Super-resolution imaging and real-time tracking lysosome in living cells by a fluorescent probe

Lysosomes function as important organelles within cells and their movement associates with diverse biological events, hence the real-time tracking of lysosomal movement is of great significance. However, since most lysosome fluorescent probes suffer from relatively unsatisfactory photostability, tracking lysosomal movement in real-time remains challenging. Here, we report that a naphthalimide-based fluorescent compound, namely NIMS, is a quite promising probe for lysosome imaging. The visualizing mechanism lies in the selective accumulation of NIMS in lysosomes via a protonation reaction, followed by the fluorescence enhancement due to the interactions of NIMS with proteins. Owing to its high selectivity and good photostability, NIMS was successfully applied to capture super-resolution fluorescence images of lysosomes. More importantly, real-time tracking of lysosome movement in a single living cell by NIMS was realized with a confocal laser scanning microscope. Surprisingly, even in normal culture conditions, around 2/3 of the captured lysosomes were observed to move within 5 min, indicative of the highly dynamic features of lysosomes. Thus, this probe may facilitate the understanding of the lysosome dynamics in physiological or pathological conditions.     

Theranostic Iridium(III) Complexes as One‐ and Two‐Photon Phosphorescent Trackers to Monitor Autophagic Lysosomes

During autophagy, the intracellular components are captured in autophagosomes and delivered to lysosomes for degradation and recycling. Changes in lysosomal trafficking and contents are key events in the regulation of autophagy, which has been implicated in many physiological and pathological processes. In this work, two iridium(III) complexes ( LysoIr1 and LysoIr2 ) are developed as theranostic agents to monitor autophagic lysosomes. These complexes display lysosome‐activated phosphorescence and can specifically label lysosomes with high photostability. Simultaneously, they can induce autophagy potently without initiating an apoptosis response. We demonstrate that LysoIr2 can effectively implement two functions, namely autophagy induction and lysosomal tracking, in the visualization of autophagosomal–lysosomal fusion. More importantly, they display strong two‐photon excited fluorescence (TPEF), which is favorable for live cell imaging and in vivo applications.     

A novel acidotropic pH indicator and its potential application in labeling acidic organelles of live cells.

BACKGROUND: Ratio imaging has received intensive attention in the past few decades. The growing potential of ratio imaging is significantly limited, however, by the lack of appropriate fluorescent probes, for acidic organelles in particular. The classic fluorescent dyes (such as fluoresceins, rhodamines and coumarins) are not suitable for studying acidic organelles (such as lysosomes) because their fluorescence is significantly decreased under neutral or acidic conditions. This has motivated us to develop probes that can be used in ratio imaging that are strongly fluorescent even in acidic media. RESULTS: The compound 2-(4-pyridyl)-5-((4-(2-dimethylaminoethyl-aminocarbamoyl) methoxy)phenyl)oxazole (PDMPO) was prepared and characterized as a new acidotropic dual-excitation and dual-emission pH indicator. It emits intense yellow fluorescence at lower pH and gives intense blue fluorescence at higher pH. This unique pH-dependent fluorescence property was readily explored to selectively stain lysosomes and to determine the pH of the organelle in an emission-ratio-imaging mode. PDMPO is selectively localized to lysosomes and exhibits a pH-dependent dual excitation and emission. CONCLUSIONS: PDMPO selectively labels acidic organelles (such as lysosomes) of live cells and the two distinct emission peaks can be used to monitor the pH fluctuations of live cells in ratio measurements. Additionally, the very large Stokes shift and excellent photostability of PDMPO make the compound an ideal fluorescent acidotropic probe. The unique fluorescence properties of PDMPO might give researchers a new tool with which to study acidic organelles of live cells.     

PHOTOOXIDATIVE DAMAGE TO LYSOSOMES OF CULTURED MACROPHAGES BY ACRIDINE ORANGE

Cultured cells accumulate acridine orange (AO), which is a weak basic dye and a photosensitizer, in lysosomes and other acidic compartments. During exposure to blue light, AO-loaded macrophages show decreasing red granular fluorescence and increasing green diffuse fluorescence. This is hypothesized to represent peroxidative damage to lysosomal membranes resulting in an impaired proton gradient with deprotonation of the AO to its uncharged form and subsequent leakage of the dye. Further damage to the lysosomal membranes will result in release of lytic enzymes from the lysosomal compartment into the cytosol, leading to degeneration and finally cell death. The survival of AO-loaded and light-exposed macrophages is controllable by varying the exposure times to blue light. Inhibition of lysosomal proteases by E-64 results in increased cell survival after AO and blue light-mediated damage, indicating a role of proteolytic enzymes in this type of damage. Morphological analysis shows 'rounding up' with formation of retraction fibrils and pronounced plasma membrane blebbing. The formation of autophagic vacuoles is an early and pronounced event. After protease inhibition, however, all these phenomena are inhibitable to a considerable degree. We have thus directed photooxidative damage selectively to lysosomal membranes and their contents. This technique will allow further detailed studies of the role of lysosomes in degeneration-regeneration processes.     

STED Imaging the Dynamics of Lysosomes by Dually Fluorogenic Si-Rhodamine

Super-resolution microscopy (SRM) imaging of the finite subcellular structures and subtle bioactivities inside organelles delivers abundant cellular information with high fidelity to unravel the intricate biological processes. An ideal fluorescent probe with precise control of fluorescence is critical in SRM technique like stimulated emission depletion (STED). Si-rhodamine was decorated with both targeting group and H⁺-receptor, affording the dually fluorogenic Si-rhodamine in which the NIR fluorescence was efficiently controlled by the coalescent of spirolactone-zwitterion equilibrium and PeT mechanism. The dually fluorogenic characters of the probe offer a perfect mutual enhancement in sensitivity, specificity and spatial resolution. Strong fluorescence only released in the existence of targeting protein at acidic lysosomal pH, ensured precisely tracking the dynamic of lysosomal structure and pH in living cells by STED.     

Organelle‐Specific Activity‐Based Protein Profiling in Living Cells

A multimodal activity‐based probe for targeting acidic organelles was developed to measure subcellular native enzymatic activity in cells by fluorescence microscopy and mass spectrometry. A cathepsin‐reactive warhead conjugated to a weakly basic amine and a clickable alkyne, for subsequent appendage of a fluorophore or biotin reporter tag, accumulated in lysosomes as observed by structured illumination microscopy (SIM) in J774 mouse macrophage cells. Analysis of in vivo labeled J774 cells by mass spectrometry showed that the probe was very selective for cathepsins B and Z, two lysosomal cysteine proteases. Analysis of starvation‐induced autophagy, a catabolic pathway involving lysosomes, showed a large increase in the number of tagged proteins and an increase in cathepsin activity. The organelle‐targeting of activity‐based probes holds great promise for the characterization of enzyme activities in the myriad diseases linked to specific subcellular locations, particularly the lysosome.     

Lysosomal pH Rise during Heat Shock Monitored by a Lysosome‐Targeting Near‐Infrared Ratiometric Fluorescent Probe

Heat stroke is a life‐threatening condition, featuring a high body temperature and malfunction of many organ systems. The relationship between heat shock and lysosomes is poorly understood, mainly because of the lack of a suitable research approach. Herein, by incorporating morpholine into a stable hemicyanine skeleton, we develop a new lysosome‐targeting near‐infrared ratiometric pH probe. In combination with fluorescence imaging, we show for the first time that the lysosomal pH value increases but never decreases during heat shock, which might result from lysosomal membrane permeabilization. We also demonstrate that this lysosomal pH rise is irreversible in living cells. Moreover, the probe is easy to synthesize, and shows superior overall analytical performance as compared to the existing commercial ones. This enhanced performance may enable it to be widely used in more lysosomal models of living cells and in further revealing the mechanisms underlying heat‐related pathology.     

Increased In Vitro Lysosomal Function in Oxidative Stress-Induced Cell Lines

Exposure of mammalian cells to oxidative stress alters lysosomal enzymes. Through cytochemical analysis of lysosomes with LysoTracker, we demonstrated that the number and fluorescent intensity of lysosome-like organelles in HeLa cells increased with exposure to hydrogen peroxide (H₂O₂), 6-hydroxydopamine (6-OHDA), and UVB irradiation. The lysosomes isolated from HeLa cells exposed to three oxidative stressors showed the enhanced antimicrobial activity against Escherichia coli. Further, when lysosomes that were isolated from HeLa cells exposed by oxidative stress were treated to normal HeLa cells, the viability of the HeLa cells was drastically reduced, suggesting increased in vitro lysosomal function (i.e., antimicrobial activity, apoptotic cell death). In addition, we also found that cathepsin B and D were implicated in increased in vitro lysosomal function when isolated from HeLa cells exposed by oxidative stress. Decrease in cathepsin B activity and increase in cathepsin D activity were observed in lysosomes isolated from HeLa cells after treatment with H₂O₂, 6-ODHA, or UVB, but cathepsin B and D were not the sole factors to induce cell death by in vitro lysosomal function. Therefore, these studies suggest a new approach to use lysosomes as antimicrobial agents and as new materials for treating cancer cell lines.     

Strategic engineering of alkyl spacer length for a pH-tolerant lysosome marker and dual organelle localization

Long-term visualization of lysosomal properties is extremely crucial to evaluate diseases related to their dysfunction. However, many of the reported lysotrackers are less conducive to imaging lysosomes precisely because they suffer from fluorescence quenching and other inherent drawbacks such as pH-sensitivity, polarity insensitivity, water insolubility, slow diffusibility, and poor photostability. To overcome these limitations, we have utilized an alkyl chain length engineering strategy and synthesized a series of lysosome targeting fluorescent derivatives namely NIMCs by attaching a morpholine moiety at the peri position of the 1,8-naphthalimide (NI) ring through varying alkyl spacers between morpholine and 1,8-naphthalimide. The structural and optical properties of the synthesized NIMCs were explored by ¹H-NMR, single-crystal X-ray diffraction, UV-Vis, and fluorescence spectroscopy. Afterward, optical spectroscopic measurements were carefully performed to identify a pH-tolerant, polarity sensitive, and highly photostable fluoroprobes for further live-cell imaging applications. NIMC6 displayed excellent pH-tolerant and polarity-sensitive properties. Consequently, all NIMCs were employed in kidney fibroblast cells (BHK-21) to investigate their applicability for lysosome targeting and probing lysosomal micropolarity. Interestingly, a switching of localization from lysosomes to the endoplasmic reticulum (ER) was also achieved by controlling the linker length and this phenomenon was subsequently applied in determining ER micropolarity. Additionally, the selected probe NIMC6 was also employed in BHK-21 cells for 3-D spheroid imaging and in Caenorhabditis elegans (C. elegans) for in vivo imaging, to evaluate its efficacy for imaging animal models.

A series naphthalimide-based fluorophores were designed by alkyl spacer length engineering to discover a pH-tolerant lysosomal marker. This approach also allows to probe lysosome-related organelles in C. elegans and communication between organelles.     

Efficacious Anticancer Drug Delivery Mediated by a pH‐Sensitive Self‐Assembly of a Conserved Tripeptide Derived from Tyrosine Kinase NGF Receptor

We present herein a short tripeptide sequence (Lys–Phe–Gly or KFG) that is situated in the juxtamembrane region of the tyrosine kinase nerve growth factor (Trk NGF) receptors. KFG self‐assembles in water and shows a reversible and concentration‐dependent switching of nanostructures from nanospheres (vesicles) to nanotubes, as evidenced by dynamic light scattering, transmission electron microscopy, and atomic force microscopy. The morphology change was associated with a transition in the secondary structure. The tripeptide vesicles have inner aqueous compartments and are stable at pH 7.4 but rupture rapidly at pH≈6. The pH‐sensitive response of the vesicles was exploited for the delivery of a chemotherapeutic anticancer drug, doxorubicin, which resulted in enhanced cytotoxicity for both drug‐sensitive and drug‐resistant cells. Efficient intracellular release of the drug was confirmed by fluorescence‐activated cell sorting analysis, fluorescence microscopy, and confocal microscopy.     

A Photostable AIEgen for Specific and Real‐time Monitoring of Lysosomal Processes

Lysosomes are recognized as advanced organelles involved in many cellular processes and are also considered as crucial regulators of cell homeostasis. The current strategies for monitoring activities of lysosomes exhibit some limitations. Herein, we synthesized a novel fluorescent probe named 2M‐DPAS with AIE characteristics, which was proved to have significant advantages of good biocompatibility, high selectivity, bright emission and excellent photostability. Based on those, 2M‐DPAS can be used to continuously monitor the dynamic changes of lysosomes, including autophagy and mitophagy, as well as to track the process of endocytosis of macromolecules in lysosomes, which are of benefit to better know about the lysosomes‐related diseases.     

Preparation and Cellular Uptake of pH‐Dependent Fluorescent Single‐Wall Carbon Nanotubes

Fluorescent single‐wall carbon nanotubes (SWCNTs) were prepared by mixing cut SWCNTs with acridine orange (AO). The optical absorbance and fluorescence characteristics of AO–SWCNT conjugates display interesting pH‐dependent properties. Fluorescence microscopy in combination with transmission electron microscopy proves that AO–SWCNTs can enter HeLa cells and are located inside lysosomes. The endocytosis‐inhibiting tests show that the clathrin‐mediated endocytosis is a key step in the internalization process. The internalized AO–SWCNTs remain inside lysosomes for more than a week and have little effect on cell proliferation. These findings may be useful in understanding the SWCNT‐based intracellular drug delivery mechanism and help to develop new intracellular drug transporters.     

Chemoenzymatic Synthesis of a Phosphorylated Glycoprotein

The majority of lysosomal enzymes are targeted to the lysosome by post‐translational tagging with N‐glycans terminating in mannose‐6‐phosphate (M6P) residues. Some current enzyme replacement therapies (ERTs) for lysosomal storage disorders are limited in their efficacy by the extent to which the recombinant enzymes bear the M6P‐terminated glycans required for effective trafficking. Chemical synthesis was combined with endo‐β‐N‐acetylglucosaminidase (ENGase) catalysis to allow the convergent synthesis of glycosyl amino acids bearing M6P residues. This approach can be extended to the remodeling of proteins, as exemplified by RNase. The powerful synergy of chemical synthesis and ENGase‐mediated biocatalysis enabled the first synthesis of a glycoprotein bearing M6P‐terminated N‐glycans in which the glycans are attached to the peptide backbone by entirely natural linkages.     

A new rhodamine B-based lysosomal pH fluorescent indicator

We designed and synthesized a new pH fluorescent probe, RCE, based on structural changes of rhodamine dye at different pH values. The probe exhibits high selectivity, high sensitivity and quick response to acidic pH, as well as low cytotoxicity, excellent photostability, reversibility and cell membrane permeability. Fluorescence intensity at 584 nm was increased more than 150-fold within pH range 7.51–3.53. This probe has pK_a value 4.71, which is valuable for studying acidic organelles. Because of its long absorption and emission wavelengths, RCE can avoid associated cell damage. The probe can selectively stain lysosomes and monitor lysosomal pH changes in living cells.     

Fluorogenic Tagging of Peptide and Protein 3-Nitrotyrosine with 4-(Aminomethyl)benzenesulfonic Acid for Quantitative Analysis of Protein Tyrosine Nitration

Protein 3-nitrotyrosine (3-NT) has been recognized as an important biomarker of nitroxidative stress associated with inflammatory and degenerative diseases, and biological aging. Analysis of protein-bound 3-NT continues to represent a challenge since in vivo it frequently does not accumulate on proteins in amounts detectable by quantitative analytical methods. Here, we describe a novel approach of fluorescent tagging and quantitation of peptide-bound 3-NT residues based on the selective reduction to 3-AT followed by reaction with 4-(aminomethyl)benzenesulfonic acid (ABS) in the presence of K₃Fe(CN)₆ to form a highly fluorescent 2-phenylbenzoxazole product. Synthetic 3-NT peptide (0.005-1 μM) upon reduction with 10 mM sodium dithionite and tagging with 2 mM ABS and 5 μM K₃Fe(CN)₆ in 0.1 M Na₂HPO₄ buffer (pH 9.0) was converted with yields >95% to a single fluorescent product incorporating two ABS molecules per 3-NT residue, with fluorescence excitation and emission maxima at 360 ± 2 and 490 ± 2 nm, respectively, and a quantum yield of 0.77 ± 0.08, based on reverse-phase LC with UV and fluorescence detection, fluorescence spectroscopy and LC–MS–MS analysis. This protocol was successfully tested for quantitative analysis of in vitro Tyr nitration in a model protein, rabbit muscle phosphorylase b, and in a complex mixture of proteins from C2C12 cultured cells exposed to peroxynitrite, with a detection limit of ca. 1 pmol 3-NT by fluorescence spectrometry, and an apparent LOD of 12 and 40 pmol for nitropeptides alone or in the presence of 100 μg digested cell proteins, respectively. LC–MS–MS analysis of ABS tagged peptides revealed that the fluorescent derivatives undergo efficient backbone fragmentations, allowing for sequence-specific characterization of protein Tyr nitration in proteomic studies. Fluorogenic tagging with ABS also can be instrumental for detection and visualization of protein 3-NT in LC and gel-based protein separations.     

Design of Potent Mannose 6‐Phosphate Analogues for the Functionalization of Lysosomal Enzymes To Improve the Treatment of Pompe Disease

Improving therapeutics delivery in enzyme replacement therapy (ERT) for lysosomal storage disorders is a challenge. Herein, we present the synthesis of novel analogues of mannose 6‐phosphate (M6P), known as AMFAs and functionalized at the anomeric position for enzyme grafting. AMFAs are non‐phosphate serum‐resistant derivatives that efficiently bind the cation‐independent mannose 6‐phosphate receptor (CI‐M6PR), which is the main pathway to address enzymes to lysosomes. One of the AMFAs was used to improve the treatment of the lysosomal myopathy Pompe disease, in which acid α‐glucosidase (GAA) is defective. AMFA grafting on a M6P‐free recombinant GAA led to a higher uptake of the GAA in adult Pompe fibroblasts in culture as compared to Myozyme, the M6P recombinant GAA. Moreover, the treatment of Pompe adult mice with the AMFA‐grafted recombinant enzyme led to a remarkable improvement, even at low doses, in muscle functionality and regeneration, whereas Myozyme had limited efficacy.     

A Ratiometric Two‐Photon Fluorescent Probe for Tracking Lysosomal ATP: Direct In Cellulo Observation of Lysosomal Membrane Fusion Processes

Vesicles exchange their contents through membrane fusion processes, kiss‐and‐run and full‐collapse fusion. Indirect observation of these fusion processes using artificial vesicles enhanced our understanding on the molecular mechanisms involved. Direct observation of the fusion processes in a real biological system, however, remains a challenge owing to many technical obstacles. We report a ratiometric two‐photon probe offering real‐time tracking of lysosomal ATP with quantitative information for the first time. By applying the probe to two‐photon live‐cell imaging, the lysosomal membrane fusion process in cells has been directly observed and the concentration of its content, lysosomal ATP, has been measured. Results show that the kiss‐and‐run process between lysosomes proceeds through repeated transient interactions with gradual content mixing, whereas the full‐fusion process occurs at once. Furthermore, it is confirmed that both the fusion processes proceed with conservation of the content. Such a small‐molecule probe exerts minimal disturbance and hence has potential for studying various biological processes associated with lysosomal ATP.     

Synthesis and characterization of a new fluorogenic substrate for alpha-galactosidase

Alpha-galactosidase A hydrolyzes the terminal alpha-galactosyl moieties from glycolipids and glycoproteins in lysosomes. Mutations in α-galactosidase cause lysosomal accumulation of the glycosphingolipid, globotriaosylceramide, which leads to Fabry disease. Small-molecule chaperones that bind to mutant enzyme proteins and correct their misfolding and mistrafficking have emerged as a potential therapy for Fabry disease. We have synthesized a red fluorogenic substrate, resorufinyl α-d-galactopyranoside, for a new α-galactosidase enzyme assay. This assay can be measured continuously at lower pH values, without the addition of a stop solution, due to the relatively low pK _a of resorufin (~6). In addition, the assay emits red fluorescence, which can significantly reduce interferences due to compound fluorescence and dust/lint as compared to blue fluorescence. Therefore, this new red fluorogenic substrate and the resulting enzyme assay can be used in high-throughput screening to identify small-molecule chaperones for Fabry disease. Zhen-Dan Shi and Omid Motabar contributed equally to this work     

Subcellular Duplex DNA and G‐Quadruplex Interaction Profiling of a Hexagonal PtII Metallacycle

Metal‐driven self‐assembly afforded a multitude of fascinating supramolecular coordination complexes (SCCs) with applications as catalysts, host–guest, and stimuli‐responsive systems. However, the interest in the biological applications of SCCs is only starting to emerge and thorough characterization of their behavior in biological milieus is still lacking. Herein, we report on the synthesis and detailed in‐cell tracking of a Pt₂L₂ metallacycle. We show that our hexagonal supramolecule accumulates in cancer cell nuclei, exerting a distinctive blue fluorescence staining of chromatin resistant to UV photobleaching selectively in nucleolar G4‐rich regions. SCC co‐localizes with epitopes of the quadruplex‐specific antibody BG4 and replaces other well‐known G4 stabilizers. Moreover, the photophysical changes accompanying the metallacycle binding to G4s in solution (fluorescence quenching, absorption enhancement) also take place intracellularly, allowing its subcellular interaction tracking.     

Structural Control of Cell Permeability with Highly Emissive Europium(III) Complexes Permits Different Microscopy Applications

Four anionic europium complexes are described based on triazacyclononane tris‐carboxylate or phosphinate ligands. In each case, the three sensitising chromophores comprise a substituted aryl–alkynyl pyridine group, with complex brightness in water falling in the range 4 to 23 mM ^?1 cm^?1. para‐Substitution of the aryl ring with carboxymethyl groups gives complexes that are taken into cells, stain the lysosomes selectively and unexpectedly permit lifetime measurements of lysosomal pH. In contrast, the introduction of sulfonate groups inhibits cell uptake enabling the Eu complex to be used as an extracellular donor for FRET applications at the membrane surface. Using time‐gated FRET microscopy, the cell membrane structure was highlighted, in which Cell Mask Deep Red was used as a membrane‐ localized FRET acceptor.