Metabolic Imaging and Applications in Protein and Lipid Homeostasis

Metabolic activity is an important functional parameter of a living cell. Microscopic techniques are demanded to resolve the heterogeneity of metabolic activity from cell to cell and among subcellular compartments. Towards this, work in the Min lab has been dedicated to developing a prevailing metabolic imaging platform that couples chemical imaging by stimulated Raman scattering (SRS) microscopy with small vibrational tags on precursor molecules. This thesis describes efforts along metabolic imaging by SRS microscopy, with focus on visualizing protein and lipid homeostasis. Chapter 1 describes the design principle of metabolic imaging, including selection of vibrational tags and setup of SRS microscopy, and an overview of successful demonstrations of metabolic imaging in protein and lipid metabolism. Chapter 2 describes adoption of such principle to visualize protein turnover with 13C-phenylalanine metabolic labeling under steady-state condition and various perturbations. The rest of this thesis (Chapters 3-6) switches focus to fatty acid metabolism and cellular lipid homeostasis. As the minimal tagging in vibrational imaging preserves the physicochemical property of lipid molecules to the largest extent, it motivated me to revisit fatty acid metabolism from a biophysical perspective. Bearing the question in mind whether the non-equilibrium metabolic activity could drive phase separation in biological membranes, I thus look into the principle of membrane organization and its implication in biological membranes in Chapter 3. Then in Chapter 4, I describe the discovery and characterization of previously unknown phase separation in endoplasmic reticulum (ER) membrane caused by lipid synthesis. In this case, metabolic imaging by SRS enables identification of solid-like domains formed by saturated fatty acid (SFA) metabolites. This observation further raises the question whether phase separation bears any functional roles in the adverse effects of SFAs (or lipotoxicity). Towards this, Chapter 5 introduces the background of lipotoxicity including its definition and models. Then I review proposed mechanisms for lipotoxicity, which point to the central role of ER in mediating the stress transduction. In Chapter 6, I present our findings that suggest the association of the observed solid-like domains with ER structural remodeling and local autophagic arrest. Together, these efforts demonstrate the valuable capability of SRS imaging to reveal metabolic heterogeneity and how this aids in the investigation of metabolic stress

Spectral tracing of deuterium for imaging glucose metabolism.

Cells and tissues often display pronounced spatial and dynamical metabolic heterogeneity. Common glucose-imaging techniques report glucose uptake or catabolism activity, yet do not trace the functional utilization of glucose-derived anabolic products. Here we report a microscopy technique for the optical imaging, via the spectral tracing of deuterium (STRIDE), of diverse macromolecules derived from glucose. Based on stimulated Raman-scattering imaging, STRIDE visualizes the metabolic dynamics of newly synthesized macromolecules, such as DNA, protein, lipids and glycogen, via the enrichment and distinct spectra of carbon-deuterium bonds transferred from the deuterated glucose precursor. STRIDE can also use spectral differences derived from different glucose isotopologues to visualize temporally separated glucose populations using a pulse-chase protocol. We also show that STRIDE can be used to image glucose metabolism in many mouse tissues, including tumours, brain, intestine and liver, at a detection limit of 10 mM of carbon-deuterium bonds. STRIDE provides a high-resolution and chemically informative assessment of glucose anabolic utilization

Live-cell quantitative imaging of proteome degradation by stimulated Raman scattering

Protein degradation is a regulatory process essential to cell viability and its dysfunction is implicated in many diseases, such as aging and neurodegeneration. In this report, stimulated Raman scattering microscopy coupled with metabolic labeling with ^(13)C-phenylalanine is used to visualize protein degradation in living cells with subcellular resolution. We choose the ring breathing modes of endogenous ^(12)C-phenylalanine and incorporated ^(13)C-phenylalanine as protein markers for the original and nascent proteomes, respectively, and the decay of the former wasquantified through ^(12)C/(^(12)C + ^(13)C) ratio maps. We demonstrate time-dependent imaging of proteomic degradation in mammalian cells under steady-state conditions and various perturbations, including oxidative stress, cell differentiation, and huntingtin protein aggregation

Live-Cell Bioorthogonal Chemical Imaging: Stimulated Raman Scattering Microscopy of Vibrational Probes

Innovations in light microscopy have tremendously revolutionized the way researchers study biological systems with subcellular resolution. In particular, fluorescence microscopy with the expanding choices of fluorescent probes has provided a comprehensive toolkit to tag and visualize various molecules of interest with exquisite specificity and high sensitivity. Although fluorescence microscopy is currently the method of choice for cellular imaging, it faces fundamental limitations for studying the vast number of small biomolecules. This is because common fluorescent labels, which are relatively bulky, could introduce considerable perturbation to or even completely alter the native functions of vital small biomolecules. Hence, despite their immense functional importance, these small biomolecules remain largely undetectable by fluorescence microscopy. To address this challenge, a bioorthogonal chemical imaging platform has recently been introduced. By coupling stimulated Raman scattering (SRS) microscopy, an emerging nonlinear Raman microscopy technique, with tiny and Raman-active vibrational probes (e.g., alkynes and stable isotopes), bioorthogonal chemical imaging exhibits superb sensitivity, specificity, and biocompatibility for imaging small biomolecules in live systems. In this Account, we review recent technical achievements for visualizing a broad spectrum of small biomolecules, including ribonucleosides and deoxyribonucleosides, amino acids, fatty acids, choline, glucose, cholesterol, and small-molecule drugs in live biological systems ranging from individual cells to animal tissues and model organisms. Importantly, this platform is compatible with live-cell biology, thus allowing real-time imaging of small-molecule dynamics. Moreover, we discuss further chemical and spectroscopic strategies for multicolor bioorthogonal chemical imaging, a valuable technique in the era of “omics”. As a unique tool for biological discovery, this platform has been applied to studying various metabolic processes under both physiological and pathological states, including protein synthesis activity of neuronal systems, protein aggregations in Huntington disease models, glucose uptake in tumor xenografts, and drug penetration through skin tissues. We envision that the coupling of SRS microscopy with vibrational probes would do for small biomolecules what fluorescence microscopy of fluorophores has done for larger molecular species

Vibrational Imaging of Glucose Uptake Activity in Live Cells and Tissues by Stimulated Raman Scattering

Glucose is a ubiquitous energy source for most living organisms. Its uptake activity closely reflects cellular metabolic demand in various physiopathological conditions. Extensive efforts have been made to specifically image glucose uptake, such as with positron emission tomography, magnetic resonance imaging, and fluorescence microscopy, but all have limitations. A new platform to visualize glucose uptake activity in live cells and tissues is presented that involves performing stimulated Raman scattering on a novel glucose analogue labeled with a small alkyne moiety. Cancer cells with differing metabolic activities can be distinguished. Heterogeneous uptake patterns are observed with clear cell-cell variations in tumor xenograft tissues, neuronal culture, and mouse brain tissues. By offering the distinct advantage of optical resolution but without the undesirable influence of fluorophores, this method will facilitate the study of energy demands of living systems with subcellular resolution

Volumetric chemical imaging by clearing-enhanced stimulated Raman scattering microscopy

Three-dimensional visualization of tissue structures using optical microscopy facilitates the understanding of biological functions. However, optical microscopy is limited in tissue penetration due to severe light scattering. Recently, a series of tissue-clearing techniques have emerged to allow significant depth-extension for fluorescence imaging. Inspired by these advances, we develop a volumetric chemical imaging technique that couples Raman-tailored tissue-clearing with stimulated Raman scattering (SRS) microscopy. Compared with the standard SRS, the clearing-enhanced SRS achieves greater than 10-times depth increase. Based on the extracted spatial distribution of proteins and lipids, our method reveals intricate 3D organizations of tumor spheroids, mouse brain tissues, and tumor xenografts. We further develop volumetric phasor analysis of multispectral SRS images for chemically specific clustering and segmentation in 3D. Moreover, going beyond the conventional label-free paradigm, we demonstrate metabolic volumetric chemical imaging, which allows us to simultaneously map out metabolic activities of protein and lipid synthesis in glioblastoma. Together, these results support volumetric chemical imaging as a valuable tool for elucidating comprehensive 3D structures, compositions, and functions in diverse biological contexts, complementing the prevailing volumetric fluorescence microscopy

Live-cell vibrational imaging of choline metabolites by stimulated Raman scattering coupled with isotope-based metabolic labeling

Choline is a small molecule that occupies a key position in the biochemistry of all living organisms. Recent studies have strongly implicated choline metabolites in cancer, atherosclerosis and nervous system development. To detect choline and its metabolites, existing physical methods such as magnetic resonance spectroscopy and positron emission tomography are often limited by the poor spatial resolution and substantial radiation dose. Fluorescence imaging, although with submicrometer resolution, requires introduction of bulky fluorophores and thus is difficult in labeling the small choline molecule. By combining the emerging bond-selective stimulated Raman scattering microscopy with metabolic incorporation of deuterated choline, herein we have achieved high resolution imaging of choline-containing metabolites in living mammalian cell lines, primary hippocampal neurons and the multicellular organism C. elegans. Different subcellular distributions of choline metabolites are observed between cancer cells and non-cancer cells, which may reveal a functional difference in the choline metabolism and lipid-mediated signaling events. In neurons, choline incorporation is visualized within both soma and neurites, where choline metabolites are more evenly distributed compared to proteins. Furthermore, choline localization is also observed in the pharynx region of C. elegans larvae, consistent with its organogenesis mechanism. These applications demonstrate the potential of isotope-based stimulated Raman scattering microscopy for future choline-related disease detection and development monitoring in vivo

pH-mediated upregulation of AQP1 gene expression through the Spi-B transcription factor

Background: Bicarbonate-based peritoneal dialysis (PD) fluids enhance the migratory capacity and damage-repair ability of human peritoneal mesothelial cells by upregulating AQP1. However, little is known about the underlying molecular mechanisms. Results: Here we used HEK-293T cells to investigate the effect of pH on AQP1 gene transcription levels. We found that AQP1 mRNA levels increases with pH. Transfection of HEK-293T cells with luciferase reporter vectors containing different regions of the AQP1 promoter identified an upstream region in the AQP1 gene between − 2200 and – 2300 bp as an enhancer required for pH-mediated regulation of AQP1 expression. Site-directed mutagenesis of this specific promoter region revealed a critical region between − 2257 and − 2251 bp, and gene knock-down experiments and ChIP assays suggested that the Spi-B transcription factor SPIB is involved in pH-mediated regulation of AQP1 expression. Conclusions: We identified an upstream region in the AQP1 gene and the transcription factor SPIB that are critically involved in pH-mediated regulation of AQP1 expression. These findings provide the basis for further studies on the pH- and buffer-dependent effects of PD fluids on peritoneal membrane integrity and function

Metabolic activity induces membrane phase separation in endoplasmic reticulum

Membrane phase behavior has been well characterized in model membranes in vitro under thermodynamic equilibrium state. However, the widely observed differences between biological membranes and their in vitro counterparts are placing more emphasis on nonequilibrium factors, including influx and efflux of lipid molecules. The endoplasmic reticulum (ER) is the largest cellular membrane system and also the most metabolically active organelle responsible for lipid synthesis. However, how the nonequilibrium metabolic activity modulates ER membrane phase has not been investigated. Here, we studied the phase behavior of functional ER in the context of lipid metabolism. Utilizing advanced vibrational imaging technique, that is, stimulated Raman scattering microscopy, we discovered that metabolism of palmitate, a prevalent saturated fatty acid (SFA), could drive solid-like domain separation from the presumably uniformly fluidic ER membrane, a previously unknown phenomenon. The potential of various fatty acids to induce solid phase can be predicted by the transition temperatures of their major metabolites. Interplay between saturated and unsaturated fatty acids is also observed. Hence, our study sheds light on cellular membrane biophysics by underscoring the nonequilibrium metabolic status of living cell