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There is increasing evidence documenting the critical role played by autophagic and autophagy-associated processes in maintaining cell homeostasis and overall systemic health. Autophagy is considered a degradative as well as a recycling pathway that relies on encapsulated intracellular components trafficking to and fusing with degradative compartments, including lysosomes. In this chapter, we describe the use of DQ™-BSA to study autophagosome–lysosome fusion as well as a means by which to analyze hybrid autophagic pathways. Such noncanonical pathways include LC3-associated phagocytosis, better known as LAP. Both autophagosomes and LAPosomes (LC3-associated phagosomes) deliver cargo for degradation. The use of fluorescent DQ™-BSA in conjugation with autophagic makers and biomarkers of hybrid autophagy offers a reliable technique to monitor the formation of autolysosomes and LAPo-lysosomes in both fixed- and live-cell studies. This technique relies on cleavage of the self-quenched DQ™ Green- or DQ™ Red BSA protease substrates in an acidic compartment to generate a highly fluorescent product.
The cell turns over its own constituents in a regulated manner to maintain homeostasis, in a process called autophagy, derived from the Greek word for “self-eating.” Numerous components of the autophagic pathway are multifunctional, forming a nexus of cross talk with other cellular processes to help define cell type-specific functions of autophagy and processes associated with autophagy-related proteins. Such processes include metabolism, hybrid phagocytosis, membrane transport, and host defense strategies.
Degradation of both intracellular and extracellular material is a crucial function of phagocytic cells. Two seemingly independent engulfment pathways, phagocytosis and autophagy, deliver material to lysosomes for degradation. Macroautophagy, hereafter referred to as autophagy, is the major catabolic pathway required for the lysosome/vacuolar degradation of cytoplasmic proteins and organelles. Through canonical autophagy, intracellular substrates are enwrapped as cargo by double membrane structures known as autophagosomes; thus allowing for bulk turnover of cytoplasmic components, enabling among other functions, the survival of nutrient-deprived cells. Upon nutrient deprivation, a serine-threonine kinase, UNC-51-like kinase (ULK), is released from its mammalian target of rapamycin (mTOR)-mediated inhibition (Jung et al., 2009; Kim, Kundu, Viollet, & Guan, 2011); in concert with the class III phosphatidylinositol-3 kinase, Vps-34 complexed with Beclin1, it recruits and activates components of the Atg5/12/16L conjugation system (Funderburk, Wang, & Yue, 2010). The Atg5/12/16L multimeric complex regulates LC3 lipidation by phosphatidylethanolamine to form lipidated LC3, called LC3II. LC3II is necessary for autophagosome elongation and closure in cargo engulfment. LC3-containing autophagosomes subsequently fuse with lysosomes in an apparent LC3II-dependent manner to facilitate degradation of intracellular components (Esclatine, Chaumorcel, & Codogno, 2009; Jahreiss, Menzies, & Rubinsztein, 2008). Details of autophagy-associated proteins and their specific functions are reviewed in Klionsky et al. (2016).
Not all LC3-containing intracellular vesicles are autophagosomes; phagosomes can be targeted by autophagy proteins in an autophagosome-independent manner. In some epithelial cells and macrophages, phagocytosis activates the Vps34/beclin1 and Atg5/12/16L conjugation systems resulting in lipidation of LC3 directly onto the single membrane (nascent) phagosome (Florey, Kim, Sandoval, Haynes, & Overholtzer, 2011; Florey & Overholtzer, 2012). In this hybrid pathway, commonly known as LC3-associated phagocytosis (LAP), the LC3II-decorated phagosome fuses with lysosomes for degradation. Autophagosome-independent, LC3-associated degradative events exhibit common themes that define the process of LAP: LC3 recruitment to phagosomes occurs under nutrient replete conditions in which mTOR is active with canonical autophagy inhibited. LAP is however dependent on Vps34/beclin1 and Atg5/12/16L. It is becoming increasingly evident that LAP processes are a means by which phagocytes monitor their contents to ensure complete degradation of ingested materials. LAP requiring processes include phagocytosis of dead cells (Martinez et al., 2011), the degradation of photoreceptors, and recycling of visual pigments (Frost et al., 2015; Frost, Mitchell, & Boesze-Battaglia, 2014; Kim et al., 2013) and pathogen degradation (Sanjuan et al., 2007). Most recently, LAP has been shown to inhibit the autoimmune response (Martinez et al., 2016).
Independent of whether LC3 decorates the inner and outer bilayers of double membrane autophagosomes or just the outside of ingested phagosomes, the ultimate fate of the contents of these structures is degradation upon fusion with lysosomes. Historically, the function of degradative compartments was often measured as proteolytic activity; highly substituted fluorescein conjugated derivatives of proteins served as biomarkers of a cell’s degradative efficiency and capacity. A limitation of such fluorescein derivatives is their pH sensitivity over physiological lysosomal pH ranges; they provide limited detection below pH 8.0. Newer fluorescent compounds have emerged including the BODIPY© family of probes which are insensitive to pH changes from pH 3 to 11. Here we provide a method by which the bovine serum albumin derivative DQ™-BSA is utilized to detect the formation of the autolysosomes and the quantitation of degradative cargo using confocal imaging.
The DQ™BSA conjugate is a derivative of BSA that is labeled to such a high degree with either the green-fluorescent BODIPY® FL or the red-fluorescent BODIPY® TR-X dye that the fluorescence is self-quenched. Spectral properties of DQ™Green and DQ™Red BSA allow for extensive utility in a variety of applications. They both provide low background fluorescence and provide high signal-to-noise ratios upon digestion. The cleavage of DQ™ Green BSA results in the release of fragments which have λex = 505 nm and λem = 515 nm. In the case of DQ™ Red BSA the proteolytic fragments have λex = 590 nm and λem = 620 nm. As the DQ™-BSA enters an acidic environment proteases cleave the previously quenched polypeptide to generate fluorescent peptide fragments as illustrated schematically in Fig. 1.
DQ-BSA probes are available as a lyophilized powder that if protected from light are stable for up to 6 months at −20°C. After reconstitution, the solution is stable for 3–4 weeks at 4°C (protected from light; wrap vial in foil). We routinely reconstitute with sterile PBS.
The methods below describe the use of DQ™ Green BSA in the visualization and semiquantitation of autolysosome formation as well as the delivery of LC3-associated cargo to lysosomes (Frost et al., 2015). Ha et al. (2010), utilized DQ™ Red BSA to measure the role of autophagic flux in Anthrax Toxin lethal factor delivery using confocal imaging and FACS. Both approaches effectively measure the convergence between an autophagic or autophagocytic compartment and a functional lysosome; in the case of ingested phosphatidylserine positive photoreceptor outer segments (POS), the autophagy-associated process is LAP (LC3-phagolysosomes) while for anthrax toxin it is autolysosome formation. Here we describe DQ™-BSA’s use for autolysosomes as well as a modification in which we measure LAP.
The individual steps for monitoring autolysosome and LC3-phagolysosome formation are described later. This protocol can be simplified for nonpolarized cells by ignoring steps in Section 3.
Monitoring of autolysosome formation requires that the DQ™-BSA traffic to lysosomes where acidic proteases generate a highly fluorescent product. Autolysosomes are defined herein as intracellular structures in which there is colocalization of cleaved DQ™-BSA fragments (a marker of lysosomes) with fluorescently tagged-LC3 or endogenous LC3 (a marker of autophagic structures).
Rapamycin acts to remove the mTOR-mediated inhibition of the Ulk1 complex thereby resulting in an increase in autophagy. Cells are incubated with 100 nM rapamycin (Enzo Life Sciences, BML-A275-0005) for 4 or 24 h and subsequently incubated with DQ™-BSA, follow steps 1–4 in Section 5.3.
To remove media wash cells three times in PBS. To serum starve change to 0.25% FBS. To control cells, add complete culture media DMEM/F12 with 1% fetal bovine serum (Sigma, 12003C) and 5% Penicillin–Streptomycin (Sigma, P4333). Incubate cells at 37°C for 4 h.
The generation of highly fluorescent proteolytic products in lysosomes allows us to monitor the delivery of autophagy-dependent cargo to these organelles. In the example below, we provide a semiquantitative method by which to analyze a hybrid phagocytosis–autophagy-dependent process; LC3-associated phagocytosis known as LAP. Here LC3-phagolysosomes are defined as intracellular structures in which there is a triple colocalization between cleaved DQ™-BSA fragments, fluorescently tagged-LC3 or endogenous LC3 and fluorescently labeled phagosomes (Frost et al., 2015).
We describe basic protocols to assay the last step of autophagy, one shared with LAP, the fusion of an LC3 positive compartment with a degradative lysosome. Incorporation of the fluorogenic DQ™Green or Red BSA probe is an easy and fast method by which to monitor starvation or pharmacologically induced autophagy in fixed or live cells. Although the colocalization of phagocytosed particles with LC3 assesses the formation of a LAPosome, it does not address whether that structure then appropriately traffics to a lysosome for degradation. Therefore, detecting the colocalization of LC3-associated cargo with DQ™-BSA is the one of the most reliable methods for monitoring fusion between a LAPosome and an active proteolytic compartment, either late-endosome or lysosome. This method is in many ways superior to colocalization studies that rely only on the presence of lysosomal enzyme. Typically, such immunofluorescence studies do not assess the compartment’s function, as the presence of a protease, for example, cathepsin D does not necessarily confirm that the compartment is functional. The use of DQ™-BSA circumvents this problem since fluorescence is only observed in a proteolytically active compartment.
This work was supported in whole or in part by the National Institutes of Health Grants EY10420, EY026525, and DE022465 (to K.B.B.) and P30EY00331 (Penn-Vision Core). The authors would like to acknowledge the use of the PDM live-cell imaging core.