Imaging Lysosomal pH Alteration in Stressed Cells with a Sensitive Ratiometric Fluorescence Sensor

The organelle-specific pH is crucial for cell homeostasis. Aberrant pH of lysosomes has been manifested in myriad diseases. To probe lysosome responses to cell stress, we herein report the detection of lysosomal pH changes with a dual colored probe (CM-ROX), featuring a coumarin domain with “always-on” blue fluorescence and a rhodamine–lactam domain activatable to lysosomal acidity to give red fluorescence. With sensitive ratiometric signals upon subtle pH changes, CM-ROX enables discernment of lysosomal pH changes in cells undergoing autophagy, cell death, and viral infection

Defining Cancer Cell Bioenergetic Profiles Using a Dual Organelle-Oriented Chemosensor Responsive to pH Values and Electropotential Changes

Cell fate is largely shaped by combined activity of different types of organelles, which often feature functionally critical parameters that succumb to pathological inducers. We herein report the analysis of cell bioenergetic profiles with a dual organelle-oriented chemosensor (RC-AMI), partitioning in mitochondria to give blue fluorescence and in lysosomes to give red fluorescence. Responsive to lysosomal pH and mitochondrial transmembrane potential (ΔΨ_m), two parameters crucial to cell bioenergetics, RC-AMI enables dual colored reporting of lysosomal acidity and ΔΨ_m, revealing upregulated ΔΨ_m and imbalance dramatically shifted favoring ΔΨ_m over lysosomal acidity in cancer cells whereas the tendency is reversed in starved cells. Complementing classical homo-organelle-specific sensors, this dual organelle-oriented and fluorescently responsive probe offers a new tool to detect imbalance between lysosomal acidity and mitochondrial ΔΨ_m, an index critical for cancer bioenergetics

Lysosomal pH Decrease in Inflammatory Cells Used To Enable Activatable Imaging of Inflammation with a Sialic Acid Conjugated Profluorophore

Inflammation causes significant morbidity and mortality, necessitating effective in vivo imaging of inflammation. Prior approaches often rely on combination of optical agents with entities specific for proteinaceous biomarkers overexpressed in inflammatory tissues. We herein report a fundamentally new approach to image inflammation by targeting lysosomes undergoing acidification in inflammatory cells with a sialic acid (Sia) conjugated near-infrared profluorophore (pNIR). Sia–pNIR contains a sialic acid domain for in vivo targeting of inflamed tissues and a pNIR domain which isomerizes into fluorescent and optoacoustic species in acidic lysosomes. Sia–pNIR displays high inflammation-to-healthy tissue signal contrasts in mice treated with Escherichia coli, Staphylococcus aureus, or lipopolysaccharide. In addition, inflammation-associated fluorescence is switched off upon antibiotics treatment in mice. This report shows the potentials of Sia–pNIR for activatable dual-modality inflammation imaging, and particularly the use of lysosomes of inflamed cells as a previously unappreciated biomarker for inflammation imaging

Optical Tracking of Phagocytosis with an Activatable Profluorophore Metabolically Incorporated into Bacterial Peptidoglycan

Phagocytosis is critical for immunity against pathogens. Prior imaging using dye-labeled synthetic beads or green fluorescent protein-expressing bacteria is limited by “always-on” signals which compromise discerning phagocytosed particles from adherent particles. Targeting cellular internalization of pathogens into acidic phagolysosomes, we herein report “turn-on” fluorescence imaging of phagocytosis with viable bacteria featuring peptidoglycans covalently modified with rhodamine-lactam responsive to acidic pH. Culturing of <i>Escherichia coli</i> (<i>E. coli</i>) and <i>Staphylococcus aureus</i> (<i>S. aureus</i>) with d-lysine conjugated rhodamine-lactam and fluorescein isocyanate (FITC) leads to efficient metabolic incorporation of FITC and rhodamine-lactam into bacterial peptidoglycan. <i>E. coli</i> and <i>S. aureus</i> become red-emissive upon phagocytosis into Raw 264.7 macrophages. With FITC as the reference signal, the mono- and dual-color emission allow efficient <i>in situ</i> distinction of ingested bacteria from extracellular bacteria. Given the ease of optical peptidoglycan labeling, the prevalence of microbial peptidoglycan and preservation of microbial surface landscape, this approach would be of use for investigation on microbial pathogenesis and high-throughput screening of immunomodulators of phagocytosis

Organelle-Directed Staudinger Reaction Enabling Fluorescence-on Resolution of Mitochondrial Electropotentials via a Self-Immolative Charge Reversal Probe

Organelles often feature parameters pertinent to functions and yet responsive to biochemical stress. The electropotential across the mitochondrial membrane (ΔΨm) is a crucial mediator of cell fates. Herein we report a bioorthogonal reaction enabled fluorescence-on probing of ΔΨm alterations featuring anionic fluorescein-triphenylphosphonium diad (F-TPP), which is released via intramitochondria Staudinger reaction triggered self-immolation of <i>o</i>-azidomethylbenzoylated F-TPP. Compared to classical cationic mitochondria-specific dyes, F-TPP is hydrophilic and negatively charged. Effectively discerning ΔΨm changes upon diverse stress inducers, the organelle-directed bioorthogonal imaging strategy offers unprecedented choices to probe mitochondrial biology with functional molecules that are otherwise inaccessible via physiological organelle-probe affinity

Inhibition of ERK- or JNK- pathway blocks zVAD- but not TNF-induced cell death.

<p>(<b>A</b>) Inhibition of ERK- or JNK- pathway blocked zVAD-induced cell death. L929 cells were untreated or treated with ERK inhibitor PD98059 (100 µM), JNK inhibitor SP600125 (10 µM), p38 inhibitor SB203580 (10 µM), and PI3K inhibitor LY294002 (100 µM), respectively. Then the cells were treated with mock or zVAD (20 µM) for 24 h and cell viabilities were measured. (<b>B</b>) Inhibition of MAPKs does not block TNF-induced cell death. L929 cells were untreated or treated with PD98059 (100 µM), SP600125 (10 µM), p38 inhibitor SB203580 (10 µM), and LY294002 (100 µM). Then the cells were treated with mock or TNF (10 ng/ml) for 24 h and cell viabilities were measured. (<b>C</b>) zVAD had little effect on MAPKs’ activation. L929 cells were treated with zVAD or zVAD+LY294002 for 0, 0.5, 1, 2, 4 h, respectively. Cell lysates were subjected to western blot analysis with antibodies against p-ERK, ERK, p-JNK, JNK, p-p38 and p38. (<b>D</b>) Inhibition of zVAD-induced cell death has no effect on MAPKs’ activation. L929 cells were treated with zVAD or zVAD+Nec-1 for 0, 0.5, 1, 2, 4, 6 h, respectively. Cell lysates were subjected to western blot analysis with the indicated antibodies. (<b>E</b>) Inhibitors of MAPKs did not affect zVAD-induced LC3 modification. L929 cells were untreated or treated with PD98059 (100 µM), SP600125 (10 µM), SB203580 (10 µM), or LY294002 (100 µM). Then the cells were treated with mock or zVAD (20 µM) for 12 h and LC3 were analyzed by western blot. (<b>F</b>) Hypothetical pathway network of zVAD-induced cell death. The main pathways of zVAD-induced cell death are autophagy and autocrine of TNF. The induction of TNF by zVAD may depend on the collective effect of a number of pathways such as ERK, JNK, and autophagy. Mechanisms of zVAD-induced L929 cells could be different due to the variation of L929 sublines. **, p<0.01.</p

Regulator of G-Protein Signaling 19 (RGS19) and Its Partner Gα-Inhibiting Activity Polypeptide 3 (GNAI3) Are Required for zVAD-Induced Autophagy and Cell Death in L929 Cells

<div><p>Autophagy has diverse biological functions and is involved in many biological processes. The L929 cell death induced by the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-(OMe)-fluoromethyl ketone (zVAD) was shown to be an autophagy-mediated death for which RIP1 and RIP3 were both required. It was also reported that zVAD can induce a small amount of TNF production, which was shown to be required for zVAD-induced L929 cell death, arguing for the contribution of autophagy in the zVAD-induced L929 cell death. In an effort to study RIP3 mediated cell death, we identified regulator of G-protein signaling 19 (RGS19) as a RIP3 interacting protein. We showed that RGS19 and its partner Gα-inhibiting activity polypeptide 3 (GNAI3) are involved in zVAD-, but not TNF-, induced cell death. The role of RGS19 and GNAI3 in zVAD-induced cell death is that they are involved in zVAD-induced autophagy. By the use of small hairpin RNAs and chemical inhibitors, we further demonstrated that zVAD-induced autophagy requires not only RIP1, RIP3, PI3KC3 and Beclin-1, but also RGS19 and GNAI3, and this autophagy is required for zVAD-induced TNF production. Collectively, our data suggest that zVAD-induced L929 cell death is a synergistic result of autophagy, caspase inhibition and autocrine effect of TNF.</p></div

Knockdown of RGS19 or GNAI3 inhibits both zVAD- and TNF-induced autophagy.

<p>(<b>A–B</b>) Knockdown of RGS19 or GNAI3 inhibited zVAD-induced modification of LC3. Control and RGS19-knockdown (<b>A</b>) or GNAI3 knockdown (<b>B</b>) L929 cells were treated with mock or zVAD (20 µM) for 12 h. Cell lysates were subjected to western blot analysis with antibodies against LC3 and β-actin. Results from RIP1- or PI3KC3-knockdown cells were included as controls. (<b>C–D</b>) Knockdown of RGS19 or GNAI3 inhibited TNF-induced modification of LC3. Control and RGS19-knockdown (<b>C</b>) or GNAI3 knockdown (<b>D</b>) L929 cells were treated with mock or TNF (1 ng/ml) for 12 h. Cell lysates were subjected to western blot analysis with the indicated antibodies. (<b>E</b>) Knockdown of RGS19 or GNAI3 impaired zVAD-induced LC3 flux. Control and RGS19-knockdown or GNAI3-konckdown L929 cells were cultured with or without chloroquine (25 µM) and treated with or without zVAD (20 µM) for 12 h. LC3 levels were measured by Western blot.</p

Blocking autophagy pathway inhibits zVAD-induced TNF production.

<p>(<b>A</b>) Knockdown of TNFR1 inhibited both TNF- and zVAD-induced cell deaths. Control and GFP- or TNFR1-knockdown L929 cells were treated with mock, TNF (10 ng/ml) or zVAD (20 µM) for 24 h. Then cell viabilities were measured. (<b>B</b>) Knockdown of TNF inhibited zVAD- but not TNF-induced cell death. Control and GFP- or TNF-knockdown L929 cells were treated with mock, TNF (10 ng/ml) or zVAD (20 µM) for 24 h. Then cell viabilities were measured. (<b>C</b>) Knockdown of RGS19, GNAI3, RIP3, or PI3KC3 all inhibited zVAD-induced increase of TNF mRNA. Control and RGS19-, GNAI3-, RIP3- or PI3KC3-knockdown L929 cells were treated with mock or zVAD (20 µM) for 3 h. TNF mRNA levels were measured by real-time PCR. (<b>D</b>) Knockdown of RGS19, GNAI3, RIP3, or PI3KC3 all inhibited zVAD-induced TNF secretion. Control and RGS19-, GNAI3-, RIP3- or PI3KC3-knockdown L929 cells were treated with mock or zVAD (20 µM) for 24 h. Then the cell culture medium was collected and concentrated 10 folds, and the TNF secretion was determined by ELISA. (<b>E</b>) Beclin-1, but not TNF or TNFR1, is required for zVAD-induced LC3 modification. Control and TNF-knockdown or TNFR-knockdown or Bclin 1-knockdown L929 cells were treated with mock or zVAD (20 µM) for 12 h. Cell lysates were subjected to Western blot analysis with antibodies against LC3 and β-actin. (<b>F</b>) Control and Bclin 1-knockdown L929 cells were treated with mock or zVAD (20 µM) for 12 h. Then TNF secretion was determined (left) and cell viabilities were measured (right). **, p<0.01.</p

RGS19 and GNAI3 interact with RIP3 and promote zVAD- but not TNF-induced cell death.

<p>(<b>A</b>) Co-IP analysis is used to study the interactions between RGS19 and RIP3, GNAI3 and RIP3, RGS19 and GNAI3. Myc-GNAI3 with Flag-RIP3 or Flag-RGS19, Flag-RIP3 with Myc-RGS19 were respectively co-expressed in 293T cells, Flag-RIP3 or Flag-RGS19 was then immunoprecipitated with anti-Flag M2-beads, and Myc-GNAI3 or Myc-RGS19 was detected in the immunoprecipitates with anti-myc antibody by Western blotting. (<b>B</b>) Knockdown of RGS19 or GNAI3 effectively blocked zVAD-induced cell death, but had no effect on TNF-induced cell death. Control and RGS19- or GNAI3-knockdown L929 cells were treated for 24 h with mock, TNF (10 ng/ml) or zVAD (20 µM). RIP1-, RIP3- or PI3KC3-knockdown L929 cells were also included. Then cell viabilities were measured (upper panel). The knockdown efficiencies were determined by relative mRNA levels calculated from real-time PCR results (lower panel). **, p<0.01.</p