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International Journal of Molecular... Feb 2023Phagocytosis is one of the most polarised of all cellular activities. Both the stimulus (the target for phagocytosis) and the response (its internalisation) are focussed... (Review)
Review
Phagocytosis is one of the most polarised of all cellular activities. Both the stimulus (the target for phagocytosis) and the response (its internalisation) are focussed at just one part of the cell. At the locus, and this locus alone, pseudopodia form a phagocytic cup around the particle, the cytoskeleton is rearranged, the plasma membrane is reorganised, and a new internal organelle, the phagosome, is formed. The effect of signals from the stimulus must, thus, both be complex and yet be restricted in space and time to enable an effective focussed response. While many aspects of phagocytosis are being uncovered, the mechanism for the restriction of signalling or the effects of signalling remains obscure. In this review, the details of the problem of restricting chemical intracellular signalling are presented, with a focus on diffusion into the cytosol and of signalling lipids along the plasma membrane. The possible ways in which simple diffusion is overcome so that the restriction of signalling and effective phagocytosis can be achieved are discussed in the light of recent advances in imaging, biophysics, and cell biochemistry which together are providing new insights into this area.
Topics: Phagocytosis; Phagosomes; Pseudopodia; Cytoskeleton; Cytosol
PubMed: 36769146
DOI: 10.3390/ijms24032825 -
The EMBO Journal Jan 2023In their recent article, Polyansky et al identify phosphatidylcholine (PC) as the most abundant lipid in the autophagosome membrane and demonstrate that eliminating de...
In their recent article, Polyansky et al identify phosphatidylcholine (PC) as the most abundant lipid in the autophagosome membrane and demonstrate that eliminating de novo PC synthesis sharply impairs autophagic processing. In the absence of PC synthesis, open cup-like structures accumulate, implicating PC as a key component in the closure of autophagosomes.
Topics: Autophagosomes; Phosphatidylcholines; Autophagy
PubMed: 36478568
DOI: 10.15252/embj.2022113046 -
Frontiers in Immunology 2023Bacterial infections still impose a significant burden on humanity, even though antimicrobial agents have long since been developed. In addition to individual severe... (Review)
Review
Bacterial infections still impose a significant burden on humanity, even though antimicrobial agents have long since been developed. In addition to individual severe infections, the f fatality rate of sepsis remains high, and the threat of antimicrobial-resistant bacteria grows with time, putting us at inferiority. Although tremendous resources have been devoted to the development of antimicrobial agents, we have yet to recover from the lost ground we have been driven into. Looking back at the evolution of treatment for cancer, which, like infectious diseases, has the similarity that host immunity eliminates the lesion, the development of drugs to eliminate the tumor itself has shifted from a single-minded focus on drug development to the establishment of a treatment strategy in which the de-suppression of host immunity is another pillar of treatment. In infectious diseases, on the other hand, the development of therapies that strengthen and support the immune system has only just begun. Among innate immunity, the first line of defense that bacteria encounter after invading the host, the molecular mechanisms of the phagolysosome pathway, which begins with phagocytosis to fusion with lysosome, have been elucidated in detail. Bacteria have a large number of strategies to escape and survive the pathway. Although the full picture is still unfathomable, the molecular mechanisms have been elucidated for some of them, providing sufficient clues for intervention. In this article, we review the host defense mechanisms and bacterial evasion mechanisms and discuss the possibility of host-directed therapy for bacterial infection by intervening in the phagolysosome pathway.
Topics: Humans; Bacterial Infections; Immunity, Innate; Anti-Infective Agents; Bacteria; Phagosomes; Communicable Diseases
PubMed: 37841276
DOI: 10.3389/fimmu.2023.1227467 -
Current Protocols in Cell Biology Jun 2020Phagocytes, notably macrophages, are critical sentinels of their environment, patrolling for and eradicating unwanted components. The ability of cells to process...
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.
Topics: Animals; Autophagy; Flow Cytometry; Humans; Macrophages; Microscopy, Confocal; Phagocytosis; Phagosomes
PubMed: 32436654
DOI: 10.1002/cpcb.104 -
Cells Dec 2019Autophagy is a highly regulated bulk degradation process that plays a key role in the maintenance of cellular homeostasis. During autophagy, a double membrane-bound... (Review)
Review
Autophagy is a highly regulated bulk degradation process that plays a key role in the maintenance of cellular homeostasis. During autophagy, a double membrane-bound compartment termed the autophagosome is formed through de novo nucleation and assembly of membrane sources to engulf unwanted cytoplasmic components and targets them to the lysosome or vacuole for degradation. Central to this process are the autophagy-related (ATG) proteins, which play a critical role in plant fitness, immunity, and environmental stress response. Over the past few years, cryo-electron microscopy (cryo-EM) and single-particle analysis has matured into a powerful and versatile technique for the structural determination of protein complexes at high resolution and has contributed greatly to our current understanding of the molecular mechanisms underlying autophagosome biogenesis. Here we describe the plant-specific ATG proteins and summarize recent structural and mechanistic studies on the protein machinery involved in autophagy initiation with an emphasis on those by single-particle analysis.
Topics: Autophagosomes; Autophagy; Autophagy-Related Proteins; Microscopy, Electron; Models, Molecular; Plant Proteins; Plants
PubMed: 31842460
DOI: 10.3390/cells8121627 -
American Journal of Physiology. Lung... Sep 2021Patients with cystic fibrosis (CF) have defective macrophage phagocytosis and efferocytosis. Several reports demonstrate that neutrophil elastase (NE), a major...
Patients with cystic fibrosis (CF) have defective macrophage phagocytosis and efferocytosis. Several reports demonstrate that neutrophil elastase (NE), a major inflammatory protease in the CF airway, impairs macrophage phagocytic function. To date, NE-impaired macrophage phagocytic function has been attributed to cleavage of cell surface receptors or opsonins. We applied an unbiased proteomic approach to identify other potential macrophage targets of NE protease activity that may regulate phagocytic function. Using the murine macrophage cell line, RAW 264.7, human blood monocyte-derived macrophages, and primary alveolar macrophages from Cftr-null and wild-type littermate mice, we demonstrated that NE exposure blocked phagocytosis of bio-particles. We performed liquid chromatography-tandem mass spectroscopy (LC-MS/MS) proteomic analysis of the conditioned media from RAW264.7 treated either with active NE or inactive (boiled) NE as a control. Out of 840 proteins identified in the conditioned media, active NE upregulated 142 proteins and downregulated 211 proteins. NE released not only cell surface proteins into the media but also cytoskeletal, mitochondrial, cytosolic, and nuclear proteins that were detected in the conditioned media. At least 32 proteins were associated with the process of phagocytosis including 11 phagocytic receptors [including lipoprotein receptor-related protein 1 (LRP1)], 7 proteins associated with phagocytic cup formation, and 14 proteins involved in phagocytic maturation (including calpain-2) and phagolysosome formation. NE had a broad effect on the proteome required for regulation of all stages of phagocytosis and phagolysosome formation. Furthermore, the NE sheddome/secretome included proteins from other macrophage cellular domains, suggesting that NE may globally regulate macrophage structure and function.
Topics: Adolescent; Adult; Animals; Child; Child, Preschool; Cystic Fibrosis Transmembrane Conductance Regulator; Female; Humans; Leukocyte Elastase; Lysosomes; Macrophages; Male; Mice; Mice, Mutant Strains; Phagocytosis; Phagosomes; RAW 264.7 Cells
PubMed: 34261337
DOI: 10.1152/ajplung.00499.2019 -
Trends in Cell Biology Feb 2020Phagocytosis is a receptor-mediated, actin-dependent process of internalization of large extracellular particles, such as pathogens or apoptotic cells. Engulfment of... (Review)
Review
Phagocytosis is a receptor-mediated, actin-dependent process of internalization of large extracellular particles, such as pathogens or apoptotic cells. Engulfment of phagocytic targets requires the activity of myosins, actin-dependent molecular motors, which perform a variety of functions at distinct steps during phagocytosis. By applying force to actin filaments, the plasma membrane, and intracellular proteins and organelles, myosins can generate contractility, directly regulate actin assembly to ensure proper phagocytic internalization, and translocate phagosomes or other cargo to appropriate cellular locations. Recent studies using engineered microenvironments and phagocytic targets have demonstrated how altering the actomyosin cytoskeleton affects phagocytic behavior. Here, we discuss how studies using genetic and biochemical manipulation of myosins, force measurement techniques, and live-cell imaging have advanced our understanding of how specific myosins function at individual steps of phagocytosis.
Topics: Animals; Biological Transport; Humans; Models, Biological; Myosins; Phagocytosis; Phagosomes; Pseudopodia
PubMed: 31836280
DOI: 10.1016/j.tcb.2019.11.002 -
The Journal of Cell Biology Jun 2021Regulation of autophagy in neurons remains unclear. In this issue, Kulkarni et al. (2021. J. Cell Biol.https://doi.org/10.1083/jcb.202002084) show with elegant live...
Regulation of autophagy in neurons remains unclear. In this issue, Kulkarni et al. (2021. J. Cell Biol.https://doi.org/10.1083/jcb.202002084) show with elegant live imaging that in dendrites, but not in axons, autophagosome motility and function is regulated by synaptic activity.
Topics: Autophagosomes; Autophagy; Axons; Neurons; Synapses
PubMed: 33988696
DOI: 10.1083/jcb.202105008 -
The EMBO Journal Dec 2022Phagocytosis is a key process in innate immunity and homeostasis. After particle uptake, newly formed phagosomes mature by acquisition of endolysosomal enzymes....
Phagocytosis is a key process in innate immunity and homeostasis. After particle uptake, newly formed phagosomes mature by acquisition of endolysosomal enzymes. Macrophage activation by interferon gamma (IFN-γ) increases microbicidal activity, but delays phagosomal maturation by an unknown mechanism. Using quantitative proteomics, we show that phagosomal proteins harbour high levels of typical and atypical ubiquitin chain types. Moreover, phagosomal ubiquitylation of vesicle trafficking proteins is substantially enhanced upon IFN-γ activation of macrophages, suggesting a role in regulating phagosomal functions. We identified the E3 ubiquitin ligase RNF115, which is enriched on phagosomes of IFN-γ activated macrophages, as an important regulator of phagosomal maturation. Loss of RNF115 protein or ligase activity enhanced phagosomal maturation and increased cytokine responses to bacterial infection, suggesting that both innate immune signalling from the phagosome and phagolysosomal trafficking are controlled through ubiquitylation. RNF115 knock-out mice show less tissue damage in response to S. aureus infection, indicating a role of RNF115 in inflammatory responses in vivo. In conclusion, RNF115 and phagosomal ubiquitylation are important regulators of innate immune functions during bacterial infections.
Topics: Animals; Mice; Bacterial Infections; Interferon-gamma; Phagocytosis; Phagosomes; Staphylococcus aureus; Ubiquitin-Protein Ligases
PubMed: 36281581
DOI: 10.15252/embj.2021108970 -
Autophagy May 2021Nearly all diseases in humans, to a certain extent, exhibit sex differences, including differences in the onset, progression, prevention, therapy, and prognosis of... (Review)
Review
Nearly all diseases in humans, to a certain extent, exhibit sex differences, including differences in the onset, progression, prevention, therapy, and prognosis of diseases. Accumulating evidence shows that macroautophagy/autophagy, as a mechanism for development, differentiation, survival, and homeostasis, is involved in numerous aspects of sex differences in diseases such as cancer, neurodegeneration, and cardiovascular diseases. Advances in our knowledge regarding sex differences in autophagy-mediated diseases have enabled an understanding of their roles in human diseases, although the underlying molecular mechanisms of sex differences in autophagy remain largely unexplored. In this review, we discuss current advances in our insight into the biology of sex differences in autophagy and disease, information that will facilitate precision medicine.: AD: Azheimer disease; AMBRA1: autophagy and beclin 1 regulator 1; APP: amyloid beta precursor protein; AR: androgen receptor; AMPK: AMP-activated protein kinase; ATG: autophagy related; ATP6AP2: ATPase H+ transporting accessory protein 2; BCL2L1: BCL2 like 1; BECN1: beclin 1; CTSD: cathepsin D; CYP19A1: cytochrome P450 family 19 subfamily A member 1; DSD: disorders of sex development; eALDI: enhancer alternate long-distance initiator; ESR1: estrogen receptor 1; ESR2: estrogen receptor 2; FYCO1: FYVE and coiled-coil domain autophagy adaptor 1; GABARAP: GABA type A receptor-associated protein; GLA: galactosidase alpha; GTEx: genotype-tissue expression; HDAC6: histone deacetylase 6; I-R: ischemia-reperfusion; LAMP2: lysosomal associated membrane protein 2; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MTOR: mechanistic target of rapamycin kinase; m6A: N6-methyladenosine; MYBL2: MYB proto-oncogene like 2; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; PSEN1: presenilin 1; PSEN2: presenilin 2; RAB9A, RAB9A: member RAS oncogene family; RAB9B, RAB9B: member RAS oncogene family; RAB40AL: RAB40A like; SF1: splicing factor 1; SOX9: SRY-box transcription factor 9; SRY: sex determining region Y; TFEB: transcription factor EB; ULK1: unc-51 like autophagy activating kinase 1; UVRAG: UV radiation resistance associated; VDAC2: voltage dependent anion channel 2; WDR45: WD repeat domain 45; XPDS: X-linked parkinsonism and spasticity; YTHDF2: YTH N6-methyladenosine RNA binding protein 2.
Topics: Apoptosis Regulatory Proteins; Autophagosomes; Autophagy; Disease; Humans; Precision Medicine; Sex Characteristics
PubMed: 32264724
DOI: 10.1080/15548627.2020.1752511