LC3-associated Phagocytosis Research Articles

Dendritic cell PIK3C3/VPS34 controls the pathogenicity of CNS autoimmunity independently of LC3-associated phagocytosis

ABSTRACT PIK3C3/VPS34 is a key player in macroautophagy/autophagy and MAP1LC3/LC3-associated phagocytosis (LAP), which play critical roles in dendritic cell (DC) function. In this study, we assessed the contribution of PIK3C3 to DC function during experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). We found that Pik3c3-deficient DCs exhibit attenuated capacity to reactivate encephalitogenic T cells in the central nervous system, leading to reduced incidence and severity of EAE in DC-specific Pik3c3-deficient mice. Additionally, animals with a DC-specific deficiency in Rb1cc1/Fip200 but not Rubcn were protected against EAE, suggesting that the EAE phenotype of DC-specific Pik3c3-deficient mice is due to defective canonical autophagy rather than LAP. Collectively, our studies have revealed a critical role of PIK3C3 in DC function and the pathogenicity of these cells during EAE, with important implications for the development of immunotherapies for autoimmune diseases such as MS.

Macrophage LC3‐associated phagocytosis is an immune defense against Streptococcus pneumoniae that diminishes with host aging

Streptococcus pneumoniae is a leading cause of pneumonia and invasive disease, particularly, in the elderly. S. pneumoniae lung infection of aged mice is associated with high bacterial burdens and detrimental inflammatory responses. Macrophages can clear microorganisms and modulate inflammation through two distinct lysosomal trafficking pathways that involve 1A/1B-light chain 3 (LC3)-marked organelles, canonical autophagy, and LC3-associated phagocytosis (LAP). The S. pneumoniae pore-forming toxin pneumolysin (PLY) triggers an autophagic response in nonphagocytic cells, but the role of LAP in macrophage defense against S. pneumoniae or in age-related susceptibility to infection is unexplored. We found that infection of murine bone-marrow-derived macrophages (BMDMs) by PLY-producing S. pneumoniae triggered Atg5- and Atg7-dependent recruitment of LC3 to S. pneumoniae-containing vesicles. The association of LC3 with S. pneumoniae-containing phagosomes required components specific for LAP, such as Rubicon and the NADPH oxidase, but not factors, such as Ulk1, FIP200, or Atg14, required specifically for canonical autophagy. In addition, S. pneumoniae was sequestered within single-membrane compartments indicative of LAP. Importantly, compared to BMDMs from young (2-mo-old) mice, BMDMs from aged (20- to 22-mo-old) mice infected with S. pneumoniae were not only deficient in LAP and bacterial killing, but also produced higher levels of proinflammatory cytokines. Inhibition of LAP enhanced S. pneumoniae survival and cytokine responses in BMDMs from young but not aged mice. Thus, LAP is an important innate immune defense employed by BMDMs to control S. pneumoniae infection and concomitant inflammation, one that diminishes with age and may contribute to age-related susceptibility to this important pathogen.

First person – Edismauro Freitas Filho

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping early-career researchers promote themselves alongside their papers. Edismauro Freitas Filho is first author on ‘ RACK1 plays a critical role in mast cell secretion and Ca2+ mobilization by modulating F-actin dynamics’, published in JCS. Edismauro conducted the research described in this article while a PhD student in Maria Célia Jamur's lab at the Department of Cell and Molecular Biology and Pathogenic Bioagents, University of São Paulo, Brazil. He is now a postdoctoral fellow in the lab of Larissa Dias da Cunha at the Department of Cell and Molecular Biology and Pathogenic Bioagents, University of São Paulo, investigating the role of signaling proteins and specific lipids in the initial events of signal transduction, cytoskeleton rearrangement and LC3-associated phagocytosis during immune cell responses.

Dendritic cell PIK3C3/VPS34 controls the pathogenicity of CNS autoimmunity independently of LC3-associated phagocytosis

Abstract The PIK3C3/VPS34 subunit of the class III phosphatidylinositol 3-kinase (PtdIns3K) complex is a key player in macroautophagy/autophagy and MAP1LC3/LC3-associated phagocytosis (LAP), both of which play critical roles in mediating dendritic cell (DC) function. In this study, we assessed the contribution of PIK3C3 to DC function in the context of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). We found that Pik3c3-deficient DCs exhibited attenuated ability in reactivating encephalitogenic T cells in the central nervous system, leading to reduced incidence and severity of EAE in DC-specific Pik3c3-deficient mice. Additionally, animals with DC-specific deficiency of Rb1cc1 (essential for autophagosome nucleation but not LAP) but not Rubcn (required for LAP but not autophagy) were protected against EAE, suggesting that the EAE phenotype of DC-specific Pik3c3-deficient mice is associated with the lack of canonical autophagy rather than LAP. Collectively, our studies have revealed a critical role of PIK3C3 in DC function and in the pathogenicity of these cells during EAE. Our findings also have important implications for the development of immunotherapies to treat autoimmune diseases such as MS by targeting PIK3C3-containing complexes.

Non-canonical autophagy drives alternative ATG8 conjugation to phosphatidylserine

SummaryAutophagy is a fundamental catabolic process that uses a unique post-translational modification, the conjugation of ATG8 protein to phosphatidylethanolamine (PE). ATG8 lipidation also occurs during non-canonical autophagy, a parallel pathway involving conjugation of ATG8 to single membranes (CASM) at endolysosomal compartments, with key functions in immunity, vision, and neurobiology. It is widely assumed that CASM involves the same conjugation of ATG8 to PE, but this has not been formally tested. Here, we discover that all ATG8s can also undergo alternative lipidation to phosphatidylserine (PS) during CASM, induced pharmacologically, by LC3-associated phagocytosis or influenza A virus infection, in mammalian cells. Importantly, ATG8-PS and ATG8-PE adducts are differentially delipidated by the ATG4 family and bear different cellular dynamics, indicating significant molecular distinctions. These results provide important insights into autophagy signaling, revealing an alternative form of the hallmark ATG8 lipidation event. Furthermore, ATG8-PS provides a specific “molecular signature” for the non-canonical autophagy pathway.

Hypoxia enhances basal autophagy of epithelial-derived ameloblastoma cells.

Ameloblastoma is a locally aggressive odontogenic tumor. Etiopathogenesis and locally aggressive growth properties of ameloblastoma can be attributed to a hypoxic microenvironment conducive to tumor cell survival. Epithelial-derived follicular ameloblastoma cells (EP-AMCs) display enhanced basal autophagy, but the interplay of hypoxia and autophagy in EP-AMCs survival and ameloblastoma recurrence is unclear. We evaluated differential expression of autophagic markers in primary and recurrent ameloblastomas and hypothesized that hypoxia-induced autophagy supports EP-AMC survival. Primary and recurrent ameloblastomas were comparatively assessed for expression levels of pan-cytokeratin, Vimentin, and autophagic markers SQSTM1/p62, LC3, and pS6. EP-AMCs compared with human odontoma-derived cells (HODCs) were subjected to severe hypoxia to determine the interplay of hypoxia and autophagic process in posthypoxia survival. Pan-cytokeratin and SQSTM1/p62 were expressed by both primary and recurrent ameloblastoma epithelial cells while the ameloblastoma connective tissues displayed weak reactivity to vimentin. Under hypoxia, EP-AMC expression levels of hypoxia-inducible factor (HIF)-1α, p62, and LC3 were increased while pS6 was decreased posthypoxia. The combined decrease in pS6 and enhanced LC3 in EP-AMCs under hypoxia indicate that EP-AMCs re-establish basal autophagy under hypoxia. Taken together, these suggest a possible role of LC3-associated phagocytosis (LAP) in ameloblastoma cell survival.

Oxidation inhibits autophagy protein deconjugation from phagosomes to sustain MHC class II restricted antigen presentation

LC3-associated phagocytosis (LAP) contributes to a wide range of cellular processes and notably to immunity. The stabilization of phagosomes by the macroautophagy machinery in human macrophages can maintain antigen presentation on MHC class II molecules. However, the molecular mechanisms involved in the formation and maturation of the resulting LAPosomes are not completely understood. Here, we show that reactive oxygen species (ROS) produced by NADPH oxidase 2 (NOX2) stabilize LAPosomes by inhibiting LC3 deconjugation from the LAPosome cytosolic surface. NOX2 residing in the LAPosome membrane generates ROS to cause oxidative inactivation of the protease ATG4B, which otherwise releases LC3B from LAPosomes. An oxidation-insensitive ATG4B mutant compromises LAP and thereby impedes sustained MHC class II presentation of exogenous Candida albicans antigens. Redox regulation of ATG4B is thereby an important mechanism for maintaining LC3 decoration of LAPosomes to support antigen processing for MHC class II presentation.

The Macroautophagy Machinery in MHC Restricted Antigen Presentation.

Autophagy-related (ATG) gene products regulate macroautophagy, LC3-associated phagocytosis (LAP) and LC3-dependent extracellular vesicle loading and secretion (LDELS). These processes also influence antigen processing for presentation on major histocompatibility complex (MHC) molecules to T cells. Here, I summarize how these different pathways use the macroautophagy machinery, contribute to MHC class I and II restricted antigen presentation and influence autoimmunity, tumor immunology and immune control of infectious diseases. Targeting these different pathways should allow the regulation of intracellular and extracellular antigen presentation to T cells to modulate protective and pathological immune responses.

About the Master.

See related article on p. 130Doug Green is the Peter C. Doherty Endowed Chair of Immunology at St. Jude Children's Research Hospital (Memphis, TN). Prior to this, he was Head of the Division of Cellular Immunology at the La Jolla Institute for Allergy and Immunology. Doug received his Bsc (1977) and PhD (1981) from Yale University (New Haven, CT), followed by postdoctoral work in experimental surgery and marine ecology, also at Yale. He joined the faculty at the University of Alberta in Edmonton in 1985, where he attained the rank of Associate Professor. He moved to what is now the La Jolla Institute of Immunology in 1990 and to St. Jude in 2005. His research has focused on the process of active cell death and cell survival, extending from the role of cell death in the regulation of cancer and immune responses in the whole organism to the fundamental molecular events directing the death of the cell. More recently, he discovered the process of LC3-associated phagocytosis, which links the autophagy pathway to phagosome maturation. Other areas of intense interest include regulated necrosis and metabolic reprogramming during asymmetric division in T lymphocytes. He has published over 600 papers, chapters, commentaries, and books and is an ISI “highly cited” investigator. In 2017, he was awarded the Jürg Tschopp Prize for research on cell death. He is an honorary fellow of Trinity College, Dublin, an honorary Einstein Professor in China, and a foreign fellow of the Royal Society of Canada and received an honorary PhD from Tor Vergata University of Rome. His most recent book is “Apoptosis and Other Cell Death Mechanisms: Means to an End,” published in 2018 by Cold Spring Harbor Laboratory Press. His wife, Rona Mogil (PhD, Yale, 1985), and daughter, Maggie Green (BA, Yale, 2014), are much smarter than he is and display great patience in putting up with him.

Listeria monocytogenes upregulates mitochondrial calcium signalling to inhibit LC3-associated phagocytosis as a survival strategy.

Mitochondria are believed to have originated ~2.5 billion years ago. As well as energy generation in cells, mitochondria have a role in defence against bacterial pathogens. Despite profound changes in mitochondrial morphology and functions following bacterial challenge, whether intracellular bacteria can hijack mitochondria to promote their survival remains elusive. We report that Listeria monocytogenes-an intracellular bacterial pathogen-suppresses LC3-associated phagocytosis (LAP) by modulation of mitochondrial Ca2+ (mtCa2+) signalling in order to survive inside cells. Invasion of macrophages by L. monocytogenes induced mtCa2+ uptake through the mtCa2+ uniporter (MCU), which in turn increased acetyl-coenzyme A (acetyl-CoA) production by pyruvate dehydrogenase. Acetylation of the LAP effector Rubicon with acetyl-CoA decreased LAP formation. Genetic ablation of MCU attenuated intracellular bacterial growth due to increased LAP formation. Our data show that modulation of mtCa2+ signalling can increase bacterial survival inside cells, and highlight the importance of mitochondrial metabolism in host-microbial interactions.

Inhibition of LC3-associated phagocytosis in COPD and in response to cigarette smoke

Introduction/Rationale:In chronic obstructive pulmonary disease (COPD), defective macrophage phagocytic clearance of cells undergoing apoptosis by efferocytosis may lead to secondary necrosis of the uncleared cells and contribute to airway inflammation. The precise mechanisms for this phenomenon remain unknown. LC3-associated phagocytosis (LAP) is indispensable for effective efferocytosis. We hypothesized that cigarette smoke inhibits the regulators of LAP pathway, potentially contributing to the chronic airways inflammation associated with COPD.Methods:Bronchoalveolar (BAL)-derived alveolar macrophages, lung tissue macrophages obtained from lung resection surgery, and monocyte-derived macrophages (MDM) were prepared from COPD patients and control participants. Lung/airway samples from mice chronically exposed to cigarette smoke were also investigated. Differentiated THP-1 cells were exposed to cigarette smoke extract (CSE). The LAP pathway including Rubicon, as an essential regulator of LAP, efferocytosis and inflammation was examined using western blot, ELISA, flow cytometry, and/or immunofluorescence.Results:Rubicon was significantly depleted in COPD alveolar macrophages compared with non-COPD control macrophages. Rubicon protein in alveolar macrophages of cigarette smoke-exposed mice and cigarette smoke-exposed MDM and THP-1 was decreased with a concomitant impairment of efferocytosis. We also noted increased expression of LC3 which is critical for LAP pathway in COPD and THP-1 macrophages. Furthermore, THP-1 macrophages exposed to cigarette smoke extract exhibited higher levels of other key components of LAP pathway including Atg5 and TIM-4. There was a strong positive correlation between Rubicon protein expression and efferocytosis.Conclusion:LAP is a requisite for effective efferocytosis and an appropriate inflammatory response, which is impaired by Rubicon deficiency. Our findings suggest dysregulated LAP due to reduced Rubicon as a result of CSE exposure. This phenomenon could lead to a failure of macrophages to effectively process phagosomes containing apoptotic cells during efferocytosis. Restoring Rubicon protein expression has unrecognized therapeutic potential in the context of disease-related modifications caused by exposure to cigarette smoke.

Measuring oxidation within LC3-associated phagosomes that optimizes MHC class II restricted antigen presentation.

LC3-associated phagocytosis (LAP) uses components of the molecular machinery of macroautophagy and is involved in the presentation of extracellular antigens by Major Histocompatibility Complex (MHC) class II molecules. It is initiated by receptor-mediated phagocytosis and results in the formation of LAPosomes: single-membrane vesicles that are decorated with the macroautophagy protein LC3B. LAPosomes have been described to prolong antigen presentation in macrophages but the molecular mechanism of this process is just beginning to be understood. Known key regulators of LAPosome formation are Reactive Oxygen Species (ROS), which can modulate the pH and the oxidative state within LAPosomes. Here, we present two complementary methods to monitor oxidation in LAPosomes and to study its function in MHC class II restricted antigen presentation, both in primary human macrophages: (I) Coating the LAP-trigger zymosan with OxyBURST allows semi-quantitative assessment of oxidation levels within LAPosomes by confocal microscopy. (II) The co-culture of macrophages with CD4+T cells to assess the effects of LAP on Candida albicans antigen presentation by measuring IL-17A and IFN-γ secretion.

Acinetobacter baumannii Targets Human Carcinoembryonic Antigen-Related Cell Adhesion Molecules (CEACAMs) for Invasion of Pneumocytes.

Multidrug-resistant Acinetobacter baumannii is regarded as a life-threatening pathogen mainly associated with nosocomial and community-acquired pneumonia. Here, we show that A. baumannii can bind the human carcinoembryonic antigen-related cell adhesion molecule (CEACAM) receptors CEACAM1, CEACAM5, and CEACAM6. This specific interaction enhances A. baumannii internalization in membrane-bound vacuoles, promptly decorated with Rab5, Rab7, and lipidated microtubule-associated protein light chain 3 (LC3). Dissecting intracellular signaling pathways revealed that infected pneumocytes trigger interleukin-8 (IL-8) secretion via the extracellular signal-regulated kinase (ERK)1/2 and nuclear factor-kappa B (NF-κB) signaling pathways for A. baumannii clearance. However, in CEACAM1-L-expressing cells, IL-8 secretion lasts only 24 h, possibly due to an A. baumannii-dependent effect on the CEACAM1-L intracellular domain. Conversely, the glycosylphosphatidylinositol-anchored CEACAM5 and CEACAM6 activate the c-Jun NH2-terminal kinase (JNK)1/2-Rubicon-NOX2 pathway, suggestive of LC3-associated phagocytosis. Overall, our data show for the first time novel mechanisms of adhesion to and invasion of pneumocytes by A. baumannii via CEACAM-dependent signaling pathways that eventually lead to bacterial killing. These findings suggest that CEACAM upregulation could put patients at increased risk of lower respiratory tract infection by A. baumannii IMPORTANCE This work shows for the first time that Acinetobacter baumannii binds to carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), CEACAM5, and CEACAM6. This binding significantly enhances A. baumannii internalization within alveolar host cell epithelia. Intracellular trafficking involves typical Rab5 and Rab7 vacuolar proteins as well as light chain 3 (LC3) and slowly progresses to bacterial killing by endosome acidification. CEACAM engagement by A. baumannii leads to distinct and specific downstream signaling pathways. The CEACAM1 pathway finely tunes interleukin-8 (IL-8) secretion, whereas CEACAM5 and CEACAM6 mediate LC3-associated phagocytosis. The present study provides new insights into A. baumannii-host interactions and could represent a promising therapeutic strategy to reduce pulmonary infections caused by this pathogen.

Macrophage LC3-associated phagocytosis is an immune defense against Streptococcus pneumoniae that diminishes with host aging

Streptococcus pneumoniae is a leading cause of pneumonia and invasive disease, particularly, in the elderly. S. pneumoniae lung infection of aged mice is associated with high bacterial burdens and detrimental inflammatory responses. Macrophages can clear microorganisms and modulate inflammation through two distinct lysosomal trafficking pathways that involve 1A/1B-light chain 3 (LC3)-marked organelles, canonical autophagy, and LC3-associated phagocytosis (LAP). The S. pneumoniae pore-forming toxin pneumolysin (PLY) triggers an autophagic response in nonphagocytic cells, but the role of LAP in macrophage defense against S. pneumoniae or in age-related susceptibility to infection is unexplored. We found that infection of murine bone-marrow-derived macrophages (BMDMs) by PLY-producing S. pneumoniae triggered Atg5- and Atg7-dependent recruitment of LC3 to S. pneumoniae-containing vesicles. The association of LC3 with S. pneumoniae-containing phagosomes required components specific for LAP, such as Rubicon and the NADPH oxidase, but not factors, such as Ulk1, FIP200, or Atg14, required specifically for canonical autophagy. In addition, S. pneumoniae was sequestered within single-membrane compartments indicative of LAP. Importantly, compared to BMDMs from young (2-mo-old) mice, BMDMs from aged (20- to 22-mo-old) mice infected with S. pneumoniae were not only deficient in LAP and bacterial killing, but also produced higher levels of proinflammatory cytokines. Inhibition of LAP enhanced S. pneumoniae survival and cytokine responses in BMDMs from young but not aged mice. Thus, LAP is an important innate immune defense employed by BMDMs to control S. pneumoniae infection and concomitant inflammation, one that diminishes with age and may contribute to age-related susceptibility to this important pathogen.

Autophagy and Lc3-Associated Phagocytosis in Zebrafish Models of Bacterial Infections.

Modeling human infectious diseases using the early life stages of zebrafish provides unprecedented opportunities for visualizing and studying the interaction between pathogens and phagocytic cells of the innate immune system. Intracellular pathogens use phagocytes or other host cells, like gut epithelial cells, as a replication niche. The intracellular growth of these pathogens can be counteracted by host defense mechanisms that rely on the autophagy machinery. In recent years, zebrafish embryo infection models have provided in vivo evidence for the significance of the autophagic defenses and these models are now being used to explore autophagy as a therapeutic target. In line with studies in mammalian models, research in zebrafish has shown that selective autophagy mediated by ubiquitin receptors, such as p62, is important for host resistance against several bacterial pathogens, including Shigella flexneri, Mycobacterium marinum, and Staphylococcus aureus. Furthermore, an autophagy related process, Lc3-associated phagocytosis (LAP), proved host beneficial in the case of Salmonella Typhimurium infection but host detrimental in the case of S. aureus infection, where LAP delivers the pathogen to a replication niche. These studies provide valuable information for developing novel therapeutic strategies aimed at directing the autophagy machinery towards bacterial degradation.

The Host Autophagy During Toxoplasma Infection.

Autophagy is an important homeostatic mechanism, in which lysosomes degrade and recycle cytosolic components. As a key defense mechanism against infections, autophagy is involved in the capture and elimination of intracellular parasites. However, intracellular parasites, such as Toxoplasma gondii, have developed several evasion mechanisms to manipulate the host cell autophagy for their growth and establish a chronic infection. This review provides an insight into the autophagy mechanism used by the host cells in the control of T. gondii and the host exploitation by the parasite. First, we summarize the mechanism of autophagy, xenophagy, and LC3-associated phagocytosis. Then, we illustrate the process of autophagy proteins-mediated T. gondii clearance. Furthermore, we discuss how the parasite blocks and exploits this process for its survival.

The Chlamydia effector CT622/TaiP targets a nonautophagy related function of ATG16L1

The obligate intracellular bacteria Chlamydia trachomatis, the causative agent of trachoma and sexually transmitted diseases, multiply in a vacuolar compartment, the inclusion. From this niche, they secrete "effector" proteins, that modify cellular activities to enable bacterial survival and proliferation. Here, we show that the host autophagy-related protein 16-1 (ATG16L1) restricts inclusion growth and that this effect is counteracted by the secretion of the bacterial effector CT622/TaiP (translocated ATG16L1 interacting protein). ATG16L1 is mostly known for its role in the lipidation of the human homologs of ATG8 (i.e., LC3 and homologs) on double membranes during autophagy as well as on single membranes during LC3-associated phagocytosis and other LC3-lipidation events. Unexpectedly, the LC3-lipidation-related functions of ATG16L1 are not required for restricting inclusion development. We show that the carboxyl-terminal domain of TaiP exposes a mimic of an eukaryotic ATG16L1-binding motif that binds to ATG16L1's WD40 domain. By doing so, TaiP prevents ATG16L1 interaction with the integral membrane protein TMEM59 and allows the rerouting of Rab6-positive compartments toward the inclusion. The discovery that one bacterial effector evolved to target ATG16L1's engagement in intracellular traffic rather than in LC3 lipidation brings this "secondary" activity of ATG16L1 in full light and emphasizes its importance for maintaining host cell homeostasis.

LC3-Associated Phagocytosis (LAP): A Potentially Influential Mediator of Efferocytosis-Related Tumor Progression and Aggressiveness.

One aim of cancer therapies is to induce apoptosis of tumor cells. Efficient removal of the apoptotic cells requires coordinated efforts between the processes of efferocytosis and LC3-associated phagocytosis (LAP). However, this activity has also been shown to produce anti-inflammatory and immunosuppressive signals that can be utilized by live tumor cells to evade immune defense mechanisms, resulting in tumor progression and aggressiveness. In the absence of LAP, mice exhibit suppressed tumor growth during efferocytosis, while LAP-sufficient mice show enhanced tumor progression. Little is known about how LAP or its regulators directly affect efferocytosis, tumor growth and treatment responses, and identifying the mechanisms involved has the potential to lead to the discovery of novel approaches to target cancer cells. Also incompletely understood is the direct effect of apoptotic cancer cells on LAP. This is particularly important as induction of apoptosis by current cytotoxic cancer therapies can potentially stimulate LAP following efferocytosis. Herein, we highlight the current understanding of the role of LAP and its relationship with efferocytosis in the tumor microenvironment with a view to presenting novel therapeutic strategies.

Role of FIP200 in inflammatory processes beyond its canonical autophagy function.

FIP200 (RB1CC1) is a critical regulator of canonical macroautophagy and has also emerged as a crucial regulator of selective autophagy as well as inflammatory processes. The illumination of FIP200's role in autophagy at the molecular level has been accompanied by studies demonstrating the importance of its autophagy function in physiological processes in mammals and pathological contexts such as cancer. However, there is an increasing appreciation that most, if not all of the autophagy genes, also play a role in other processes such as LC3-associated phagocytosis, vesicle trafficking and protein secretion. Consequently, this has led to efforts in generating specific mutants of autophagy genes that are more amenable to dissecting their autophagy versus non-autophagy functions. In this aspect, we have generated a FIP200 knock-in mouse allele that is defective for canonical macroautophagy. This has revealed a canonical-autophagy-independent function of FIP200 that is responsible for limiting pro-inflammatory signaling. In this review, we will discuss FIP200's role in this process, the implications with regards to cancer immunotherapy and highlight key prospective avenues to specifically dissect the distinct functions of FIP200.

Detection of LC3-Associated Phagocytosis (LAP).

Phagocytes, notably macrophages, are critical sentinels of their environment, patrolling for and eradicating unwanted components. The ability of cells to process extracellular cargo in an appropriate manner is important for both clearance of the cargo and eventual return to homeostasis. Although the evolutionarily conserved pathway of autophagy involves the degradation and recycling of unnecessary or dysfunctional cellular components during starvation, we now appreciate that the reach of autophagy extends beyond nutrient deprivation, notably including cellular quality control (e.g., mitophagy) and host defense against internalized pathogens (i.e., xenophagy). Despite being seemingly disparate, autophagic functions are unified as conserved mechanisms for containment and immunosuppression, suggesting an original immune function for autophagy. A recently described pathway called LC3‐associated phagocytosis (LAP) marries the ancient concepts of phagocytosis and autophagy, revealing new ways in which the autophagy machinery, in a molecularly distinct pathway, contributes to the inflammatory response. In this article, protocols to detect LAP by electron microscopy, immunofluorescence, flow cytometry, and phagosome purification are described, allowing the user to detect multiple characteristics of LAP in both qualitative and quantitative manners. Published 2020. U.S. Government. Basic Protocol 1: Detection of LAP by electron microscopy Basic Protocol 2: Detection of LAP by confocal microscopy of LC3‐GFP‐expressing cells Alternate Protocol 1: Detection of LAP by confocal microscopy using immunofluorescence Basic Protocol 3: Detection of LAP using flow cytometry of LC3‐GFP‐expressing cells Alternate Protocol 2: Detection of LAP using antibody staining and flow cytometry Basic Protocol 4: Detection of LAP by western blot of purified LAPosomes