http://www.springerprotocols.com/cdp/access/showWebFeedListing This feed provides the latest 25 protocols in the given category. http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-157-4_1 Autophagy and phagocytosis are evolutionarily ancient processes functioning in capture and digestion of material found in the cellular interior and exterior, respectively. In their most primordial form, both processes are involved in cellular metabolism and feeding, supplying cells with externally obtained particulate nutrients or using portions of cell’s own cytoplasm to generate essential nutrients and energy at times of starvation. Although autophagy and phagocytosis are commonly treated as completely separate biological phenomena, they are topologically similar and can be, at least morphologically, viewed as different manifestations of a spectrum of related processes. Autophagy is the process of sequestering portions of cellular interior (cytosol and intracellular organelles) into a membranous organelle (autophagosome), whereas phagocystosis is its topological equivalent engaged in sequestering cellular exterior. Both autophagosomes and phagosomes mature into acidified, degradative organelles, termed autolysosomes and phagolysosomes, respectively. The basic role of autophagy as a nutritional process, and that of phagocytosis where applicable, has survived in present-day organisms ranging from yeast to man. It has in addition evolved into a variety of specialized processes in metazoans, with a major role in cellular/cytoplasmic homeostasis. In humans, autophagy has been implicated in many health and disease states, including cancer, neurodegeneration, aging and immunity, while phagocytosis plays a role in immunity and tissue homeostasis. Autophagy and phagocytosis cooperate in the latter two processes. In this chapter, we briefly review the regulatory and execution stages of both autophagy and phagocytosis. 2008-05-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-157-4_10 The methods described are designed to enable the assignment of an intracellular localization of secretory proteins, either soluble or membrane associated, to later secretory compartments, such as the trans-Golgi network (TGN) or endosome. These two subcellular compartments are closely linked through extensive protein trafficking, in both an anterograde and a retrograde direction. These compartments are likely to be important in the formation of autophagosomes during the process of autophagy. Our current knowledge of how autophagosomes form is scarce, and further investigation into the role that other subcellular compartments have in this process is needed. 2008-05-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-157-4_11 Sphingolipids are constituents of biological membranes. Ceramide and sphingosine 1-phosphate (S1P) also act as second messengers and are part of a rheostat system, in which ceramide promotes cell death and growth arrest, and S1P induces proliferation and maintains cell survival. As macroautophagy is a lysosomal catabolic mechanism involved in determining the duration of the lifetime of cells, we raised the question of its regulation by sphingolipid messengers. Using chemical and genetic methods, we have shown by GFP-LC3 staining and analysis of the degradation of long-lived proteins that both ceramide and S1P stimulate autophagy. 2008-05-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-157-4_12 Macroautophagy (herein referred to as autophagy) contributes to the control of life and death throughout the animal and plant kingdoms. Bilateral links have been found between apoptosis and autophagy where inducers of apoptosis also induce autophagy and vice versa. In some cases, autophagy delays the onset of apoptosis and thus prolongs life although it may also promote apoptosis and other forms of cell death. It is thus of great biological and medical interest to understand the molecular connections between these two pathways, and try to utilize—or block—them selectively to aid induction of cell death (e.g., cancer cells) or inhibit death (e.g., in degenerative disorders). This chapter describes methods for studying apoptotic induction of autophagy and its effects on cell function. We also discuss potential pitfalls. Although cell lines are used as model systems, the substances and methods described here can be applied to primary cells and tissues. 2008-05-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-157-4_13 The accumulation of mutant aggregate-prone proteins is a feature of several human disorders, collectively referred to as protein conformation disorders or proteinopathies. We have shown that autophagy, a cytosolic, non-specific bulk degradation system, is an important clearance route for many cytosolic toxic, aggregate-prone proteins, like mutant huntingtin and mutant ${\bf \alpha}$ -synucleins. Induction of autophagy enhances the clearance of both soluble and aggregated forms of the mutant protein, and protects against toxicity caused by these mutations in cell, fly, and mouse models. Inhibition of autophagy has opposite effects. Thus, the autophagic pathway may represent a possible therapeutic target in the treatment of certain protein conformation disorders. 2008-05-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-157-4_14 Intracellular antigens can be presented on major histocompatibility complex (MHC) class II molecules after degradation via macroautophagy. To enhance MHC class II presentation of potential vaccine antigens, we have developed a method to target antigens for autophagic degradation via fusion to the Atg8/LC3 protein: Atg8/LC3 is specifically incorporated into autophagosomes via coupling to phosphatidylethanolamine, and subsequently degraded in MHC class II loading compartments (MIICs). Antigens fused to the N-terminus of Atg8/LC3 follow the same pathway and get preferentially presented on MHC class II molecules. The localization of Atg8/LC3 fusion antigens in MIICs can be visualized by confocal microscopy, and MHC class II presentation can be quantified in a presentation assay with antigen-specific CD4+ T-cell clones. These assays are good measures of autophagosome formation and lysosomal degradation of macroautophagy cargo and therefore are useful for studying regulation of the autophagic pathway under various experimental conditions and physiological perturbations. 2008-05-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-157-4_15 Chaperone-mediated autophagy (CMA) is the only type of autophagy in mammalian cells able to selectively degrade cytosolic proteins in lysosomes. CMA is maximally activated in response to stressors such as prolonged starvation, exposure to toxic compounds, or oxidative stress. We have found that CMA activity decreases in aging and in some age-related disorders such as Parkinson’s disease. Impaired CMA under these conditions may be responsible for the accumulation of damaged proteins inside cells and for their higher vulnerability to stressors. In contrast to other forms of autophagy, where substrates are engulfed or sequestered along with other cytosolic components, CMA substrates are translocated one-by-one across the lysosomal membrane. Changes in the levels/activity of the lysosomal components required for substrate translocation can be used to stimulate CMA activity. However, the most unequivocal method to measure CMA is by directly tracking the translocation of substrate proteins into isolated lysosomes. 2008-05-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-157-4_16 Microautophagy involves direct invagination and fission of the vacuolar/lysosomal membrane under nutrient limitation. In Saccharomyces cerevisiae microautophagic uptake of soluble cytosolic proteins occurs via an autophagic tube, a highly specialized vacuolar membrane invagination. At the tip of an autophagic tube vesicles (autophagic bodies) pinch off into thevacuolar lumen for degradation. Formation of autophagic tubes is topologically equivalent to other budding processes directed away from the cytosolic environment, e.g., the invagination of multivesicular endosomes, retroviral budding, piecemeal microautophagy of the nucleus and micropexophagy. This clearly distinguishes microautophagy from other membrane fission events following budding toward the cytosol. Such processes are implicated in transport between organelles like the plasma membrane, the endoplasmic reticulum (ER), and the Golgi. Over many years microautophagy only could be characterized microscopically. Recent studies provided the possibility to study the process in vitro and have identified the first molecules that are involved in microautophagy 2008-05-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-157-4_17 Electron microscopy (EM) is the only technique that can combine sensitive protein-detection methods with detailed information on the substructure of cellular compartments. The purpose of this chapter is to describe some of the methods at the EM level that can be used to analyze the spatial organization of cell organelles with respect to phagosomes or vacuoles in which pathogens are sequestered, characterize the compartment in which pathogens are harbored, ie immature phagosomes, phagolysosomes, autophagosomes, and ER-derived vacuoles, to cite a few, and decipher the molecular mechanisms involved in survival of pathogens within infected host cells. 2008-05-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-157-4_18 Phosphoinositide signaling is essential for successful phagocytosis. Phosphoinositides regulate processes such as actin assembly and the recruitment of molecular motors required for ingestion, as well as fusion events required for the maturation of the phagosome. Phosphoinositides not only serve as substrates for the generation of second messengers, but also function to anchor to the membrane cytosolic proteins that contain phosphoinositide-binding motifs. Conventional methods for the detection of phosphoinositides involve their extraction from the cells and separation by chromatographic procedures. These approaches are laborious and expensive and fail to provide spatio-temporal information, which is critical when analyzing localized and transient phenomena like phagocytosis. In this chapter we describe a method to monitor phosphoinositides dynamically by transfection of fluorescently tagged probes (biosensors) into cultured macrophages. These biosensors are based on the fusion of phosphoinositide-binding protein domains with fluorescent proteins. Some specifications for live cell imaging of such phosphoinositide-specific probes are also provided. 2008-05-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-157-4_19 Phagolysosome biogenesis plays a pivotal role in elimination of foreign particles and pathogens by leukocytes. This process is achieved by multiple cycles of membrane fusion between the phagosome and the endosomal system. In vitro reconstitution of phagosome fusion with endosomes is instrumental in studying this intricate process. Such an assay is also invaluable for the identification of effectors produced by intracellular pathogens that may hamper the pathway. In this chapter we describe a highly sensitive and relatively rapid method to measure fusion between phagosomes and early, as well as late, endosomal compartments. The assay is based on the formation of a stable biotin–streptavidin complex upon fusion between biotinylated–peroxidase loaded endosomes and magnetic streptavidin conjugated bead-containing phagosomes. Fusion is quantified using a fluorescence-based detection method that measures the peroxidase activity associated with the beads. 2008-05-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-157-4_2 This chapter describes the electron microscopic fine structure of early and late autophagic vacuoles in mammalian cells. Detailed instructions are given for the preparation of cells for conventional electron microscopy and for the identification of autophagic vacuoles by morphology. Electron microscopy remains one of the most accurate methods for quantitation of autophagic vacuole accumulation. Therefore, quantitation of autophagic vacuoles by electron microscopy and point counting is also described. Finally, a short description is given for preparation of ultra thin cryosections for immunogold labeling of autophagic vacuoles. 2008-05-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-157-4_20 The ultimate goal of phagosomal maturation is the delivery of internalized, particulate cargo to acidic, hydrolytically competent compartments capable of mediating its degradation. Here we outline in detail three fluorometric techniques that allow the study of phagosomal maturation in macrophages by quantifying functionally important features of the lumenal environment of the developing phagosome in real time. The first assay utilizes a particle-restricted, pH-sensitive fluorochrome to measure the acidification of the phagosome. The second reports on the development of the proteolytic capacity of the phagosome byfollowing the hydrolysis of a fluorogenic, generic proteinase substrate. The third quantifies the accumulation of lysosomal constituents within the phagosome by measuring the fluorescence resonance energy transfer (FRET) efficiency between a particle-restricted, donor fluor and a fluid phase acceptor fluor that had been chased previously into lysosomes. The assays aredescribed as population-based methodologies utilizing a spectrofluorometer but, alternatively, can be adapted readily to confocal-based technologies for single phagosomal measurements. 2008-05-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-157-4_21 The amoeba Dictyostelium discoideum is an established model to study phagocytosis. The sequence of events leading to the internalization and degradation of a particle is conserved in D. discoideum compared to metazoan cells. As its small haploid genome has been sequenced, it is now amenable to genome-wide analysis including organelle proteomics. Therefore, we adapted to Dictyostelium the classical protocol to purify phagosomes formed by ingestion of latex beads particles. The pulse-chase protocol detailed here gives easy access to pure, intact, and synchronized phagosomes from representative stages of the entire process of phagosome maturation. Recently, this protocol was used to generate individual temporal profiles of proteins and lipids during phagosome maturation generating a proteomic fingerprint of six maturation stages (1). In addition, immunolabeling of phagosomes on a coverslip was developed to visualize and quantitate antigen distribution at the level of individual phagosomes. 2008-05-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-157-4_22 2008-05-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-157-4_23 Phagocytic antigen-presenting cells (APCs) are involved in innate and adaptive immune responses to bacteria. Adaptive responses to bacteria involve processing of bacterial antigens for presentation by class II major histocompatibility complex (MHC II) molecules and class I MHC (MHC I) molecules to stimulate CD4+ and CD8+ T cells, respectively. To examine the role of phagosomes in processing of antigens for presentation by MHC II molecules to CD4+ T cells, phagosomes have been biochemically and functionally analyzed by a variety of techniques that include flow analysis (flow organellometry), SDS-PAGE/Western blotting, and an antigen-presenting organelle assay. Using these techniques, we have demonstrated that phagosomes containing latex beads or Mycobacterium tuberculosis (MTB) contain components of the MHC II processing pathway and support the formation of peptide–MHC II complexes. 2008-05-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-157-4_24 A unique feature of the intracellular life cycle of Legionella pneumophila is the interaction between the vacuole in which L. pneumophila resides and the endoplasmic reticulum of the host cell. This interaction is crucial for L. pneumophila to establish a niche in which the bacteria can replicate intracellularly. Microscopic analysis of endoplasmic reticulum (ER) markers during infection yields information regarding the nature of the recruited vesicles as well as the kinetics of their recruitment. The recruitment of YFP-KDEL, GFP-p58, calnexin, and myc-Sec22b to the L. pneumophila – containing vacuole can be assessed by fluorescence microscopy. Methods for detection of these various ER markers during infection of mammalian cells by L. pneumophila are described. 2008-05-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-157-4_25 Coxiella burnetii is a bacterial obligate intracellular pathogen that replicates within a spacious parasitophorous vacuole (PV) with lysosomal characteristics. The pathogen actively participates in the biogenesis of its PV by synthesizing proteins that mediate vesicular interactions. Both C. burnetii and host factors that regulate PV formation are likely localized to the PV membrane, and their identification would be aided by an efficient method for isolating the C. burnetii vacuole. To this end, we developed a method to separate intact PV from host cell material that relies on fusion of the vacuole with latex bead-containing phagosomes (LBP). Sequestration of latex beads by the C. burnetii PV increases the vacuole’s buoyant density and facilitates its fractionation on a sucrose step gradient. Transmission electron microscopy confirms the isolation of intact PV-containing latex beads from infected MH-S murine alveolar macrophage-like cells. Immunoblotting demonstrates that C. burnetii PV lysates are dramatically enriched for the late endosome/lysosome markers LAMP-1 and LAMP-2 when compared to total host cell lysates. Conversely, PV preparations are devoid of p62 and GM130, markers of the nucleus and Golgi apparatus, respectively, indicating effective separation of the vacuole from these host cell compartments. Two-dimensional gel electrophoresis and immunoblotting reveal distinct protein differences between C. burnetii PV and LBP. Identification of proteins unique to the PV membrane will yield important insight into C. burnetii–host interactions. 2008-05-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-157-4_26 Interferon-gamma IFN-γ)–induced remodeling of the bacterial phagosome for pathogen clearance elicits the aid of a new family of GTPases termed the p47 IRGs. Members of this group reside primarily on ER-Golgi membranes before translocating to the nascent phagosome within minutes of bacterial uptake. Recruitment of p47 IRGs coincides with the acquisition of phagosome maturation and autophagy markers as well as enhanced acidification of this organelle. Here we describe a simple spectrofluorometric assay to measure luminal acidification of the bacterial phagosome within intact cells such as macrophages. This method can be applied to study the phagosomal pH pH_pg) of activated cells infected with a variety of infectious microorganisms and the roles played by members of the p47 IRG family in auto)phagolysosome biogenesis. 2008-05-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-157-4_27 Phagocytosis is a process by which invading organisms are taken up by macrophages and targeted to the lysosomes, where they are degraded. However, many pathogens modulate this central process of macrophage-mediated killing by inhibiting their transport to the lysosomes through a variety of pathogen-derived mechanisms. Given the importance of Rab proteins in the regulation of intracellular transport pathways, we investigated the role of different host endocytic Rabs on the maturation of Salmonella-containing phagosomes in macrophages. Initially, we have developed a ligand mixing assay to measure the transport of the Salmonella-containing phagosomes to lysosomes. Using this assay we have shown that Salmonella decline their transport to the lysosomes. In order to determine whether inhibition of Salmonella transport to lysosomes is due to their sustained fusion with early endosomes, we have developed an in vitro fusion assay between Salmonella-containing phagosomes and early endosomes. Here, we have discussed how these methodologies are helpful to determine the mechanism of evasion of Salmonella transport to the lysosomes. 2008-05-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-157-4_28 Tuberculosis is currently the most devastating human bacterial disease, causing millions of deaths annually and infecting an overwhelming percentage of the global population. Its success as a scourge lies in the ability of Mycobacterium tuberculosis to prevent normal phagolysosome biogenesis, essential to the destruction of invading microorganisms, inside macrophages. Recent work has identified host GTPases involved in the block of normal phagolysosome biogenesis during mycobacterial infection and has provided a set of methods, in particular efficient macrophage transfection, which will prove essential in examining the role of host effectors in this process. 2008-05-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-157-4_3 Autophagic (or type 2) cell death is characterized by the massive accumulation of autophagic vacuoles (autophagosomes) in the cytoplasm of cells that lack signs of apoptosis (type 1 cell death). Here we detail and critically assess a series of methods to promote and inhibit autophagy via pharmacological and genetic manipulations. We also review the techniques currently available to detect autophagy, including transmission electron microscopy, half-life assessments of long-lived proteins, detection of LC3 maturation/aggregation, fluorescence microscopy, and colocalization of mitochondrion- or endoplasmic reticulum–specific markers with lysosomal proteins. Massive autophagic vacuolization may cause cellular stress and represent a frustrated attempt of adaptation. In this case, cell death occurs with (or in spite of) autophagy. When cell death occurs through autophagy, on the contrary, the inhibition of the autophagic process should prevent cellular demise. Accordingly, we describe a strategy for discriminating cell death with autophagy from cell death through autophagy. 2008-05-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-157-4_4 Microtubule-associated protein 1A/1B-light chain 3 (LC3) is a soluble protein with a molecular mass of ∼17 kDa that is distributed ubiquitously in mammalian tissues and cultured cells. During autophagy, autophagosomes engulf cytoplasmic components, including cytosolic proteins and organelles. Concomitantly, a cytosolic form of LC3 (LC3-I) is conjugated to phosphatidylethanolamine to form LC3-phosphatidylethanolamine conjugate (LC3-II), which is recruited to autophagosomal membranes. Autophagosomes fuse with lysosomes to form autolysosomes, and intra-autophagosomal components are degraded by lysosomal hydrolases. At the same time, LC3-II in autolysosomal lumen is degraded. Thus, lysosomal turnover of the autophagosomal marker LC3-II reflects starvation-induced autophagic activity, and detecting LC3 by immunoblotting or immunofluorescence has become a reliable method for monitoring autophagy and autophagy-related processes, including autophagic cell death. Here we describe basic protocols to assay for endogenous LC3-II by immunoblotting, immunoprecipitation, and immunofluorescence. 2008-05-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-157-4_5 Amino acids are not only substrates for various metabolic pathways, but can also serve as signaling molecules controlling signal transduction pathways. One of these signaling pathways is mTOR-dependent and is activated by amino acids (leucine in particular) in synergy with insulin. Activation of this pathway inhibits autophagy. Because activation of mTOR-mediated signaling also stimulates protein synthesis, it appears that protein synthesis and autophagic protein degradation are reciprocally controlled by the same signaling pathway. Recent developments indicate that amino acid–stimulated mTOR-dependent signaling is subject to complex regulation. The mechanism by which amino acids stimulate mTORdependent signaling (and other signaling pathways), and its molecular connection with the autophagic machinery, is still unknown. 2008-05-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-157-4_6 Autophagy is a cellular homeostasis pathway used to sustain cellular anabolic needs during times of nutrient or energy deprivation. Autophagosomes sequester cytoplasmic constituents, including macromolecules such as long-lived proteins. Upon fusion of autophagosomes with lysosomes, the engulfed cargo is degraded. The proteolysis of longlived proteins by macroautophagy is a standard, specific measure of autophagic degradation and represents an end-point assay for the pathway. The assay is based on a pulse-chase approach, whereby cellular proteins are radiolabeled by an isotopically marked amino acid, the short-lived, rapidly turned over, proteins are allowed to be degraded during a long chase period, and then the remaining, stable radiolabeled proteins are subjected to autophagic degradation. The classical application of this method has been in hepatocytes, but the recent growth of interest in autophagy has necessitated adaptation of this method in nonliver cells. Here we describe a protocol to quantify autophagic degradation of longlived proteins in macrophages. This chapter details the method of analyzing autophagic proteolysis in RAW264.7 mouse macrophages. 2008-05-01T04:00:00Z