Chemical Control of Fluorescence Lifetime towards Multiplexing Imaging.

Fluorescence lifetime imaging has been a powerful tool for biomedical research. Recently, fluorescence lifetime-based multiplexing imaging has expanded imaging channels by using probes that harbor the same spectral channels and distinct excited state lifetime. While it is desirable to control the excited state lifetime of any given fluorescent probes, the rational control of fluorescence lifetimes remains a challenge. Herein, we chose boron dipyrromethene (BODIPY) as a model system and provided chemical strategies to regulate the fluorescence lifetime of its derivatives with varying spectral features. We find electronegativity of structural substituents at the 8' and 5' positions is important to control the lifetime for the green-emitting and red-emitting BODIPY scaffolds. Mechanistically, such influences are exerted via the photo-induced electron transfer and the intramolecular charge transfer processes for the 8' and 5' positions of BODIPY, respectively. Based on these principles, we have generated a group of BODIPY probes that enable imaging experiments to separate multiple targets using fluorescence lifetime as a signal. In addition to BODIPY, we envision modulation of electronegativity of chemical substituents could serve as a feasible strategy to achieve rational control of fluorescence lifetime for a variety of small molecule fluorophores.

Detecting protein-protein interaction during liquid-liquid phase separation using fluorogenic protein sensors.

The formation of cellular condensates, akin to membraneless organelles, is typically mediated by liquid-liquid phase separation (LLPS), during which proteins and RNA molecules interact with each other via multivalent interactions. Gaining a comprehensive understanding of these interactions holds significance in unraveling the mechanisms underlying condensate formation and the pathology of related diseases. In an attempt toward this end, fluorescence microscopy is often used to examine the colocalization of target proteins/RNAs. However, fluorescence colocalization is inadequate to reliably identify protein interaction due to the diffraction limit of traditional fluorescence microscopy. In this study, we achieve this goal through adopting a novel chemical biology approach via the dimerization-dependent fluorescent proteins (ddFPs). We succeeded in utilizing ddFPs to detect protein interaction during LLPS both in vitro and in living cells. The ddFPs allow us to investigate the interaction between two important LLPS-associated proteins, FUS and TDP-43, as cellular condensates formed. Importantly, we revealed that their interaction was associated with RNA binding upon LLPS, indicating that RNA plays a critical role in mediating interactions between RBPs. More broadly, we envision that utilization of ddFPs would reveal previously unknown protein-protein interaction and uncover their functional roles in the formation and disassembly of biomolecular condensates.

Micropolarity governs the structural organization of biomolecular condensates.

Membraneless organelles within cells have unique microenvironments that play a critical role in their functions. However, how microenvironments of biomolecular condensates affect their structure and function remains unknown. In this study, we investigated the micropolarity and microviscosity of model biomolecular condensates by fluorescence lifetime imaging coupling with environmentally sensitive fluorophores. Using both in vitro and in cellulo systems, we demonstrated that sufficient micropolarity difference is key to forming multilayered condensates, where the shells present more polar microenvironments than the cores. Furthermore, micropolarity changes were shown to be accompanied by conversions of the layered structures. Decreased micropolarities of the granular components, accompanied by the increased micropolarities of the dense fibrillar components, result in the relocation of different nucleolus subcompartments in transcription-stalled conditions. Our results demonstrate the central role of the previously overlooked micropolarity in the regulation of structures and functions of membraneless organelles.

Nuclear bodies protect phase separated proteins from degradation in stressed proteome.

RNA-binding proteins (RBPs) containing intrinsically disordered domains undergo liquid-liquid phase separation to form nuclear bodies under stress conditions. This process is also connected to the misfolding and aggregation of RBPs, which are associated with a series of neurodegenerative diseases. However, it remains elusive how folding states of RBPs changes upon the formation and maturation of nuclear bodies. Here, we describe SNAP-tag based imaging methods to visualize the folding states of RBPs in live cells via time-resolved quantitative microscopic analyses of their micropolarity and microviscosity. Using these imaging methods in conjunction with immunofluorescence imaging, we demonstrate that RBPs, represented by TDP-43, initially enters the PML nuclear bodies in its native state upon transient proteostasis stress, albeit it begins to misfolded during prolonged stress. Furthermore, we show that heat shock protein 70 co-enters the PML nuclear bodies to prevent the degradation of TDP-43 from the proteotoxic stress, thus revealing a previously unappreciated protective role of the PML nuclear bodies in the prevention of stress-induced degradation of TDP-43. In summary, our imaging methods described in the manuscript, for the first time, reveal the folding states of RBPs, which were previously challenging to study with conventional methods in nuclear bodies of live cells. This study uncovers the mechanistic correlations between the folding states of a protein and functions of nuclear bodies, in particular PML bodies. We envision that the imaging methods can be generally applied to elucidating the structural aspects of other proteins that exhibit granular structures under biological stimulus.

Micropolarity governs the structural organization of biomolecular condensates.

Microenvironment is critical to the function of cells and organisms. One example is provided by biomolecular condensates, whose microenvironment can be vastly different from the surrounding cellular environments to engage unique biological functions. How microenvironments of biomolecular condensates affect their structure and function remains unknown. Here, we show that the arrangements and partitioning of biomolecules are dictated by the differences between the micropolarity of each subcompartment. Sufficient difference in micropolarity results in layered structures with the exterior shell presenting a more polar microenvironment than the interior core. Accordingly, micropolarity inversion is accompanied by conversions of the layered structures. These findings demonstrated the central role of the previously overlooked microenvironment in regulating the structural organization and function of membraneless organelles.

Xanthone-based solvatochromic fluorophores for quantifying micropolarity of protein aggregates.

Proper three-dimensional structures are essential for maintaining the functionality of proteins and for avoiding pathological consequences of improper folding. Misfolding and aggregation of proteins have been both associated with neurodegenerative disease. Therefore, a variety of fluorogenic tools that respond to both polarity and viscosity have been developed to detect protein aggregation. However, the rational design of highly sensitive fluorophores that respond solely to polarity has remained elusive. In this work, we demonstrate that electron-withdrawing heteroatoms with (d-p)-π* conjugation can stabilize lowest unoccupied molecular orbital (LUMO) energy levels and promote bathochromic shifts. Guided by computational analyses, we have devised a novel series of xanthone-based solvatochromic fluorophores that have rarely been systematically studied. The resulting probes exhibit superior sensitivity to polarity but are insensitive to viscosity. As proof of concept, we have synthesized protein targeting probes for live-cell confocal imaging intended to quantify the polarity of misfolded and aggregated proteins. Interestingly, our results reveal several layers of protein aggregates in a way that we had not anticipated. First, microenvironments with reduced polarity were validated in the misfolding and aggregation of folded globular proteins. Second, granular aggregates of AgHalo displayed a less polar environment than aggregates formed by folded globular protein represented by Htt-polyQ. Third, our studies reveal that granular protein aggregates formed in response to different types of stressors exhibit significant polarity differences. These results show that the solvatochromic fluorophores solely responsive to polarity represent a new class of indicators that can be widely used for detecting protein aggregation in live cells, thus paving the way for elucidating cellular mechanisms of protein aggregation as well as therapeutic approaches to managing intracellular aggregates.

Entecavir competitively inhibits deoxyguanosine and deoxyadenosine phosphorylation in isolated mitochondria and the perfused rat heart.

Deoxyguanosine kinase (dGK) is reported responsible for the phosphorylation of deoxyadenosine (dA) and deoxyguanosine (dG) in the mitochondrial purine salvage pathway. Antiviral nucleoside analogs known as nucleoside reverse transcriptase inhibitors (NRTIs) must be phosphorylated by host enzymes for the analog to become active. We address the possibility that NRTI purine analogs may be competitive inhibitors of dGK. From a group of such analogs, we demonstrate that entecavir (ETV) competitively inhibited the phosphorylation of dG and dA in rat mitochondria. Mitochondria from the brain, heart, kidney, and liver showed a marked preference for phosphorylation of dG over dA (10-30-fold) and ETV over dA (2.5-4-fold). We found that ETV inhibited the phosphorylation of dG with an IC50 of 15.3 ± 2.2 µM and that ETV and dG were both potent inhibitors of dA phosphorylation with IC50s of 0.034 ± 0.007 and 0.028 ± 0.006 µM, respectively. In addition, the phosphorylation of dG and ETV followed Michaelis-Menten kinetics and each competitively inhibited the phosphorylation of the other. We observed that the kinetics of dA phosphorylation were strikingly different from those of dG phosphorylation, with an exponentially lower affinity for dGK and no effect of dA on dG or ETV phosphorylation. Finally, in an isolated heart perfusion model, we demonstrated that dG, dA, and ETV were phosphorylated and dG phosphorylation was inhibited by ETV. Taken together, these data demonstrate that dGK is inhibited by ETV and that the primary role of dGK is in the phosphorylation of dG rather than dA.

Visualizing the Multistep Process of Protein Aggregation in Live Cells.

Protein aggregation is a biological phenomenon in which aberrantly processed or mutant proteins misfold and assemble into a variety of insoluble aggregates. Decades of studies have delineated the structure, interaction, and activity of proteins in either their natively folded structures or insoluble aggregates such as amyloid fibrils. However, a variety of intermediate species exist between these two extreme states in the protein folding landscape. Herein, we collectively term these intermediate species as misfolded protein oligomers, including soluble oligomers and preamyloid oligomers that are formed by unfolded or misfolded proteins. While extensive tools have been developed to study folded proteins or amyloid fibrils, research to understand the properties and activities of misfolded protein oligomers has been limited by the lack of methods to detect and interrogate these species in live cells.In this Account, we describe our efforts in the development of chemical methods that allow for the characterization of the multistep protein aggregation process, in particular the misfolded protein oligomers, in living cells. As the start of this journey, we attempted to develop a fluorogenic method wherein the misfolded oligomers could turn on the fluorescence of chemical probes that are conjugated to the protein-of-interest (POI). To this end, we produced a series of destabilized HaloTag variants, formulating the primary component of the AgHalo sensor, which misfolds and aggregates when cells are subjected to stress. When AgHalo is covalently conjugated with a solvatochromic fluorophore, misfolding of the AgHalo conjugate would activate fluorescence, resulting in the observation of misfolded oligomers. Following this work, we extended the scope of detection from AgHalo to any protein-of-interest via the AggTag method, wherein the POIs are genetically fused to self-labeling protein tags (HaloTag or SNAP-tag). Focusing on the molecular rotor-based fluorophores, we applied the modulated fluorescent protein (FP) chromophore core as a prototype for the AggTag probes, to enable the fluorogenic detection of misfolded soluble oligomers of multiple proteins in live cells. Next, we further developed the AggTag method to distinguish insoluble aggregates from misfolded oligomers, using two classes of probes that activate different fluorescence emission toward these two conformations. To enable this goal, we applied physical organic chemistry and computational chemistry to discover a new category of triode-like fluorophores, wherein the π orbitals of either an electron density regulator or the donor-acceptor linkages are used to control the rotational barriers of fluorophores in the excited states. This mechanism allows us to rationally design molecular rotor-based fluorophores that have desired responses to viscosity, thus extending the application of the AggTag method.In summary, our work allows the direct monitoring of the misfolded protein oligomers and differentiation of insoluble aggregates from other conformations in live cells, thus enabling studies of many currently unanswered questions in protein aggregation. Future directions are to develop methods that enable quantitative analyses of the protein aggregation process. Further, new methods are needed to detect and to quantify the formation and maturation of protein or RNA condensates that form membraneless organelles.

N-terminal Domain of TDP43 Enhances Liquid-Liquid Phase Separation of Globular Proteins.

Liquid-liquid phase separation (LLPS) of proteins is involved in a growing number of cellular processes. Most proteins with LLPS harbor intrinsically disordered regions (IDR), which serve as a guideline to search for cellular proteins that potentially phase separate. Herein, we reveal that oligomerization lowers the barriers for LLPS and could act as a general mechanism to enhance LLPS of proteins domains independent of IDR. Using TDP43 as a model system, we found that deleting its IDR resulted in LLPS that was dependent on the oligomerization of the N-terminal domain (NTD). Replacing TDP43's NTD with other oligomerization domains enhanced the LLPS proportionately to the state of oligomerization. In addition to TDP43, fusing NTD to other globular proteins without known LLPS behavior also drove their phase separation in a manner dependent on oligomerization. Finally, we demonstrate that heterooligomers composed of NTD-fused proteins can be driven into droplets through NTD interactions. Our results potentiate a new paradigm for using oligomerization domains as a signature to systematically identify cellular proteins with LLPS behavior, thus broadening the scope of this exciting research field.

AggFluor: Fluorogenic Toolbox Enables Direct Visualization of the Multi-Step Protein Aggregation Process in Live Cells.

Aberrantly processed or mutant proteins misfold and assemble into a variety of soluble oligomers and insoluble aggregates, a process that is associated with an increasing number of diseases that are not curable or manageable. Herein, we present a chemical toolbox, AggFluor, that allows for live cell imaging and differentiation of complex aggregated conformations in live cells. Based on the chromophore core of green fluorescent proteins, AggFluor is comprised of a series of molecular rotor fluorophores that span a wide range of viscosity sensitivity. As a result, these compounds exhibit differential turn-on fluorescence when incorporated in either soluble oligomers or insoluble aggregates. This feature allows us to develop, for the first time, a dual-color imaging strategy to distinguish unfolded protein oligomers from insoluble aggregates in live cells. Furthermore, we have demonstrated how small molecule proteostasis regulators can drive formation and disassembly of protein aggregates in both conformational states. In summary, AggFluor is the first set of rationally designed molecular rotor fluorophores that evenly cover a wide range of viscosity sensitivities. This set of fluorescent probes not only change the status quo of current imaging methods to visualize protein aggregation in live cells but also can be generally applied to study other biological processes that involve local viscosity changes with temporal and spatial resolutions.

Methylene blue decreases mitochondrial lysine acetylation in the diabetic heart.

Diabetic cardiomyopathy is preceded by mitochondrial alterations, and progresses to heart failure. We studied whether treatment with methylene blue (MB), a compound that was reported to serve as an alternate electron carrier within the mitochondrial electron transport chain (ETC), improves mitochondrial metabolism and cardiac function in type 1 diabetes. MB was administered at 10 mg/kg/day to control and diabetic rats. Both echocardiography and hemodynamic studies were performed to assess cardiac function. Mitochondrial studies comprised the measurement of oxidative phosphorylation and specific activities of fatty acid oxidation enzymes. Proteomic studies were employed to compare the level of lysine acetylation on cardiac mitochondrial proteins between the experimental groups. We found that MB facilitates NADH oxidation, increases NAD+, and the activity of deacetylase Sirtuin 3, and reduces protein lysine acetylation in diabetic cardiac mitochondria. We identified that lysine acetylation on 83 sites in 34 proteins is lower in the MB-treated diabetic group compared to the same sites in the untreated diabetic group. These changes occur across critical mitochondrial metabolic pathways including fatty acid transport and oxidation, amino acid metabolism, tricarboxylic acid cycle, ETC, transport, and regulatory proteins. While the MB treatment has no effect on the activities of acyl-CoA dehydrogenases, it decreases 3-hydroxyacyl-CoA dehydrogenase activity and long-chain fatty acid oxidation, and improves cardiac function. Providing an alternative route for mitochondrial electron transport is a novel therapeutic approach to decrease lysine acetylation, alleviate cardiac metabolic inflexibility, and improve cardiac function in diabetes.

Effects of Zidovudine Treatment on Heart mRNA Expression and Mitochondrial DNA Copy Number Associated with Alterations in Deoxynucleoside Triphosphate Composition in a Neonatal Rat Model.

The prevention of mother-to-child transmission (MTCT) of HIV is a crucial component in HIV therapy. Nucleoside reverse transcriptase inhibitors (NRTIs), primarily 3'-azido-3'-thymidine (AZT [zidovudine]), have been used to treat both mothers and neonates. While AZT is being replaced with less toxic drugs in treating mothers in MTCT prevention, it is still commonly used to treat neonates. Problems related to mitochondrial toxicity and potential mutagenesis associated with AZT treatment have been reported in treated cohorts. Yet little is known concerning the metabolism and potential toxicity of AZT on embryonic and neonatal tissues, especially considering that the enzymes of nucleoside metabolism change dramatically as many tissues convert from hyperplastic to hypertrophic growth during this period. AZT is known to inhibit thymidine phosphorylation and potentially alter deoxynucleoside triphosphate (dNTP) pools in adults. This study examines the effects of AZT on dNTP pools, mRNA expression of deoxynucleoside/deoxynucleotide metabolic enzymes, and mitochondrial DNA levels in a neonatal rat model. Results show that AZT treatment dramatically altered dNTP pools in the first 7 days of life after birth, which normalized to age-matched controls in the second and third weeks. Additionally, AZT treatment dramatically increased the mRNA levels of many enzymes involved in deoxynucleotide synthesis and mitochondrial biogenesis during the first week of life, which normalized to age-matched controls by the third week. These results were correlated with depletion of mitochondrial DNA noted in the second week. Taken together, results demonstrated that AZT treatment has a powerful effect on the deoxynucleotide synthesis pathways that may be associated with toxicity and mutagenesis.

Heart mitochondrial TTP synthesis and the compartmentalization of TMP.

The primary pathway of TTP synthesis in the heart requires thymidine salvage by mitochondrial thymidine kinase 2 (TK2). However, the compartmentalization of this pathway and the transport of thymidine nucleotides are not well understood. We investigated the metabolism of [(3)H]thymidine or [(3)H]TMP as precursors of [(3)H]TTP in isolated intact or broken mitochondria from the rat heart. The results demonstrated that [(3)H]thymidine was readily metabolized by the mitochondrial salvage enzymes to TTP in intact mitochondria. The equivalent addition of [(3)H]TMP produced far less [(3)H]TTP than the amount observed with [(3)H]thymidine as the precursor. Using zidovudine to inhibit TK2, the synthesis of [(3)H]TTP from [(3)H]TMP was effectively blocked, demonstrating that synthesis of [(3)H]TTP from [(3)H]TMP arose solely from the dephosphorysynthase pathway that includes deoxyuridine triphosphatelation of [(3)H]TMP to [(3)H]thymidine. To determine the role of the membrane in TMP metabolism, mitochondrial membranes were disrupted by freezing and thawing. In broken mitochondria, [(3)H]thymidine was readily converted to [(3)H]TMP, but further phosphorylation was prevented even though the energy charge was well maintained by addition of oligomycin A, phosphocreatine, and creatine phosphokinase. The failure to synthesize TTP in broken mitochondria was not related to a loss of membrane potential or inhibition of the electron transport chain, as confirmed by addition of carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone and potassium cyanide, respectively, in intact mitochondria. In summary, these data, taken together, suggest that the thymidine salvage pathway is compartmentalized so that TMP kinase prefers TMP synthesized by TK2 over medium TMP and that this is disrupted in broken mitochondria.