Interactions between autophagic and endo-lysosomal markers in endothelial cells

Autophagic and endo-lysosomal degradative pathways are essential for cell homeostasis. Availability of reliable tools to interrogate these pathways is critical to unveil their involvement in physiology and pathophysiology. Although several probes have been recently developed to monitor autophagic or lysosomal compartments, their specificity has not been validated through co-localization studies with well-known markers. Here, we evaluate the selectivity and interactions between one lysosomal (Lyso-ID) and one autophagosomal (Cyto-ID) probe under conditions modulating autophagy and/or endo-lysosomal function in live cells. The probe for acidic compartments Lyso-ID was fully localized inside vesicles positive for markers of late endosome-lysosomes, including Lamp1-GFP and GFP-CINCCKVL. Induction of autophagy by amino acid deprivation in bovine aortic endothelial cells caused an early and potent increase in the fluorescence of the proposed autophagy dye Cyto-ID. Cyto-ID-positive compartments extensively co-localized with the autophagosomal fluorescent reporter RFP-LC3, although the time and/or threshold for organelle detection was different for each probe. Interestingly, use of Cyto-ID in combination with Lysotracker Red or Lyso-ID allowed the observation of structures labeled with either one or both probes, the extent of co-localization increasing upon treatment with protease inhibitors. Inhibition of the endo-lysosomal pathway with chloroquine or U18666A resulted in the formation of large Cyto-ID and Lyso-ID-positive compartments. These results constitute the first assessment of the selectivity of Cyto-ID and Lyso-ID as probes for the autophagic and lysosomal pathways, respectively. Our observations show that these probes can be used in combination with protein-based markers for monitoring the interactions of both pathways in live cells.     

Development of a novel method for quantification of autophagic protein degradation by AHA labeling

Autophagy is a catabolic process during which cellular components including protein aggregates and organelles are degraded via a lysosome-dependent process to sustain metabolic homeostasis during nutrient or energy deprivation. Measuring the rate of proteolysis of long-lived proteins is a classical assay for measurement of autophagic flux. However, traditional methods, such as a radioisotope labeling assay, are technically tedious and have low sensitivity. Here, we report a novel method for quantification of long-lived protein degradation based on L-azidohomoalanine (AHA) labeling in mouse embryonic fibroblasts (MEFs) and in human cancer cells. AHA is a surrogate for L-methionine, containing a bio-orthogonalazide moiety. When added to cultured cells, AHA is incorporated into proteins during active protein synthesis. After a click reaction between an azide and an alkyne, the azide-containing proteins can be detected with an alkyne-tagged fluorescent dye, coupled with flow cytometry. Induction of autophagy by starvation or mechanistic target of rapamycin (MTOR) inhibitors was able to induce a significant reduction of the fluorescence intensity, consistent with other autophagic markers. Coincidently, inhibition of autophagy by pharmacological agents or by Atg gene deletion abolished the reduction of the fluorescence intensity. Compared with the classical radioisotope pulse-labeling method, we think that our method is sensitive, quantitative, nonradioactive, and easy to perform, and can be applied to both human and animal cell culture systems.     

A new probe to measure autophagic flux in vitro and in vivo

Macroautophagy is a catabolic process that delivers cytoplasmic components via the autophagosome to lysosomes for degradation. Measuring autophagic activity is critical to dissect molecular mechanisms and functions of autophagy but remains challenging due to the lack of a definitive method. We have recently developed a new fluorescent probe, GFP-LC3-RFP-LC3ΔG, to assess autophagic flux. Upon intracellular expression, the probe is cleaved by ATG4 family proteases into equimolar amounts of GFP-LC3 and RFP-LC3ΔG. The former is degraded by autophagy while the latter persists as an internal control in the cytosol. Autophagic flux can thus be quantified by obtaining the ratio of GFP:RFP signals. Using this method, we have identified several autophagy-modulating drugs by screening an approved drug library. We have also demonstrated that induced and basal autophagic flux can be monitored in zebrafish and mice.     

Sorting cells for basal and induced autophagic flux by quantitative ratiometric flow cytometry

We detail here a protocol using tandem-tagged mCherry-EGFP-LC3 (C-G-LC3) to quantify autophagic flux in single cells by ratiometric flow cytometry and to isolate subpopulations of cells based on their relative levels of autophagic flux. This robust and sensitive method measures autophagic flux rather than autophagosome number and is an important addition to the autophagy researcher’s array of tools for measuring autophagy. Two crucial steps in this protocol are i) generate cells constitutively expressing C-G-LC3 with low to medium fluorescence and low fluorescence variability, and ii) correctly set up gates and voltage/gain on a properly equipped flow cytometer. We have used this method to measure autophagic flux in a variety of cell types and experimental systems using many different autophagy stimuli. On a sorting flow cytometer, this technique can be used to isolate cells with different levels of basal autophagic flux, or cells with variable induction of flux in response to a given stimulus for further analysis or experimentation. We have also combined quantification of autophagic flux with methods to measure apoptosis and cell surface proteins, demonstrating the usefulness of this protocol in combination with other flow cytometry labels and markers.     

Development of a novel method for quantification of autophagic protein degradation by AHA labeling

Autophagy is a catabolic process during which cellular components including protein aggregates and organelles are degraded via a lysosome-dependent process to sustain metabolic homeostasis during nutrient or energy deprivation. Measuring the rate of proteolysis of long-lived proteins is a classical assay for measurement of autophagic flux. However, traditional methods, such as a radioisotope labeling assay, are technically tedious and have low sensitivity. Here, we report a novel method for quantification of long-lived protein degradation based on L-azidohomoalanine (AHA) labeling in mouse embryonic fibroblasts (MEFs) and in human cancer cells. AHA is a surrogate for L-methionine, containing a bio-orthogonalazide moiety. When added to cultured cells, AHA is incorporated into proteins during active protein synthesis. After a click reaction between an azide and an alkyne, the azide-containing proteins can be detected with an alkyne-tagged fluorescent dye, coupled with flow cytometry. Induction of autophagy by starvation or mechanistic target of rapamycin (MTOR) inhibitors was able to induce a significant reduction of the fluorescence intensity, consistent with other autophagic markers. Coincidently, inhibition of autophagy by pharmacological agents or by Atg gene deletion abolished the reduction of the fluorescence intensity. Compared with the classical radioisotope pulse-labeling method, we think that our method is sensitive, quantitative, nonradioactive, and easy to perform, and can be applied to both human and animal cell culture systems.     

Discovery of pan autophagy inhibitors through a high-throughput screen highlights macroautophagy as an evolutionarily conserved process across 3 eukaryotic kingdoms

Due to the involvement of macroautophagy/autophagy in different pathophysiological conditions such as infections, neurodegeneration and cancer, identification of novel small molecules that modulate the process is of current research and clinical interest. In this work, we developed a luciferase-based sensitive and robust kinetic high-throughput screen (HTS) of small molecules that modulate autophagic degradation of peroxisomes in the budding yeast Saccharomyces cerevisiae. Being a pathway-specific rather than a target-driven assay, we identified small molecule modulators that acted at key steps of autophagic flux. Two of the inhibitors, Bay11 and ZPCK, obtained from the screen were further characterized using secondary assays in yeast. Bay11 inhibited autophagy at a step before fusion with the vacuole whereas ZPCK inhibited the cargo degradation inside the vacuole. Furthermore, we demonstrated that these molecules altered the process of autophagy in mammalian cells as well. Strikingly, these molecules also modulated autophagic flux in a novel model plant, Aponogeton madagascariensis. Thus, using small molecule modulators identified by using a newly developed HTS autophagy assay, our results support that macroautophagy is a conserved process across fungal, animal and plant kingdoms.     

PML-RARα enhances constitutive autophagic activity through inhibiting the Akt/mTOR pathway

Autophagy is a highly conserved, closely regulated homeostatic cellular activity that allows for the bulk degradation of long-lived proteins and cytoplasmic organelles. Its roles in cancer initiation and progression and in determining the response of tumor cells to anticancer therapy are complicated, and only limited investigation has been conducted on the potential significance of autophagy in the pathogenesis and therapeutic response of acute myeloid leukemia. Here we demonstrate that the inducible or transfected expression of the acute promyelocytic leukemia (APL)-specific PML-RARα, but not PLZF-RARα or NPM-RARα, fusion protein upregulates constitutive autophagy activation in leukemic and nonleukemic cells, as evaluated by hallmarks for autophagy including transmission electron microscopy. The significant increase in autophagic activity is also found in the leukemic cells-infiltrated bone marrow and spleen from PML-RARα-transplanted leukemic mice. The autophagy inhibitor 3-methyladenine significantly abrogates the autophagic events upregulated by PML-RARα, while the autophagic flux assay reveals that the fusion protein induces autophagy by increasing the on-rate of autophagic sequestration. Furthermore, this modulation of autophagy by PML-RARα is possibly mediated by a decreased activation of the Akt/mTOR pathway. Finally, we also show that autophagy contributes to the anti-apoptotic function of the PML-RARα protein. Given the critical role of the PML-RARα oncoprotein in APL pathogenesis, this study suggests an important role of autophagy in the development and treatment of this disease.     

BCR-ABL1-induced leukemogenesis and autophagic targeting by arsenic trioxide

We have recently shown that arsenic trioxide (As₂O₃) is a potent inducer of autophagic degradation of the BCR-ABL1 oncoprotein, which is the cause of chronic myeloid leukemia (CML) and Ph+ acute lymphoid leukemia (Ph+ ALL). Our recently published work has shown that pharmacological inhibition of autophagy or molecularly targeting of elements of the autophagic machinery partially reverses the suppressive effects of As₂O₃ on primitive leukemic precursors from CML patients. Altogether, our studies have provided direct evidence that arsenic-induced, autophagy-mediated, degradation of BCR-ABL1 is an important mechanism for the generation of the effects of As₂O₃ on BCR-ABL1 transformed leukemic progenitors. These studies raise the potential of future clinical-translational efforts employing combinations of arsenic trioxide with autophagy-modulating agents to promote elimination of early leukemic progenitors and, possibly, leukemia-initiating stem cells.     

Monitoring autophagic flux by an improved tandem fluorescent-tagged LC3 (mTagRFP-mWasabi-LC3) reveals that high-dose rapamycin impairs autophagic flux in cancer cells

Monitoring autophagic flux is important for the analysis of autophagy. Tandem fluorescent-tagged LC3 (mRFP-EGFP-LC3) is a convenient assay for monitoring autophagic flux based on different pH stability of EGFP and mRFP fluorescent proteins. However, it has been reported that there is still weak fluorescence of EGFP in acidic environments (pH between 4 and 5) or acidic lysosomes. So it is possible that autolysosomes are labeled with yellow signals (GFP⁺RFP⁺ puncta), which results in misinterpreting autophagic flux results. Therefore, it is desirable to choose a monomeric green fluorescent protein that is more acid sensitive than EGFP in the assay of autophagic flux. Here, we report on an mTagRFP-mWasabi-LC3 reporter, in which mWasabi is more acid sensitive than EGFP and has no fluorescence in acidic lysosomes. Meanwhile, mTagRFP-mWasabi-LC3ΔG was constructed as the negative control for this assay. Compared with mRFP-EGFP-LC3, our results showed that this reporter is more sensitive and accurate in detecting the accumulation of autophagosomes and autolysosomes. Using this reporter, we find that high-dose rapamycin (30 μM) will impair autophagic flux, inducing many more autophagosomes than autolysosomes in HeLa cells, while low-dose rapamycin (500 nM) has an opposite effect. In addition, other chemical autophagy inducers (cisplatin, staurosporine and Z18) also elicit much more autophagosomes at high doses than those at low doses. Our results suggest that the dosage of chemical autophagy inducers would obviously influence autophagic flux in cells.     

Shining a light on autophagy in neurodegenerative diseases

Small-molecule modulators of autophagy have been widely investigated as potential therapies for neurodegenerative diseases. In a recent issue of JBC, Safren et al. described a novel assay that uses a photoconvertible fusion protein to identify compounds that alter autophagic flux. Autophagy inducers identified using this assay were found to either alleviate or exacerbate neurotoxicity in different cellular models of amyotrophic lateral sclerosis, challenging the notion that autophagy stimulation can be used as a one-size-fits-all therapy for neurodegenerative disease.     

Development of a specific live-cell assay for native autophagic flux

Autophagy is an evolutionarily conserved pathway mediating the breakdown of cellular proteins and organelles. Emphasizing its pivotal nature, autophagy dysfunction contributes to many diseases; nevertheless, development of effective autophagy modulating drugs is hampered by fundamental deficiencies in available methods for measuring autophagic activity or flux. To overcome these limitations, we introduced the photoconvertible protein Dendra2 into the MAP1LC3B locus of human cells via CRISPR/Cas9 genome editing, enabling accurate and sensitive assessments of autophagy in living cells by optical pulse labeling. We used this assay to perform high-throughput drug screens of four chemical libraries comprising over 30,000 diverse compounds, identifying several clinically relevant drugs and novel autophagy modulators. A select series of candidate compounds also modulated autophagy flux in human motor neurons modified by CRISPR/Cas9 to express GFP-labeled LC3. Using automated microscopy, we tested the therapeutic potential of autophagy induction in several distinct neuronal models of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). In doing so, we found that autophagy induction exhibited discordant effects, improving survival in disease models involving the RNA binding protein TDP-43, while exacerbating toxicity in neurons expressing mutant forms of UBQLN2 and C9ORF72 associated with familial ALS/FTD. These studies confirm the utility of the Dendra2-LC3 assay, while illustrating the contradictory effects of autophagy induction in different ALS/FTD subtypes.     

Autophagy impairment with lysosomal and mitochondrial dysfunction is an important characteristic of oxidative stress-induced senescence

Macroautophagy/autophagy has profound implications for aging. However, the true features of autophagy in the progression of aging remain to be clarified. In the present study, we explored the status of autophagic flux during the development of cell senescence induced by oxidative stress. In this system, although autophagic structures increased, the degradation of SQSTM1/p62 protein, the yellow puncta of mRFP-GFP-LC3 fluorescence and the activity of lysosomal proteolytic enzymes all decreased in senescent cells, indicating impaired autophagic flux with lysosomal dysfunction. The influence of autophagy activity on senescence development was confirmed by both positive and negative autophagy modulators; and MTOR-dependent autophagy activators, rapamycin and PP242, efficiently suppressed cellular senescence through a mechanism relevant to restoring autophagic flux. By time-phased treatment of cells with the antioxidant N-acetylcysteine (NAC), the mitochondria uncoupler carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and ambroxol, a reagent with the effect of enhancing lysosomal enzyme maturation, we found that mitochondrial dysfunction plays an initiating role, while lysosomal dysfunction is more directly responsible for autophagy impairment and senescence. Interestingly, the effect of rapamycin on autophagy flux is linked to its role in functional revitalization of both mitochondrial and lysosomal functions. Together, this study demonstrates that autophagy impairment is crucial for oxidative stress-induced cell senescence, thus restoring autophagy activity could be a promising way to retard senescence.     

Direct quantification of autophagic flux by a single molecule-based probe

Macroautophagy/autophagy remains a rapidly advancing research topic, and there continues to be a need for constantly evolving methodology to investigate this pathway at each individual step. Accordingly, new assays to measure autophagic flux in a robust and reliable manner are essential to understand the mechanism and physiological roles of autophagy. Kaizuka et al. recently reported a new fluorescence probe, GFP-LC3-RFP-LC3ΔG to directly demonstrate autophagic flux without being combined with lysosomal inhibitors (see the Kaizuka et al. punctum in this issue of the journal). When expressed in cells, the probe is cleaved into a degradable/quenchable part, GFP-LC3, and stable/cytosolic part, RFP-LC3ΔG. The latter serves as an internal control and a decrease of the GFP:RFP ratio indicates the occurrence of autophagy. Since the key index of this probe is the degradation of GFP-LC3, it can be used to measure the cumulative effect of basal autophagy. The assay is applicable to high-throughput drug discovery as well as in vivo analysis.     

Identification of novel autophagy regulators by a luciferase-based assay for the kinetics of autophagic flux

Macroautophagy (hereafter referred to as autophagy) has recently emerged as an attractive target for the treatment of various degenerative diseases and cancer. The discovery of effective pharmaceutical regulators of autophagy has, however, been hindered by a lack of feasible assay systems for autophagic flux. Here, we present a luciferase-based reporter assay that measures autophagic flux in real time in living cells and demonstrate that this assay system is apt for the detection of dose- and stimulus-dependent differences in autophagy kinetics. Furthermore, by screening a small molecule kinase inhibitor library containing 80 compounds we identified 12 compounds as inducers of autophagic flux. Importantly, six inhibitors of the class I phosphoinositide 3-kinase – protein kinase B – mammalian target of rapamycin complex 1 axis, the central signaling pathway repressing autophagy, scored as autophagy inducers adequately validating the screen. We conclude that the assay system presented here allows easy and rapid monitoring of autophagy kinetics and is suitable for screening of small molecule libraries.     

A Revised Assay for Monitoring Autophagic Flux in Arabidopsis thaliana Reveals Involvement of AUTOPHAGY-RELATED9 in Autophagy

Autophagy targets cytoplasmic cargo to a lytic compartment for degradation. Autophagy-related (Atg) proteins, including the transmembrane protein Atg9, are involved in different steps of autophagy in yeast and mammalian cells. Functional classification of core Atg proteins in plants has not been clearly confirmed, partly because of the limited availability of reliable assays for monitoring autophagic flux. By using proUBQ10-GFP-ATG8a as an autophagic marker, we showed that autophagic flux is reduced but not completely compromised in Arabidopsis thaliana atg9 mutants. In contrast, we confirmed full inhibition of auto-phagic flux in atg7 and that the difference in autophagy was consistent with the differences in mutant phenotypes such as hypersensitivity to nutrient stress and selective autophagy. Autophagic flux is also reduced by an inhibitor of phosphatidylinositol kinase. Our data indicated that atg9 is phenotypically distinct from atg7 and atg2 in Arabidopsis, and we proposed that ATG9 and phosphatidylinositol kinase activity contribute to efficient autophagy in Arabidopsis.     

A quantitative TR-FRET plate reader immunoassay for measuring autophagy

Autophagy involves the isolation and targeting of unwanted cellular components to lysosomes for their digestion and reuse. Autophagic dysregulation has recently been implicated in a wide range of disease processes, yet facile methods for quantifying autophagy have been lacking in the field. Here we describe the generation of a quantitative plate reader assay for measuring the autophagic activity of cells. One of the best characterized autophagy markers is the protein LC3B, which normally resides in the cytosol (LC3B-I) but upon induction of autophagy becomes lipidated and embedded in autophagosomal membranes (LC3B-II). To quantify autophagy, we reasoned that GFP-tagged LC3B could serve as a time-resolved fluorescence resonance energy transfer (TR-FRET) acceptor upon cell lysis in the presence of terbium-labeled LC3B antibodies. Using this TR-FRET immunoassay approach, we screened a panel of LC3B antibodies and identified an antibody that exhibits strong preferential affinity toward autophagosome-associated LC3B-II and thereby facilitates specific detection of autophagic activity. The plate reader format provides both a quantitative and an objective result, thus overcoming some of the key limitations of the traditional immunoblotting and imaging approaches used to monitor autophagy. Moreover, since the assay step requires only a single addition of cell lysis buffer containing the detection antibody its simple workflow is both automation-friendly and scalable, which renders it suitable for high-throughput screening. We demonstrate how this TR-FRET immunoassay for GFP-tagged LC3B-II can be applied to quantitatively detect changes in the autophagy activity of cells, including estimating effects on autophagic flux.     

Differential effects of N-TiO2 nanoparticle and its photo-activated form on autophagy and necroptosis in human melanoma A375 cells

The manipulation of autophagy provides a new opportunity for highly effective anticancer therapies. Recently, we showed that photodynamic therapy (PDT) with nitrogen-doped titanium dioxide (N-TiO₂) nanoparticles (NPs) could promote the reactive oxygen species (ROS)-dependent autophagy in leukemia cells. However, the differential autophagic effects of N-TiO₂ NPs in the dark and light conditions and the potential of N-TiO_2-based PDT for the treatment of melanoma cells remain unknown. Here we show that depending on the visible-light condition, the autophagic response of human melanoma A375 cells to N-TiO₂ NPs switches between two different statuses (ie., flux or blockade) with the opposite outcomes (ie., survival or death). Mechanistically, low doses of N-TiO₂ NPs (1-100 µg/ml) stimulate a nontoxic autophagy flux response in A375 cells, whereas their photo-activation leads to the impairment of the autophagosome-lysosome fusion, the blockade of autophagy flux and consequently the induction of RIPK1-mediated necroptosis via ROS production. These results confirm that photo-controllable autophagic effects of N-TiO₂ NPs can be utilized for the treatment of cancer, particularly melanoma.     

PML-RARα enhances constitutive autophagic activity through inhibiting the Akt/mTOR pathway

Autophagy is a highly conserved, closely regulated homeostatic cellular activity that allows for the bulk degradation of long-lived proteins and cytoplasmic organelles. Its roles in cancer initiation and progression and in determining the response of tumor cells to anticancer therapy are complicated, and only limited investigation has been conducted on the potential significance of autophagy in the pathogenesis and therapeutic response of acute myeloid leukemia. Here we demonstrate that the inducible or transfected expression of the acute promyelocytic leukemia (APL)-specific PML-RARα, but not PLZF-RARα or NPM-RARα, fusion protein upregulates constitutive autophagy activation in leukemic and nonleukemic cells, as evaluated by hallmarks for autophagy including transmission electron microscopy. The significant increase in autophagic activity is also found in the leukemic cells-infiltrated bone marrow and spleen from PML-RARα-transplanted leukemic mice. The autophagy inhibitor 3-methyladenine significantly abrogates the autophagic events upregulated by PML-RARα, while the autophagic flux assay reveals that the fusion protein induces autophagy by increasing the on-rate of autophagic sequestration. Furthermore, this modulation of autophagy by PML-RARα is possibly mediated by a decreased activation of the Akt/mTOR pathway. Finally, we also show that autophagy contributes to the anti-apoptotic function of the PML-RARα protein. Given the critical role of the PML-RARα oncoprotein in APL pathogenesis, this study suggests an important role of autophagy in the development and treatment of this disease.