LA for 5 min, followed by a 24-h incubation with SERS probes. (A) averaged SERS spectrum of HEK GPR120 cells (n = ∼25) with five levels of LA pretreatment including peak height variation (Inset). (B) Averaged SERS spectrum of HEK CD36 cells (n = ∼25) with five levels of LA pretreatment including peak height variation (Inset). (C) Averaged SERS spectrum of TBDc1 (n = ∼25) with five levels of LA pretreatment. (D) Variation of peak height with five levels of LA stimulation in TBDc1.

6of10 | www.pnas.org/cgi/doi/10.1073/pnas.1916238117 Zhang et al. Downloaded by guest on September 28, 2021 APPLIED BIOLOGICAL SCIENCES

Fig. 6. Application of nanoprobes on primary taste bud cells collected from CV papillae and FF papillae. (A) White image and corresponding mapping image of primary taste bud cells. (B) Raman spectra of the specific spot indicated in the mapping image. (C) Comparison of SERS intensity with and without LA (60 μM) treatment in primary cells (n = 19 or 20) from CV papillae. (D) Comparison of SERS intensity with and without LA (60 μM) treatment in primary cells (n = 10 or 11) from FF papillae. *P < 0.05. (Scale bar: 10 μm.)

− − GPR120 (1,078 cm 1 from MBA), and CD36 (1,328 cm 1 from Due to the fact that both LA and SERS probe could recognize DTNB). Completing the collection of the spectra in this scan range and interact with different domains of the receptors (GPR120 takes only a few seconds per spectrum in the static scan mode and and CD36), it would be particularly interesting to examine the a few minutes for spectral mapping on single cells. This unique cell responses based on the order of LA and SERS probe capability of simultaneously detecting a cell spectral marker and treatments. If the SERS probes were added first (e.g., at 24 h), two functional membrane receptors in real time in single living the antibodies from the SERS probe would bind with the specific cells (Fig. 3) provides highly sensitive monitoring of receptor–FA regions or domains of the receptor, and subsequent treatment interaction without the need for complicated sample preparation with LA even within a short time (e.g., 5 min) would lead to and cell fixation. changes in receptor conformation. This type of change can be FA ligand binding with receptors would affect the conforma- captured by monitoring of the SERS signals. From the data, it is tion of GPR120 or CD36 receptor (from inactive to active clear that as more LA molecules are introduced, more extra- structure), which is highly correlated with the downstream sig- cellular binding domains of the receptors become available and naling pathways and the FA uptake process (29, 54). The initial accessible for more free SERS probes to bind (Fig. 7A). This will ligand binding of GPR120 is followed by intramolecular rear- give rise to increases in the SERS signal over a series of LA rangement, which may occur in intracellular and extracellular concentrations up to 150 μM (Fig. 3), along with an enriched receptor compartments (29). The electrostatic interactions be- receptor presence and distribution on the cell surface, as shown tween the carboxyl group of FA with Lys-164 of the CD36 re- in Raman mapping (Fig. 4). ceptor can also alter the receptor membrane protein conformation However, when the order of treatment is altered by introducing with functional consequences (18). LA at a certain concentration first (LA activation), HEK293 cells

Zhang et al. PNAS Latest Articles | 7of10 Downloaded by guest on September 28, 2021 expressing GRP120 or CD36 and TBDc1 cells respond differently SERS signal was decreased to a much greater degree than the during the subsequent 24-h SERS probe treatment period. (Cells CD36 SERS signal. were rinsed by buffer to remove nonspecific SERS probes be- The difference in functional cellular components and the re- fore Raman measurement.) SERS signals were found to in- quired cellular uptake machinery and pathways may be attributed crease in HEK 293 cells (Fig. 5 A and B), which we hypothesize to the observed SERS signal differences between engineered HEK may be due to the LA activation inducing conformation changes in 293 cells and TBDc1 cells. Long-chain FA can diffuse across the the receptor structure, making more extracellular binding domains cell membrane, but overwhelming evidence has demonstrated that available to antibody-based SERS probes (Fig. 7B). On the other FA uptake by mammalian cells is facilitated by the integral or hand, in TBDc1 cells, which express the normal, complete array of membrane-associated proteins (19), such as the plasma membrane cellular pathways, the decrease in SERS signals (Fig. 5 C and D)is FA-binding protein caveline 1, acyl-CoA synthetase long-chain due, we believe, to receptor-mediated endocytosis and internali- family member 1, FA translocase CD36, and the FA transport zation (Fig. 7C), a LA concentration-dependent process. Our data protein family (55, 56). The ability to detect SERS signal differ- showing decreases in both GPR120 and CD36-SERS probes ences between the engineered HEK 293 cells and TBDc1 cells interacting with TBDc1 cells (Fig. 5 C and D)suggestthatLA here implies the potential application of these receptor-specific activated receptors may be internalized in these cells, but not in SERS probes, in conjunction with other methods and techniques engineered HEK293 cells. In addition, in response to LA treat- (57), to study the underlying mechanisms of FA cellular uptake. ment, the CD36 SERS signal was decreased to a greater extent Our projected model of the mechanism of FA internalization than the GPR120 signal (Fig. 5D). However, treatment with the process is illustrated in Fig. 7 C and D. CD36 has been shown to GPR120-specific agonist GW9508 in TBDc1 cells (SI Appendix, participate in FA uptake and subsequent internalization inside Fig. S9D) had the opposite effect, as expected; that is, the GPR120 the cell, followed by a recycling back to the cell surface (20, 21).

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t = 6 min Y YY

Y Y

Y YY High [LA] Y U

Y Y Y High [GW9508] ubiquinaon site ß ß arresn 2 GPR120 GPR120 CD36 Cell GPR120 CD36 GPR120 3 1.00 CD36 0.9 0 µM 60 µM 0.9 0 µM 6 min 2 0.75 0.6

1 0.6 Intensity Intensity Intensity Intensity 0 µM 0 min 60 µM 60 µM 0 0.50 0.3 0.3 1000 1050 1300 1350 1400 1000 1100 1200 1300 1400 1000 1050 1300 1350 1400 1000 1050 1300 1350 1400 -1 -1 Raman shift (cm-1) Raman shift (cm-1) Raman shift (cm ) Raman shift (cm )

Fig. 7. Schematic illustration of the FA sensing and internalization process. (A) Real-time monitoring of SERS peak intensity in first 6 min on LA treatment after a 24-h SERS probe incubation. (B) SERS peak intensity changes after the cells were treated by LA for 5 min at various concentrations, followed by a 24-h SERS probe incubation. HEK293-GPR120 cells were taken as an example; similar results can be seen in HEK293-CD36 cells. (C) Potential internalization mechanism in TBDc1 in the process of LA uptake. CD36 serves as a transporter to carry LA into cell and then recycles back to the membrane in lower LA concentration. The unloading process was impeded at high LA concentration. Intracellular LA can also bind with ubiquitination sites to internalize CD36. (D) Potential internalization mechanism in TBDc1 cells in the process of agonist (GW9508) stimuli. See Internalization of GPR120 and CD36 for additional details.

8of10 | www.pnas.org/cgi/doi/10.1073/pnas.1916238117 Zhang et al. Downloaded by guest on September 28, 2021 The C-terminal of CD36 located inside the cell membrane has GPCR responsive to long-chain FAs that has been attracting ubiquitination sites that are sensitive to excessive intercellular considerable attention as a potential novel therapeutic target for FA, further regulating CD36 sorting and internalization (19). In metabolic diseases, including type II diabetes and obesity (17, 39). the case of low LA concentration (e.g., 5 μM), CD36 can quickly GPR120 functions as a receptor by regulating the secretion of transport and unload LA into the cell and then resensitize on the glucagon-like peptide-1 and suppressing the inflammatory effects surface (a process requiring only minutes) (21). This contributes (64). GPR120 knockout mice exhibit a lower oro-gustatory pref- to the relatively high SERS signals after 24 h of incubation with erence for FFAs and severe signs of insulin resistance (12). The the SERS probe seen in the present study. However, in the activation of GPR120 by synthetic agonists appears to improve condition of high LA concentration, the intracellular FA can tissue insulin sensitivity and regulate lipid metabolism in adipose further induce the internalization of CD36 by ubiquitination, tissue, which may help prevent obesity and diabetes (17). There- resulting in a reduced SERS signal (Fig. 5C). fore, the development and quantitative assessment of new agonists GPR120 often acts as the first messenger on ligand or agonist to GPR120 becomes an urgent task that potentially may help stimulation. The intracellular C-terminal region of GPR120 is control the overwhelming prevalence of obesity. important for interacting with G proteins (e.g., Gα, Gq) or scaffold This study demonstrates that the successful application of Raman β proteins (e.g., -arrestin-2) (12), which activate second messenger multiplex imaging with the capability of real-time monitoring of pathways. After agonist (e.g., GW9508) binding with GPR120, multiple membrane receptors on single cells in near-physiological β recruited -arrestin can desensitize GPR120 by binding to its cy- conditions. The fabricated receptor-specific SERS probes can tosolic sites and mediating dynamin-dependent endocytosis via serve as a cell-based assay and screening tool that will benefit the clathrin-coated pits (58), involving endocytic machinery including development of new agonists and antagonists to GPCRs. In ad- β N clathrin, 2-adaptin, and -ethylmaleimide-sensitive fusion protein dition to cell-based assays, the SERS technique has been suc- (59), which leads to translocation from the plasma membrane to D cessfully expanded in biological tissue measurements, such as the cytosol (Fig. 7 ). Consequently, the GPR120 SERS signal was normal and cancerous thyroid tissue (65), healthy brain tissue, significantly inhibited in taste bud cells with GW9508 treatment SI Appendix D and tumors (66). Our SERS mapping results of primary taste ( , Fig. S9 ). In GPR120 and CD36-coexpressing cells bud cells (Fig. 6) and fixed tissue samples (SI Appendix, Fig. S10) (TBDc1), LA-GPR120 binding events not only facilitate the provides a proof-of- concept application of this approach. CD36-mediated LA transport, also may phosphorylate GPR120 In summary, the extracellular interaction between receptors (especially in high concentration) via protein kinase C and and FAs was monitored by the variation in the SERS spectrum G protein-coupled receptor kinase 6 at Thr347,Ser350, and Ser357 covering peaks representing the cell, GPR120, and CD36 in real in the C-terminal tail (60). This further increases the affinity of APPLIED BIOLOGICAL SCIENCES time, whose activities were validated with calcium imaging. The GPR120 to β-arrestin-2, followed by internalization of GPR120 alteration of the spatial distribution of GPR120 and CD36 on (61). As a result, GPR120 SERS signals were also decreased in LA stimulation was visualized by SERS mapping. The potential high LA concentrations (Fig. 5 C and D). Moreover, the activation 2+ FA internalization process demonstrates the respective roles of of GPR120 by agonist would increase the intracellular Ca level GPR120 and CD36 in FA uptake. Our findings are consistent via PLCβ pathway, which may in turn induce the membrane lo- calization of CD36 (18), resulting in a decreased CD36 SERS with the idea that GPR120 acts as a primary receptor and works signal at high GW9508 levels (SI Appendix,Fig.S9D). synergistically with CD36 to transfer the signal and begin the Compared with long-chain FAs, the cellular metabolism of transport process based primarily on CD36 activation. In the short- and medium-chain FAs (such as CA in our study) depends future, the specific application of these SERS probes to FA- to a lesser extent on FA-binding proteins (49). Therefore, SERS responsive cells and/or tissues isolated from the wild type (control) signals were not influenced in all levels of CA treatment (SI and FA-receptor knockouts or dietary-induced obesity models Appendix, Fig. S8 C and D), indicating that CA can neither be may enhance our understanding of the role of FFAR dynamics recognized nor transported by GPR120 and CD36. in nutrient signaling pathways and the development of nutrient- All our findings are consistent with the interpretation that related disorders. In a broader context, the effectiveness of these GPR120 primarily works as the first messenger and CD36 acts imaging nanoprobes will prove useful for improving our under- more as a transporter in absorption of FAs. These two receptors standing of the roles of receptor-mediated signaling in the large are generally believed to have their own independent pathways, family of GPCRs and other receptor types and how their spatio- signaling mechanisms, and processes related to their movement in temporal dynamics influence cell signaling in general. and out of the cell membrane. Whether these pathways between GPR120 and CD36 intersect at some point or influence one an- Materials and Methods other needs to be further addressed (62). Nonetheless, while it was Cell samples were grown on MgF2 for Raman spectroscopy. All procedures far beyond the scope of the present study to elucidate the details involving the care and use of animals in this study were approved by the of these processes, it is clear that the SERS probes were able to Institutional Animal Care and Use Committee at Utah State University. Ad- monitor the spatiotemporal properties of these receptors in ditional information on the materials and methods used in this study is provided in SI Appendix. real time, providing a means of addressing these long-standing questions. Data Availability. The raw data generated and analyzed in this study are GPCRs are the most intensively studied drug targets. By 2017, available at Zenodo (http://dx.doi.org/10.5281/zenodo.3560297). approximately 34% of all drugs approved by the US Food and Drug Administration act on GPCRs, in addition to approximately ACKNOWLEDGMENTS. This research was supported by NSF Grant 1264498 20% of 321 targets currently in clinical trials (63). GPR120 is a and NIH Grant R01 DC013318.

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