Signal-sustained imaging of mitophagy with an Enzyme-Activatable Metabolic Lipid-Labeling Probe.

Imaging of mitophagy is of significance as aberrant mitophagy is engaged in multiple diseases. Mitophagy has been imaged with synthetic or biotic pH sensors by reporting pH acidification en route delivery into lysosomes. To circumvent uncertainty of acidity-dependent signals, we herein report an enzyme-activatable probe covalently attached on mitochondrial inner membrane (ECAM) for signal-persist mitophagy imaging. ECAM is operated via ΔΨm-driven accumulation of Mito-proGreen in mitochondria and covalent linking of the trapped probe with azidophospholipids metabolically incorporated into the mitochondrial inner membrane. Upon mitophagy, ECAM is delivered into lysosomes and hydrolyzed by LNPEP/leucyl aminopeptidase, yielding turn-on green fluorescence that is immune to lysosomal acidity changes and stably retained in fixed cells. With ECAM, phorbol-12-myristate-13-acetate (PMA) was identified as a highly potent inducer of mitophagy. Overcoming signal susceptibility of pH probes and liability of ΔΨm probes to dissipation from stressed mitochondria, ECAM offers an attractive tool to study mitophagy and mitophagy-inducing therapeutic agents.

A dual biomarker-targeting probe enables signal-on surface labeling of Staphylococcus aureus.

Imaging or killing of a specific pathogen is of significance for precise therapy. Staphylococcus aureus (S. aureus) is an infectious gram-positive bacteria relying on Sortase A (SrtA) to anchor cell surface protein on peptidoglycan. We herein report signal-on labeling of S. aureus with self-quenched optical probes featuring vancomycin-conjugated SrtA substrate that is flanked by a dabcyl moiety paired with either fluorescein or eosine photosensizer (PS). SrtA-mediated cleavage of the substrate motif releases the dabcyl quencher, leading to covalent labeling of peptidoglycan with fluorescein or PS of restored photophysical property. The dual biomarked-enabled peptidoglycan labeling enables signal-on imaging and effective photodynamic destruction of S. aureus, suggesting a protheranostic approch activatable to SrtA-positive bacteria engaged in myriad diseases.

Fluorescence-On Imaging of Reticulophagy Enabled by an Acidity-Reporting Solvatochromic Probe.

Aberrant autophagy of the endoplasmic reticulum (reticulophagy) is engaged in diverse pathological disorders. Herein, we reported sensitive imaging of reticulophagy with ER-Green-proRed, a diad combining a solvatochromic entity of trifluoromethylated naphthalimide for long-term ER tracking by green fluorescence and an entity of rhodamine-lactam fluorogenic to lysosomal acidity. Stringently accumulated in the ER to give green fluorescence, ER-Green-proRed exhibits robust red fluorescence upon codelivery with the ER subdomain into lysosomes. The relevance of turn-on red fluorescence to reticulophagy was validated by reticulophagy modulated by starvation, reticulophagic receptors, and autophagy inhibition. This imaging method was successfully employed to discern reticulophagy induced by various pharmacological agents. These results show the potential of ER-targeted pH probes, as exemplified by ER-Green-proRed, to image reticulophagy and to identify reticulophagy inducers.

Peptidoglycan-Directed Chemical Ligation for Selective Inhibition on Gram-Positive Bacteria.

Microbicides with distinct antibacterial mechanisms show potential to combat multi-drug resistance bacteria. We herein report peptidoglycan-directed chemical ligation (PGCL) between alkyne-bearing vancomycin and an azide-tagged cationic polymer. The former binds peptidoglycan and inhibits peptidoglycan crosslinking, while the latter interferes the integrity of the bacterial membrane. PGCL results in enhanced bactericidal activity against Gram-positive Staphylococcus aureus (S. aureus) over Gram-negative Escherichia coli (E. coli). These data indicate the potential of PGCL to selectively and synergistically inhibit Gram-positive pathogens via dual modality antibacterial mechanisms of peptidoglycan-inhibiting antibiotics and bacterial membrane-disrupting polycations.

Sensitive imaging of Endoplasmic reticulum (ER) autophagy with an acidity-reporting ER-Tracker.

Macroautophagic/autophagic turnover of endoplasmic reticulum (reticulophagy) is critical for cell health. Herein we reported a sensitive fluorescence-on imaging of reticulophagy using a small molecule probe (ER-proRed) comprised of green-emissive fluorinated rhodol for ER targeting and nonfluorescent rhodamine-lactam prone to lysosome-triggered red fluorescence. Partitioned in ER to exhibit green fluorescence, ER-proRed gives intense red fluorescence upon co-delivery with ER into acidic lysosomes. Serving as the signal of reticulophagy, the turning on of red fluorescence enables discernment of reticulophagy induced by starvation, varied levels of reticulophagic receptors, and chemical agents such as etoposide and sodium butyrate. These results show ER probes optically activatable in lysosomes, such as ER-proRed, offer a sensitive and simplified tool for studying reticulophagy in biology and diseases.Abbreviations: Baf-A1, bafilomycin A1; CCCP, carbonyl cyanide m-chlorophenyl hydrazone; CQ, chloroquine diphosphate; ER, endoplasmic reticulum; FHR, fluorinated hydrophobic rhodol; GFP, green fluorescent protein; Reticulophagy, selective autophagy of ER; RFP, red fluorescent protein; ROX, X-rhodamine; UPR, unfolded protein response.

Biocapture-Directed Chemical Labeling for Discerning Stressed States of Organelles.

Lysosomal rupture engaged in diverse diseases remains poorly discerned from lysosomal membrane permeabilization (LMP). We herein reported biocapture-directed chemical labeling (BCCL) for the discern of lysosomal rupture by tracking the release of optically labeled cathepsins from damaged lysosomes into the cytosol. BCCL entails covalent anchoring of an azide-tagged suicide substrate (Epo-LeuTyrAz) to the enzyme active site and bioorthogonal ligation of the introduced azide with DBCORC, a ratiometric sensor featuring an acidity-reporting red emissive X-rhodamine-lactam (ROX), blue emissive coumarin (CM) inert to pH, and DBCO reactive to azide. Aided with fluorescein isocyanate-labeled sialic acid (FITC-Sia), a probe remained in pH-elevated lysosomes but dissipated from LMP+ lysosomes, BCCL enables optical discern of four states of lysosomes: ruptured lysosomes (blue in cytosol), LMP+ lysosomes (blue in lysosomes), pH-elevated lysosomes (blue and green in lysosomes), and physiological lysosomes (blue, green and red in lysosomes). This approach could find applicability to study lysosome rupture over LMP in diseases and to evaluate lysosome rupture-inducing drugs.

In cellulo synthesis of dendrimeric sensors for fluorescence-on imaging of bacterial phagocytosis.

Methods for optical tracking of pathogen-host interactions are of biomedical significance. We herein have reported a high molecular weight pH sensor (Den-pH) that is assembled in bacteria and then stably trapped in bacteria irrespective of bacterial membrane potentials. Endowed with acidity-triggered red fluorescence, Den-pH allows signal-on tracking of S. aureus in phagocytosis by macrophages. Intra-bacterial formation of multifunctional optical probes, which offers the advantage of overcoming the liability of conventional potential-sensitive dyes to dissipate from stressed bacteria, offers a new tool to study stressed pathogens.

Metabolic installation of macrophage-recruiting glycan ligand on tumor cell surface for in vivo tumor suppression.

Synthetic probes that could direct immune cells against tumors are potential immunotherapeutics. We herein report in vivo tumor suppression via an intravenously injected abiotic sialic acid (TCCSia) that could be metabolically incorporated into tumor cell surface to yield of a high affinity ligand (TCCSiaα2,3-Gal) of Siglec-1 specifically expressed on macrophages. We observed marked suppression of pulmonary metastasis and subcutaneous tumor growth of B16F10 melanoma cells in mice with TCCSia, suggesting the utility of abiotic sialic acid to modulate tumor immunity via recruiting Siglec+ immune cells.

Imaging of Mitophagy Enabled by an Acidity-Reporting Probe Anchored on the Mitochondrial Inner Membrane.

Classical chemical probes are prone to dissipation from stressed organelles, as evidenced by the incapability of mitochondrial dyes to image mitophagy linked to multiple diseases. We herein reported mitophagy imaging via covalent anchoring of a lysosomal probe to the mitochondrial inner membrane (CALM). Utilizing DBCORC-TPP, an azide-conjugatable probe with acidity-triggered fluorescence, CALM is operated via ΔΨm-promoted probe accumulation in mitochondria and thereby bioorthogonal ligation of the trapped probe with azido-choline (Azcholine) metabolically installed on the mitochondrial membrane. Overcoming the limitation of synthetic probes to dissipate from stressed organelles, CALM enables signal-on fluorescence imaging of mitophagy induced by starvation and is further employed to reveal mitophagy in ferroptosis. These results suggest the potential of CALM as a new tool to study mitophagy.

Galectin Trafficking Pathway-Enabled Color-Switchable Detection of Lysosomal Membrane Permeabilization.

Lysosomal membrane permeabilization (LMP) engaged in multiple human diseases is accompanied by relocation of cytosolic galectin into LMP+ lysosomes. We herein reported a galectin trafficking-targeted method to image LMP using two kinds of glyco-dendrimers, a sialic acid-terminated dendrimer labeled with pH-inert rhodamine and a lactose-terminated dendrimer labeled with fluorescein that becomes green-emissive in pH-elevated lysosomes. Albeit both accumulated in physiological lysosomes, the former is released from LMP+ lysosomes while the latter binds to galectin accumulated in LMP+ lysosomes and thus trapped in LMP+ lysosomes. Accordingly, LMP+ lysosomes exhibit loss of red fluorescence and turn-on green fluorescence due to loss of lysosomal acidity. This red-to-green color switch enables discernment of LMP+ lysosomes from physiological lysosomes and pH-elevated lysosomes and can be further utilized to detect LMP in distinct cell death pathways. These results suggest the utility of galectin trafficking pathway-integrated synthetic probes for detection of LMP, a key factor for diseased cells.

Installation of high-affinity Siglec-1 ligand on tumor surface for macrophage-engaged tumor suppression.

Siglecs that binds cell surface sialoglycans are a family of immunomodulatory receptors, of which, Siglec-7 expressed on natural killer (NK) cells promotes tumor immunoevation while the role of Siglec-1 expressed on macrophages on tumor development remains largely unexplored. Herein, we selectively introduced high affinity sialoside ligands of Siglec-1 and Siglec-7 to tumor cell surface via in vivo Strain-promoted Azide-Alkyne cyclization of TCCSiaα2,3-Lactose or FITCSiaα2,6-Lactose with 9-azido sialic acid (AzSia) metabolically installed on tumor cell surface. We found that TCCSiaα2,3-Lactose conjugated on tumor surface moderately inhibited tumor growth while FITCSiaα2,6-Lactose promote tumor growth. These results suggest high-affinity ligand of Siglec-1 dispalyed on tumors surface provide a new perspective for tumor immunotherapy.

A fluorescence-activatable tumor-reporting probe for precise photodynamic therapy.

Approaches that could enable precise photodynamic therapy (PDT) are of therapeutic potential. We herein report a trifunctional probe (Glu-RdEB) that could be activated to generate fluorescent rhodamine species to pinpoint tumor foci. The probe contains a γ-glutaminyl moiety cleavable to γ-glutamyl transpeptidase (GGT) overexpressed in multiple tumors, an entity of an ENBS photosensitizer for PDT, and an entity of rhodamine fluorescently quenched by ENBS. Upon activation by tumor-associated GGT, the probe releases highly fluorescent rhodamine that is selectively confined in tumors whereby light irradiation leads to effective tumor regression in mice. These results indicate the feasibility of a fluorescently quenched dye-photosensitizer pair to yield tumor-activatable fluorescence to direct PDT.

An organelle-directed chemical ligation approach enables dual-color detection of mitophagy.

Dysfunctional organelles and defective turnover of organelles are engaged in multiple human diseases, but are elusive to image with conventional organelle probes. To overcome this, we developed intra-mitochondrial CLICK to assess mitophagy (IMCLAM), using a pair of conventional ΔΨm probes, where each probe alone fails to track dysfunctional mitochondria. The in situ formed optical triad is stably trapped in mitochondria without resorting to ΔΨm. Utilizing an acidity-responsive ΔΨm probe, IMCLAM enabled fluorescence-on detection of mitophagy by sensing pH acidification upon delivery of mitochondria into lysosomes. Moreover, we applied IMCLAM to assay mitophagy induced by pharmacological compounds in living cells and wild-type zebrafish embryos. Thus, IMCLAM offers a simplified tool to study mitochondria and mitophagy and provide a basis for screening mitophagy-inducing compounds. Abbreviations: CCCP, carbonyl cyanide m-chlorophenylhydrazone; IMCLAM, intra-mitochondrial CLICK to assess mitophagy; ROX, X-rhodamine; SPAAC, stain-promoted azide-alkyne Click Chemistry; TPP, triphenylphosphonium.

Activatable Dual ROS-Producing Probe for Dual Organelle-Engaged Photodynamic Therapy.

Photodynamic therapy (PDT) necessitates approaches capable of increasing antitumor effects while decreasing nonspecific photodamage. We herein report an activatable probe (Glu-PyEB) comprising two distinct photosensitizers with mutually suppressed photodynamics. Activation by tumor-associated γ-glutamyltranspeptidase gives rise to a generator of superoxide radical (O2-•) accumulated in lysosomes and a producer of singlet oxygen (1O2) enriched in mitochondria. This enables light-irradiation-triggered damage of lysosomes and mitochondria, robust cell death, and tumor retardation in vivo, showing the use of paired photosensitizers subjected to reciprocally suppressed photodynamics for activatable PDT.

Organelle-Directed Metabolic Glycan Labeling and Optical Tracking of Dysfunctional Lysosomes Thereof.

Metabolic glycan labeling (MGL) has been employed for diverse purposes, such as cell surface glycan imaging and tumor surface engineering. We herein reported organelle-specific MGL (OMGL) for selective tagging of the inner limiting membrane of lysosomes over the cell surface. This is operated via acidity-promoted accumulation of optical probes in lysosomes and bioorthogonal ligation of the trapped probes with 9-azidosialic acid (AzSia) metabolically installed on lysosomal membrane proteins. Overcoming the limitation of classical organelle probes to dissipate from stressed organelles, OMGL enables optical tracking of pH-elevated lysosomes in exocytosis and membrane-permeabilized lysosomes in different cell death pathways. Thus, OMGL offers a new tool to study lysosome biology.

Imaging stressed organelles via sugar-conjugated color-switchable pH sensors.

Stressed organelles are often challenging to image with canonical organelle probes owing to their propensity to dissipate from stressed organelles. We herein report the imaging of stressed lysosomes with color-switchable glyco-probes that contain an entity of mannose-6-carboxylate or sialic acid for targeting to and long-term retention in stressed lysosomes, and a diad of fluorescein/rhodamine-X-lactam exhibiting dramatic red-to-green fluorescence shift upon pH elevation. Relative to acidotropic dyes prone to dissipate from pH-elevated lysosomes, both glyco-probes are stably trapped in lysosomes without resorting to lysosomal acidity. Importantly, these probes are red emissive in acidic lysosomes (pH 4.5-5.8), but switched to green fluorescence in lysosomes of pH 6.0-7.4, allowing dual color discrimination of lysosomal pH alterations in cell starvation. These results support the use of sugar-armed sensors to investigate stressed lysosomes in biology and disease.

Organelle-Redirected Chameleon Sensor-Enabled Live Cell Imaging of Mitochondrial DNA.

Mitochondrial DNA (mtDNA) plays important roles in diverse physiological processes and myriad diseases. We herein report mtDNA imaging with a chameleon sensor containing a cationic rhodamine B (RB) entity for mitochondria targeting and a fluorogenic SYBR Green-I (SG) entity for DNA sensing. SG-RB selectively binds to mtDNA and gives green SG fluorescence in mitochondria of living cells but gives red RB fluorescence upon delivery of mitochondria into lysosomes in mitophagy. With the dual-color imaging, mtDNA aggregation and elevated mitophagy were identified in HeLa cells stressed with anticancer doxorubicin. These results suggest the utility of organelle-redirected DNA sensors for live cell imaging of mtDNA involved in myriad pathological disorders.

An in cellulo-activated multicolor cell labeling approach used to image dying cell clearance.

Dying cell clearance is critical for myriad biological processes such as tissue homeostasis. We herein report an enzyme-activated fluorescence cell labeling approach and its use for multicolor imaging of dying cell clearance. Diacetylated 4-hydroxymandelic acid (DHA)-conjugated dyes give rise to reactive quinone methides upon deacetylation in live cells, which in turn covalently labels cellular proteins. With partner cells tagged with distinct fluorescence, apoptotic cell clearance by Raw 264.7 macrophages and epithelial HeLa cells was captured by confocal microscopy, showing the potential of DHA-based cell labeling for investigating cell-cell interactions.

Inactivation of Cyclic AMP Response Element Transcription Caused by Constitutive p38 Activation Is Mediated by Hyperphosphorylation-Dependent CRTC2 Nucleocytoplasmic Transport.

The p38 signal transduction pathway can be activated transiently or constitutively, depending on the contexts in which the activation occurs. However, the biological consequence of constitutive activation of p38 is largely unknown. After screening 300 transcriptional cofactors, we identified CRTC2 as a downstream substrate of constitutively activated p38. Constitutive, rather than transient, activation of p38 led to hyperphosphorylation of CRTC2, resulting in CRTC2 cytosolic relocation and subsequent inactivation of cyclic AMP response element (CRE)-mediated transcription. Interestingly, the cytosolic translocation of CRTC2 depended on phosphorylation accumulation at multiple sites (≥11 phosphoserine/phosphothreonine residues) but not on specific sites. The hyperphosphorylation-driven nucleocytoplasmic transport of CRTC2 may not be a rare case of nuclear export of proteins, as we also observed that constitutively activated p38 promoted FOS nuclear export in a hyperphosphorylation-dependent manner. Collectively, our study uncovered a previously unknown mechanism of inactivation of selected transcription, which results from hyperphosphorylation-driven nucleocytoplasmic transport of cofactors or transcription factors mediated by constitutively active kinase.

On the use of abiotic sialic acids to attenuate cell inflammation.

Sialic acid (Sia) residues on cell surface are critical for myriad cellular events such as immunity and inflammation. We herein reported the use of abiotic Sia to raise the thresholds of inflammatory cell responses. Identified from a panel of structurally diversified Sia analogs via a cell inflammation assay, Sia-2, with N-butyryl moiety at C-5, markedly lowered LPS-stimulated NF-κB activity in macrophages. Further analysis shows that Sia-2 attenuates phosphorylation of IκB and Erk1/2/p38/JNK, critical for NF-κB signaling and MAPK signaling, and lowers gene transcription of proinflammatory interleukin-6. These results support the use of abiotic Sia as promising agents to modulate cell surface Sia-pertinent cell signaling.