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Research Techniques Made Simple: Analysis of Autophagy in the Skin

  • David Hill
    Affiliations
    Faculty of Health Sciences and Wellbeing, University of Sunderland, Sunderland, United Kingdom
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  • Ioana Cosgarea
    Affiliations
    Translation and Clinical Research Institute, Newcastle University, Newcastle upon Tyne, United Kingdom

    Newcastle Dermatology, Newcastle Hospitals NHS Trust, Newcastle upon Tyne, United Kingdom

    Newcastle Oncology, Newcastle Hospitals NHS Trust, Newcastle upon Tyne, United Kingdom
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  • Nick Reynolds
    Affiliations
    Translation and Clinical Research Institute, Newcastle University, Newcastle upon Tyne, United Kingdom

    Newcastle Dermatology, Newcastle Hospitals NHS Trust, Newcastle upon Tyne, United Kingdom
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  • Penny Lovat
    Affiliations
    Translation and Clinical Research Institute, Newcastle University, Newcastle upon Tyne, United Kingdom

    AMLo Biosciences Ltd, Newcastle University, Newcastle Upon Tyne, United Kingdom
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  • Jane Armstrong
    Correspondence
    Correspondence: Jane Armstrong, Faculty of Health Sciences and Wellbeing, University of Sunderland, Sciences Complex, City Campus, Sunderland SR1 3SD, United Kingdom.
    Affiliations
    Faculty of Health Sciences and Wellbeing, University of Sunderland, Sunderland, United Kingdom
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      Autophagy is required for normal skin homeostasis and its disordered regulation is implicated in a range of cutaneous diseases. Several well-characterized biomarkers of autophagy are used experimentally to quantify autophagic activity or clinically to correlate autophagy with disease progression. This article discusses the advantages and limitations of different approaches for measuring autophagy as well as the techniques for modulating autophagy. These include analysis of endogenous LC3, a central autophagy regulatory protein, and measurement of LC3 flux using a dual-fluorescent reporter, which provides a quantitative readout of autophagy in cell culture systems in vitro and animal models in vivo. Degradation of SQSTM1/p62 during autophagy is proposed as an alternative biomarker allowing the analysis of autophagy both experimentally and clinically. However, the complex regulation of individual autophagy proteins and their involvement in multiple pathways means that several proteins must be analyzed together, preferably over a time course to accurately interpret changes in autophagic activity. Genetic modification of autophagy proteins can be used to better understand basic autophagic mechanisms contributing to health and disease, whereas small molecule inhibitors of autophagy regulatory proteins, lysosomal inhibitors, or activators of cytotoxic autophagy have been explored as potential treatments for skin disorders where autophagy is defective.

      Abbreviations:

      mRFP (monomeric RFP), PI3K (phosphatidylinositol-3-kinase), ULK1 (unc-51-like kinase 1)

      Introduction

      Autophagy is a key cellular mechanism for the degradation and recycling of proteins and organelles and is defined according to three main classifications. Macroautophagy is the canonical form of autophagy involving sequestration of cellular components within a double-membrane autophagosome, which fuses with a lysosome to form a degradative autolysosome (Figure 1). Chaperone-mediated autophagy is a selective process in which individual proteins and/or aggregates are directly sequestered into the lysosome, whereas microautophagy is the direct engulfment of cellular contents by lysosomes or endosomes without the involvement of a secondary phagosome.
      Figure thumbnail gr1
      Figure 1Simplified schematic of autophagy. Autophagy is induced in response to cellular stress, such as nutrient deprivation or infection, and acts to provide nutrients for cellular processes as well as to maintain cellular health by removing harmful components. The process of autophagy progresses in five distinct phases (bold words), each of which refers to the stages of autophagosome maturation. Analysis of LC3 and p62 is a common approach to the assessment of autophagy, whereas autophagy can be inhibited by targeting autophagosome formation or lysosome fusion. CQ, chloroquine; PI3K, phosphatidylinositol-3-kinase; PI3KC3, class III PI3K; ULK1, unc-51-like kinase 1.
      In eukaryotes, autophagy regulatory proteins control homeostasis by interacting with a multitude of other signaling pathways, which allows adaptation to stress, regulation of differentiation, or activation of apoptosis. In the skin, autophagy is required for a range of normal physiological activities such as barrier formation, pigmentation, and antigen presentation (
      • Sil P.
      • Wong S.W.
      • Martinez J.
      More than skin deep: autophagy is vital for skin barrier function.
      ), whereas deregulation of autophagy is implicated in a variety of skin pathologies including infection, cancer, and aging. As such, a number of research techniques have been developed to study autophagy in cell culture systems, animal models, and patient samples, and these are increasingly being applied to the skin and skin disease. These models primarily focus on detecting and quantifying several important autophagy regulatory proteins, which serve as biomarkers of autophagy.

      Summary Points

      • Autophagy is a dynamic process essential for skin homeostasis.
      • Autophagy can be measured at a single time point by observation of autophagy proteins or autophagy structures using techniques such as western blotting, immunofluorescence, immunohistochemistry, or electron microscopy.
      • Multiple methods should be used to enable accurate interpretation of autophagy in vitro
      • Autophagy can be measured dynamically by the introduction of fluorescent-tagged markers into cultured cells in vitro.
      • Autophagy can be modulated in cultured cells or model systems using small molecule inhibitors of autophagy proteins or inhibitors of lysosomal function.

      Limitations

      • Accumulation of LC3-II may reflect the activation of autophagy or decreased LC3 turnover.
      • Changes in SQSTM1/p62 protein levels can be independent of autophagic activity.
      • It is not currently feasible to monitor autophagic flux in clinical samples.

      Biomarkers of Autophagy

      The initiation of macroautophagy (referred to as autophagy in the remainder of this paper) is mediated through the unc-51-like kinase 1 (ULK1) complex in response to several signals such as nutrient deprivation and stress (resulting in the accumulation of damaged proteins). The ULK1 complex phosphorylates the components of the class III phosphatidylinositol-3-kinase (PI3K) complex, which triggers the nucleation of a double-membrane structure to create a phagophore (Figure 1). Several proteins, including Beclin-1 and VPS34, are contained within the class III PI3K complex and have become important biomarkers for identifying, measuring, and modulating autophagy in an experimental setting. The early stages of autophagy initiation are regulated at multiple points by the AMBRA1 protein, which plays a multifunctional role in determining when and where autophagosome formation occurs. AMBRA1 is therefore a critical control protein for the regulation of autophagic activity and is also emerging as a useful biomarker of autophagy. The class III PI3K complex stimulates the production of phosphatidylinositol-3-phosphate within the phagophore membrane, which recruits WIPI proteins and the ATG12 complex (composed of ATG12, ATG5, and ATG16L1). The WIPIs and the ATG12 complex promote phagophore elongation through lipid conjugation of ATG8 proteins, such as LC3, which become anchored to the developing autophagosome membranes. During selective autophagy, cellular components (e.g., damaged proteins or mitochondria) are labeled with an eat-me signal (often ubiquitin), which interacts with selective autophagy receptors such as SQSTM1/p62 (referred to as p62 in the remaining part of this paper) to target cargo for sequestration into the autophagosome by binding with LC3. When the autophagosome subsequently fuses with the lysosome, p62 is degraded along with its cargo, making it an attractive candidate to be a qualitative and potentially quantitative biomarker of autophagy.

      Measuring Autophagic Activity

      Research techniques for measuring autophagy or correlating autophagy with disease status often rely on capturing a snapshot of autophagy regulatory protein expression. However, because most autophagy regulatory proteins are not specific for autophagy, multiple biomarkers need to be considered together and preferably analyzed over a time course to accurately measure autophagic activity. In this section, we describe some of the techniques used for measuring autophagy in the skin and explain the advantages and limitations of each approach.
      Given the pivotal role of LC3 in the autophagic process, this central autophagy regulatory protein is often used as a biomarker of autophagy, with studies showing that the endogenous expression of the LC3B isoform correlates with the progression of melanoma skin cancer as well as poor patient outcome (
      • Lazova R.
      • Camp R.L.
      • Klump V.
      • Siddiqui S.F.
      • Amaravadi R.K.
      • Pawelek J.M.
      • et al.
      Punctate LC3B expression is a common feature of solid tumors and associated with proliferation, metastasis, and poor outcome.
      ). Immunofluorescent staining of 446 melanoma tumors for LC3B demonstrated that LC3B levels were significantly higher in nodal, visceral, and cutaneous metastases. The same study also showed that LC3B expression strongly correlated with Ki-67 staining. Similarly, a variable LC3A expression pattern was observed in cutaneous squamous cell carcinoma, with increased numbers of densely stained LC3-positive structures correlating with tumor thickness (
      • Sivridis E.
      • Giatromanolaki A.
      • Karpathiou G.
      • Karpouzis A.
      • Kouskoukis C.
      • Koukourakis M.I.
      LC3A-positive “stone-like” structures in cutaneous squamous cell carcinomas.
      ). These data collectively indicate that LC3 expression increases with tumor progression and is associated with increased proliferation. In this study, LC3 is a biomarker of disease severity, but additional information is required to accurately determine the status of autophagic activity. To interpret such data, it is important to understand the mechanisms associated with LC3 expression and post-translational processing. Nascent pro-LC3 is proteolytically processed within the cytosol to LC3-I and subsequently conjugated with phosphatidylethanolamine to form LC3-II (Figure 1). This lipid subunit allows LC3-II to be incorporated into autophagosomal membranes where it is later degraded or deconjugated back to LC3-I after fusion with the lysosome. LC3-II is therefore a marker for autophagosomes and can be measured by western blotting for LC3-II or immunofluorescence to detect LC3-positive puncta. An important caveat is that changes in LC3 levels and puncta formation may arise as a consequence of reduced autophagosome degradation or independently to autophagic activity (
      • Klionsky D.J.
      • Abdelmohsen K.
      • Abe A.
      • Abedin M.J.
      • Abeliovich H.
      • Acevedo Arozena A.
      • et al.
      Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition).
      ). Therefore, the amount of LC3-II at a single time point does not reflect autophagic flux (the complete process of autophagy); flux can be measured by following LC3 turnover using inhibitors of lysosome-mediated degradation of LC3-II, such as chloroquine, which prevents autophagosome‒lysosome fusion and therefore inhibits the degradation of LC3-II. Increased accumulation of LC3-II in cells treated with an autophagy inducer in the presence of a lysosomal inhibitor, compared with either treatment alone, can indicate autophagic flux. Care must be taken when measuring endogenous LC3 because antibodies may have a different affinity for LC3-I and LC3-II or different specificities to LC3 isoforms, which can create artifacts in analysis (
      • Klionsky D.J.
      • Abdelmohsen K.
      • Abe A.
      • Abedin M.J.
      • Abeliovich H.
      • Acevedo Arozena A.
      • et al.
      Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition).
      ). Alternatively, tandem fluorescent‒labeled LC3 reporter probes (where both a monomeric RFP [mRFP] or mCherry and GFP are fused with LC3) are available for monitoring LC3 turnover in various experimental models, including live cells or in vivo, to provide a more accurate measure of autophagic activity (
      • Kimura S.
      • Noda T.
      • Yoshimori T.
      Dissection of the autophagosome maturation process by a novel reporter protein, tandem fluorescent-tagged LC3.
      ). This assay is based on the sensitivity of the GFP signal to the acidic conditions of the autolysosome, whereas the mRFP and/or mCherry signal is stable. In fluorescence microscopy, autophagosomes appear as a yellow signal (merged green and red fluorescence), whereas autolysosomes appear red (Figure 2). Although these reporters are not applicable to the clinical analysis of autophagy in patient tissue, they are commonly used for the preclinical analysis of autophagic activity.
      Figure thumbnail gr2
      Figure 2Analysis of autophagy using LC3. (a) After introduction into cells, tandem fluorescent‒labeled mRFP and/or mCherry-GFP-LC3 is incorporated into the autophagosome membrane during autophagy. Colocalization of green and red fluorescent signals (resulting in yellow fluorescence) indicates an autophagosome, whereas a red signal alone corresponds to an autolysosome. (b) Normal human epidermal keratinocytes expressing mCherry-EGFP-LC3B were treated with vehicle (DMSO) or rapamycin (10 nM), or (c) CCD-1106 keratinocytes were treated with vehicle (DMSO) or dithranol (1 μM) in the absence or presence of chloroquine (10 μM) for 24 hours, followed by immunostaining for LC3B. Representative fluorescent micrographs are shown. Bar = 10 μm. CQ, chloroquine; mRFP, monomeric RFP.
      An alternative biomarker for quantifying autophagy in clinical as well as preclinical samples is the autophagy receptor p62 because it is degraded alongside sequestered substrate when autophagosomes are broken down. Therefore, reduced p62 protein levels would appear to be indicative of high autophagic activity, whereas conversely, an increase in p62 expression likely indicates autophagy inhibition causing p62-containing protein aggregates to accumulate. Immunohistochemical staining of 121 primary cutaneous melanoma tumors for p62 demonstrated that tumors with low levels of p62 (<20% cells were positive for p62) were associated with significantly reduced disease-free survival (
      • Ellis R.A.
      • Horswell S.
      • Ness T.
      • Lumsdon J.
      • Tooze S.A.
      • Kirkham N.
      • et al.
      Prognostic impact of p62 expression in cutaneous malignant melanoma.
      ), suggesting that aggressive tumors have a high level of autophagic activity. Nevertheless, p62 analysis must be interpreted with caution because p62 changes can be independent of autophagy (
      • Klionsky D.J.
      • Abdelmohsen K.
      • Abe A.
      • Abedin M.J.
      • Abeliovich H.
      • Acevedo Arozena A.
      • et al.
      Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition).
      ). Conversely, in early-stage melanoma, autophagy regulatory proteins such as ATG5 are downregulated, which is associated with decreased autophagy (
      • Liu H.
      • He Z.
      • von Rütte T.
      • Yousefi S.
      • Hunger R.E.
      • Simon H.U.
      Down-regulation of autophagy-related protein 5 (ATG5) contributes to the pathogenesis of early-stage cutaneous melanoma.
      ). This approach of using immunostaining to measure the levels of autophagy regulatory proteins adds to the body of evidence of temporal regulation of autophagic activity during cancer progression (
      • Galluzzi L.
      • Pietrocola F.
      • Bravo-San Pedro J.M.
      • Amaravadi R.K.
      • Baehrecke E.H.
      • Cecconi F.
      • et al.
      Autophagy in malignant transformation and cancer progression.
      ). Collectively, these data suggest that autophagy is lost during melanoma initiation to avoid induction of endogenous tumor-suppressor mechanisms, but as the disease progresses, autophagy is reactivated in advanced stages of melanoma to support the high metabolic demands of cancer cells, often associated with a poor patient outcome (
      • Lazova R.
      • Camp R.L.
      • Klump V.
      • Siddiqui S.F.
      • Amaravadi R.K.
      • Pawelek J.M.
      • et al.
      Punctate LC3B expression is a common feature of solid tumors and associated with proliferation, metastasis, and poor outcome.
      ).
      In addition to the analysis of autophagy regulatory proteins, transmission electron microscopy is also often used in a research setting where sample preparation can be optimized to demonstrate autophagosome formation. The presence and number of double-membrane autophagic vesicles (autophagosomes) within a cell relative to other organelles is an accurate measure of autophagic activity. However, this technique is labor intensive and requires extensive training to operate, analyze, and interpret correctly, which is why its suitability for determining autophagic activity in patient tissue or in high-throughput studies is limited.

      Modulation of Autophagy Regulatory Proteins

      In recent years, several molecular tools have been developed to modulate autophagy regulatory proteins with the aim of understanding the autophagic mechanisms that contribute to normal cutaneous biology and develop more effective therapeutic interventions for disorders caused by defective autophagy. However, research has revealed a dual role for autophagy in the development and progression of skin cancer, creating problems for the development of treatments aimed at modulating autophagy. Autophagy is a critical tumor-suppressor mechanism that signals with multiple other mechanisms to prevent cancer development. Therefore, the influence of the tumor-suppressor function of autophagy is thought to be silenced in the early stages of cancer development. However, because tumor progression is an energy-intensive process, the growth-promoting function of autophagy is a survival advantage and is often reactivated to drive the growth of advanced tumors (
      • Galluzzi L.
      • Pietrocola F.
      • Bravo-San Pedro J.M.
      • Amaravadi R.K.
      • Baehrecke E.H.
      • Cecconi F.
      • et al.
      Autophagy in malignant transformation and cancer progression.
      ). Therapy aimed at modulating autophagy to kill cancer cells faces a double-edged sword because although inhibiting autophagy may slow the growth of advanced cancers by cutting off a vital energy supply, it could also promote the progression of early-stage tumors by removing autophagy-induced tumor suppression. Alternatively, some drugs may activate a cytotoxic form of autophagy, potentially of both early- and late-stage tumors.
      Experimentally, it is possible to block autophagy using RNA interference‒mediated knockdown of autophagy regulatory genes by transfecting cells in culture with a silencing RNA. Using this technique, it was shown that the inhibition of autophagosome formation by knockdown of ATG5 in mouse embryonic fibroblasts prevented the elimination of skin-resident group A Streptococcus bacteria, revealing a critical role for autophagy in our innate immune response to cutaneous infections (
      • Nakagawa I.
      • Amano A.
      • Mizushima N.
      • Yamamoto A.
      • Yamaguchi H.
      • Kamimoto T.
      • et al.
      Autophagy defends cells against invading group A Streptococcus.
      ). Alternatively, autophagy can be blocked by chemical inhibition using drugs such as chloroquine and hydroxychloroquine. In a meta-analysis of seven clinical trials, these drugs have been shown to improve the response of cancer when administered in combination with chemotherapy or radiotherapy compared with therapy without autophagy inhibition (
      • Xu R.
      • Ji Z.
      • Xu C.
      • Zhu J.
      The clinical value of using chloroquine or hydroxychloroquine as autophagy inhibitors in the treatment of cancers: a systematic review and meta-analysis.
      ). Similarly, small molecule inhibitors of the autophagy regulatory proteins can be used in vitro and in vivo to investigate the function of autophagy as well as to model potential clinical utility. For example, the use of 3-methyladenine (a class III PI3K inhibitor) in a mouse model of wound healing demonstrated the potential of autophagy inhibition as a strategy to improve wound healing in patients with diabetes (
      • Guo Y.
      • Lin C.
      • Xu P.
      • Wu S.
      • Fu X.
      • Xia W.
      • et al.
      AGEs induced autophagy impairs cutaneous wound healing via stimulating macrophage polarization to M1 in diabetes.
      ). Furthermore, targeted inhibition of VPS34 results in a block in autophagy evidenced by the accumulation of LC3 and p62, sensitizes metastatic melanoma cells to inhibition of MAPK signaling, and reduces the invasion of melanoma cells in vivo (
      • Verykiou S.
      • Alexander M.
      • Edwards N.
      • Plummer R.
      • Chaudhry B.
      • Lovat P.E.
      • et al.
      Harnessing autophagy to overcome mitogen-activated protein kinase inhibitor-induced resistance in metastatic melanoma.
      ) (Figure 3). Therefore, autophagy inhibitors may have a role in the clinical management of patients with advanced-stage skin cancer. However, an alternative approach may be to activate autophagy-induced cell death. In a mouse xenograft model of human metastatic melanoma, the cannabinoids Δ9-tetrahydrocannabinol and cannabidiol increased immunofluorescent punctate staining of LC3B in treated tumors, which correlated with reduced Ki-67 and increased TUNEL staining, indicating that these drugs induce cytotoxic autophagy within tumor cells (
      • Armstrong J.L.
      • Hill D.S.
      • McKee C.S.
      • Hernandez-Tiedra S.
      • Lorente M.
      • Lopez-Valero I.
      • et al.
      Exploiting cannabinoid-induced cytotoxic autophagy to drive melanoma cell death.
      ). This response also appears to be specific for cancer cells because corresponding nontransformed cell types (e.g., melanocytes in the case of melanoma) did not undergo apoptosis on exposure to the same cannabinoid concentrations in vitro, suggesting that cannabinoids, which are currently undergoing clinical trials in other cancer types, may be a safe and effective approach for the treatment of some patient subgroups. Nevertheless, further research is required to elucidate the mechanisms mediating the antitumor effects of cannabinoids. There is also a potential for the use of cannabinoids or other drugs that activate autophagy such as dithranol (unpublished data and Figure 2) for the treatment of other skin diseases including psoriasis, where autophagy is also defective (
      • Akinduro O.
      • Sully K.
      • Patel A.
      • Robinson D.J.
      • Chikh A.
      • McPhail G.
      • et al.
      Constitutive autophagy and nucleophagy during epidermal differentiation.
      ;
      • Mahil S.K.
      • Twelves S.
      • Farkas K.
      • Setta-Kaffetzi N.
      • Burden A.D.
      • Gach J.E.
      • et al.
      AP1S3 mutations cause skin autoinflammation by disrupting keratinocyte autophagy and up-regulating IL-36 production.
      ).
      Figure thumbnail gr3
      Figure 3Effect of autophagy inhibition on the invasion of metastatic melanoma cells. Fluorescence microscopy images of 5-day-old zebrafish embryos with GFP-expressing endothelial blood vessels (green), which were injected with DiI-labeled A375 metastatic melanoma cells (red) at 2 days after fertilization and treated for 72 hours with either DMSO or 5 μM PIK-III (a small molecule VPS34 inhibitor;
      • Verykiou S.
      • Alexander M.
      • Edwards N.
      • Plummer R.
      • Chaudhry B.
      • Lovat P.E.
      • et al.
      Harnessing autophagy to overcome mitogen-activated protein kinase inhibitor-induced resistance in metastatic melanoma.
      ).
      In summary, autophagy is central to skin homeostasis, and dysfunctional autophagy is now recognized as a key feature of a range of cutaneous pathologies. Multiple techniques must be utilized to accurately evaluate autophagic activity, its role in the pathogenesis of skin disease, and the potential to exploit this process for therapeutic intervention.

      Conflict of Interest

      The authors state no conflict of interest.

      Acknowledgments

      We thank the University of Sunderland (Sunderland, United Kingdom), the Psoriasis Association , the NC3Rs, Forsogsdyrenes Vaern & Alternativfondet, The Newcastle Hospitals Healthcare Charity (Newcastle upon Tyne, United Kingdom), Cancer Research United Kingdom (London, United Kingdom), Melanoma Focus, and the German Research Foundation (Bonn, Germany) for funding relating to this work.

      Author Contributions

      Conceptualization: DH, IC, NR, PL, JA; Writing - Original Draft Preparation: DH, IC, JA; Writing - Review and Editing: JA, PL, NR

      Multiple Choice Questions

      • 1.
        Autophagy disruption is implicated in which of the following skin pathologies?
        • A.
          Infection
        • B.
          Cancer
        • C.
          Aging
        • D.
          All of the above
      • 2.
        Autolysosome formation proceeds through the stages of initiation, nucleation, expansion, closure, fusion.
        • A.
          True
        • B.
          False
      • 3.
        Using immunolabeling of LC3 and fluorescence microscopy, increased levels of LC3-positive puncta could be caused by
        • A.
          An accumulation of autophagosomes.
        • B.
          A reduction in autophagosome degradation.
        • C.
          Mechanisms independent of autophagy.
        • D.
          All of the above.
      • 4.
        What color is the tandem fluorescent‒labeled LC3 reporter protein when it is localized to the autophagosome?
        • A.
          Green
        • B.
          Yellow
        • C.
          Red
        • D.
          Blue
      • 5.
        Which two drugs are able to inhibit autophagy in vivo?
        • A.
          Rapamycin
        • B.
          Δ9-tetrahydrocannabinol
        • C.
          VPS34 inhibitor
        • D.
          Chloroquine

      Detailed Answers

      • 1.
        Autophagy disruption is implicated in which of the following skin pathologies?
      • CORRECT ANSWER: D. All of the above
      • Autophagy is central to skin homeostasis and plays vital roles in pigmentation, differentiation, barrier formation, antigen presentation, pathogen clearance, resolution of inflammation, and wound healing. Dysfunctional autophagy has now been implicated in a number of pathological conditions, including infection, skin cancer, psoriasis, and skin aging.
      • 2.
        Autolysosome formation proceeds through the stages of initiation, nucleation, expansion, closure, fusion.
      • CORRECT ANSWER: A. True
      • Autophagy is a catabolic pathway in which cellular components are delivered to lysosomes for degradation and recycling. Central to this process is the formation of an autophagosome, in which cellular material destined for degradation are transported to lysosomes. Various cellular signals can activate autophagy (initiation), which leads to the formation of a cup-shaped membrane structure (termed the phagophore; nucleation) generated by the action of ATG proteins, which are also required for elongation (expansion) and sealing (closure) of the membrane to form a double-membraned vesicle, the autophagosome. In the final stage, the autophagosome matures and fuses with a lysosome (fusion) forming the autolysosome.
      • 3.
        Using immunolabeling of LC3 and fluorescence microscopy, increased levels of LC3-positive puncta could be caused by
      • CORRECT ANSWER: D. All of the above
      • In cells undergoing autophagy, LC3 is conjugated to a lipid (forming LC3-II) and localized to the autophagosome membrane, creating LC3-positive puncta, which can be detected by immunolabeling and fluorescence microscopy. However, whereas LC3 is a marker for autophagic structures, the accumulation of LC3 puncta could also be due to increased synthesis of LC3, a reduction in autophagosome degradation, formation of LC3 aggregates independently of autophagy, or association of LC3 with nonautophagic structures. Analysis of LC3 should therefore not be used alone to measure autophagy and additional assays of autophagic flux (LC3-II turnover in the presence and absence of inhibitors of lysosomal degradation or using tandem fluorescent-labeled LC3) used to measure the dynamic activity of autophagy.
      • 4.
        What color is the tandem fluorescent‒labeled LC3 reporter protein when it is localized to the autophagosome?
      • CORRECT ANSWER: B. Yellow
      • LC3 is incorporated into the autophagosome membrane during autophagy. Colocalization of green and red fluorescent signals (resulting in yellow) indicate an autophagosome, whereas a red signal alone corresponds to an autolysosome.
      • 5.
        Which two drugs are able to inhibit autophagy in vivo?
      • CORRECT ANSWER: C. VPS34 inhibitor D. Chloroquine
      • Rapamycin and Δ9-tetrahydrocannabinol are able to activate autophagy, whereas both the VPS34 inhibitor PIK-III and chloroquine are autophagy inhibitors. PIK-III inhibits VSP34 with 100-fold greater selectivity than kinases and reduces autophagic activity by inhibition of de novo lipidation of LC3. Chloroquine increases lysosomal pH and blocks fusion between the autophagosome and the lysosome, thereby preventing lysosomal degradation and autophagic activity.

      Supplementary Material

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