Promoters and Antagonists of Phagocytosis: A Plastic and Tunable Response.

Recent observations indicate that, rather than being an all-or-none response, phagocytosis is finely tuned by a host of developmental and environmental factors. The expression of key phagocytic determinants is regulated via transcriptional and epigenetic means that confer memory on the process. Membrane traffic, the cytoskeleton, and inside-out signaling control the activation of phagocytic receptors and their ability to access their targets. An exquisite extra layer of complexity is introduced by the coexistence of distinct "eat-me" and "don't-eat-me" signals on targets and of corresponding "eat" and "don't-eat" receptors on the phagocyte surface. Moreover, assorted physical barriers constitute "don't-come-close-to-me" hurdles that obstruct the engagement of ligands by receptors. The expression, mobility, and accessibility of all these determinants can be modulated, conferring extreme plasticity on phagocytosis and providing attractive targets for therapeutic intervention in cancer, atherosclerosis, and dementia.

Transmembrane Pickets Connect Cyto- and Pericellular Skeletons Forming Barriers to Receptor Engagement.

Phagocytic receptors must diffuse laterally to become activated upon clustering by multivalent targets. Receptor diffusion, however, can be obstructed by transmembrane proteins ("pickets") that are immobilized by interacting with the cortical cytoskeleton. The molecular identity of these pickets and their role in phagocytosis have not been defined. We used single-molecule tracking to study the interaction between Fcγ receptors and CD44, an abundant transmembrane protein capable of indirect association with F-actin, hence likely to serve as a picket. CD44 tethers reversibly to formin-induced actin filaments, curtailing receptor diffusion. Such linear filaments predominate in the trailing end of polarized macrophages, where receptor mobility was minimal. Conversely, receptors were most mobile at the leading edge, where Arp2/3-driven actin branching predominates. CD44 binds hyaluronan, anchoring a pericellular coat that also limits receptor displacement and obstructs access to phagocytic targets. Force must be applied to traverse the pericellular barrier, enabling receptors to engage their targets.

SnapShot:Macropinocytosis.

Macropinocytosis is the bulk ingestion of extracellular fluids via large endocytic vacuoles. This SnapShot provides an overview of physiological macropinocytosis in immune surveillance and its pathogenic contribution during infection and cancer proliferation.

Integrins Form an Expanding Diffusional Barrier that Coordinates Phagocytosis.

Phagocytosis is initiated by lateral clustering of receptors, which in turn activates Src-family kinases (SFKs). Activation of SFKs requires depletion of tyrosine phosphatases from the area of particle engagement. We investigated how the major phosphatase CD45 is excluded from contact sites, using single-molecule tracking. The mobility of CD45 increased markedly upon engagement of Fcγ receptors. While individual CD45 molecules moved randomly, they were displaced from the advancing phagocytic cup by an expanding diffusional barrier. By micropatterning IgG, the ligand of Fcγ receptors, we found that the barrier extended well beyond the perimeter of the receptor-ligand engagement zone. Second messengers generated by Fcγ receptors activated integrins, which formed an actin-tethered diffusion barrier that excluded CD45. The expanding integrin wave facilitates the zippering of Fcγ receptors onto the target and integrates the information from sparse receptor-ligand complexes, coordinating the progression and ultimate closure of the phagocytic cup.

Determinants, maintenance, and function of organellar pH.

The protonation state of soluble and membrane-associated macromolecules dictates their charge, conformation, and functional activity. In addition, protons (H+ or their equivalents) partake in numerous metabolic reactions and serve as a source of electrochemical energy to drive the transmembrane transport of both organic and inorganic substrates. Stringent regulation of the intracellular pH is therefore paramount to homeostasis. Although the regulation of the cytosolic pH has been studied extensively, our understanding of the determinants of the H+ concentration ([H+]) of intracellular organelles has developed more slowly, limited by their small size and inaccessibility. Recently, however, targeting of molecular probes to the organellar lumen together with advances in genomic, proteomic, and electrophysiological techniques have led to the identification and characterization of unique pumps, channels, and transporters responsible for the establishment and maintenance of intraorganellar pH. These developments and their implications for cellular function in health and disease are the subject of this review.

Cytoskeletal control of CD36 diffusion promotes its receptor and signaling function.

The mechanisms that govern receptor coalescence into functional clusters--often a critical step in their stimulation by ligand--are poorly understood. We used single-molecule tracking to investigate the dynamics of CD36, a clustering-responsive receptor that mediates oxidized LDL uptake by macrophages. We found that CD36 motion in the membrane was spatially structured by the cortical cytoskeleton. A subpopulation of receptors diffused within linear confinement regions whose unique geometry simultaneously facilitated freedom of movement along one axis while increasing the effective receptor density. Co-confinement within troughs enhanced the probability of collisions between unligated receptors and promoted their clustering. Cytoskeleton perturbations that inhibited diffusion in linear confinement regions reduced receptor clustering in the absence of ligand and, following ligand addition, suppressed CD36-mediated signaling and internalization. These observations demonstrate a role for the cytoskeleton in controlling signal transduction by structuring receptor diffusion within membrane regions that increase their collision frequency.

Lysosomal swelling and lysis mediate delivery of C3 toxins to their cytoplasmic targets.

Unlike other cholera-like toxins that contain separate binding/translocation and catalytic subunits, C3-like mono-ADP-ribosyltransferases consist of a single subunit that serves both functions. The manner whereby C3 toxins reach the host cell cytoplasm is poorly understood and was addressed in this study by monitoring the fate of fluorescently labeled C3larvinA. Following binding to the macrophage membrane in a discontinuous punctate pattern, the toxin was internalized, traversing the endocytic pathway to reach lysosomes. Strikingly, the lysosomes of C3larvinA-treated cells underwent massive swelling over the course of 1-4 h. Lysosomal swelling preceded the extensive rearrangement of the cellular F-actin caused by ADP-ribosylation of cytosolic Rho-GTPases. This suggested that lysosome swelling might be required for the escape of the toxin into the cytoplasm where the GTPases reside. Accordingly, preventing swelling by osmotic manipulation or by arresting macropinocytosis precluded the F-actin rearrangement. Toxin-induced swelling was associated with leakage of sulforhodamine B and dextran from the lysosomes, implying membrane rupture or activation of mechano-sensitive pores, enabling the toxin itself to reach the cytosol. Finally, comparison of the cellular traffic and actin remodeling activities of C3larvinA with that of two related toxins, C3larvintrunc and Plx2A, highlighted the importance of the N-terminal α1 -helix for lysosomal swelling and successful intoxication.

Rab5 regulates macropinocytosis by recruiting the inositol 5-phosphatases OCRL and Inpp5b that hydrolyse PtdIns(4,5)P2.

Rab5 is required for macropinosome formation, but its site and mode of action remain unknown. We report that Rab5 acts at the plasma membrane, downstream of ruffling, to promote macropinosome sealing and scission. Dominant-negative Rab5, which obliterates macropinocytosis, had no effect on the development of membrane ruffles. However, Rab5-containing vesicles were recruited to circular membrane ruffles, and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-dependent endomembrane fusion was necessary for the completion of macropinocytosis. This fusion event coincided with the disappearance of PtdIns(4,5)P2 that accompanies macropinosome closure. Counteracting the depletion of PtdIns(4,5)P2 by expression of phosphatidylinositol-4-phosphate 5-kinase impaired macropinosome formation. Importantly, we found that the removal of PtdIns(4,5)P2 is dependent on Rab5, through the Rab5-mediated recruitment of the inositol 5-phosphatases OCRL and Inpp5b, via APPL1. Knockdown of OCRL and Inpp5b, or APPL1, prevented macropinosome closure without affecting ruffling. We therefore propose that Rab5 is essential for the clearance of PtdIns(4,5)P2 needed to complete the scission of macropinosomes or to prevent their back-fusion with the plasmalemma.

Phosphatidylinositol-4-kinase IIα licenses phagosomes for TLR4 signaling and MHC-II presentation in dendritic cells.

Toll-like receptor (TLR) recruitment to phagosomes in dendritic cells (DCs) and downstream TLR signaling are essential to initiate antimicrobial immune responses. However, the mechanisms underlying TLR localization to phagosomes are poorly characterized. We show herein that phosphatidylinositol-4-kinase IIα (PI4KIIα) plays a key role in initiating phagosomal TLR4 responses in murine DCs by generating a phosphatidylinositol-4-phosphate (PtdIns4P) platform conducive to the binding of the TLR sorting adaptor Toll-IL1 receptor (TIR) domain-containing adaptor protein (TIRAP). PI4KIIα is recruited to maturing lipopolysaccharide (LPS)-containing phagosomes in an adaptor protein-3 (AP-3)-dependent manner, and both PI4KIIα and PtdIns4P are detected on phagosomal membrane tubules. Knockdown of PI4KIIα-but not the related PI4KIIß-impairs TIRAP and TLR4 localization to phagosomes, reduces proinflammatory cytokine secretion, abolishes phagosomal tubule formation, and impairs major histocompatibility complex II (MHC-II) presentation. Phagosomal TLR responses in PI4KIIα-deficient DCs are restored by reexpression of wild-type PI4KIIα, but not of variants lacking kinase activity or AP-3 binding. Our data indicate that PI4KIIα is an essential regulator of phagosomal TLR signaling in DCs by ensuring optimal TIRAP recruitment to phagosomes.

Phagosome-endoplasmic reticulum contacts: Kissing and not running.

The role of the endoplasmic reticulum (ER) in phagocytosis has been the subject of debate for over a decade. Proteomic determinations and dynamic microscopy of live cells led to conflicting conclusions. Recent insights into the existence of a variety of membrane contact sites (MCS) may help reconcile the seemingly disparate views. Specifically, earlier results can be rationalized considering that the ER forms specialized MCS with nascent and maturing phagosomes, without undergoing fusion. The composition and function of documented ER-to-phagosome contact sites is described. In addition, we speculate about the possible existence of additional phagosomal contact sites, based on available knowledge of interactions between the ER and other endocytic compartments. The interaction between phagosomes and the ER has been the subject of debate. Earlier observations that led to the suggestion that the ER fuses with the phagosomal membrane can now be explained in the light of recent evidence that intimate contacts form between the two organelles.

Sensors and regulators of intracellular pH.

Protons dictate the charge and structure of macromolecules and are used as energy currency by eukaryotic cells. The unique function of individual organelles therefore depends on the establishment and stringent maintenance of a distinct pH. This, in turn, requires a means to sense the prevailing pH and to respond to deviations from the norm with effective mechanisms to transport, produce or consume proton equivalents. A dynamic, finely tuned balance between proton-extruding and proton-importing processes underlies pH homeostasis not only in the cytosol, but in other cellular compartments as well.

Resolution of macropinosomes, phagosomes and autolysosomes: Osmotically driven shrinkage enables tubulation and vesiculation.

Macropinosomes, phagosomes and autolysosomes are comparatively large, quasi-spherical organelles that play essential functions in immunity and homeostasis. These vacuolar organelles are relatively short-lived, promptly fragmenting into smaller structures. Vacuolar resolution is mediated by tubulation and vesiculation, processes orchestrated by protein complexes that are recruited to highly curved membranes. Importantly, the surface-to-volume ratios of the tubules and vesicles generated during the resolution process are considerably larger than that of the parental vacuole. Because membranes under high hydrostatic tension resist deformation, an active, concomitant loss of volume is required to sustain the resolution process and may even initiate tubulation and vesiculation. Despite its fundamental role in membrane remodeling, the mechanisms that account for organellar volume loss are poorly understood, but are likely to involve the export of solutes followed by osmotically obliged water. In this review, we describe the principles and possible mechanisms underlying the resolution of organelles, with particular attention paid to the osmolytes they contain and the pathways mediating their exit.

Role of phospholipids in endocytosis, phagocytosis, and macropinocytosis.

Endocytosis, phagocytosis, and macropinocytosis are fundamental processes that enable cells to sample their environment, eliminate pathogens and apoptotic bodies, and regulate the expression of surface components. While a great deal of effort has been devoted over many years to understanding the proteins involved in these processes, the important contribution of phospholipids has only recently been appreciated. This review is an attempt to collate and analyze the rapidly emerging evidence documenting the role of phospholipids in clathrin-mediated endocytosis, phagocytosis, and macropinocytosis. A primer on phospholipid biosynthesis, catabolism, subcellular distribution, and transport is presented initially, for reference, together with general considerations of the effects of phospholipids on membrane curvature and charge. This is followed by a detailed analysis of the critical functions of phospholipids in the internalization processes and in the maturation of the resulting vesicles and vacuoles as they progress along the endo-lysosomal pathway.

The Role of Membrane Surface Charge in Phagocytosis.

The formation and maturation of phagosomes are accompanied by acute changes in lipid metabolism. Phosphoinositides, in particular, undergo extensive modification as part of the signaling sequence that drives cytoskeletal and membrane remodeling. Because the phosphoinositides provide much of the anionic charge of the cytosolic leaflet of the plasmalemma and phagosomal membrane, the metabolic changes associated with signaling result in marked changes of the surface charge. Here we summarize the pathways involved in lipid remodeling during phagocytosis, the resultant alterations in the surface charge of the nascent and maturing phagosomes, and the consequent effects on the association of proteins attached to the membrane by electrostatic means.

The life cycle of phagosomes: formation, maturation, and resolution.

Phagocytosis, the regulated uptake of large particles (>0.5 µm in diameter), is essential for tissue homeostasis and is also an early, critical component of the innate immune response. Phagocytosis can be conceptually divided into three stages: phagosome, formation, maturation, and resolution. Each of these involves multiple reactions that require exquisite spatial and temporal orchestration. The molecular events underlying these stages are being unraveled and the current state of knowledge is briefly summarized in this article.

Salmonella exploits host Rho GTPase signalling pathways through the phosphatase activity of SopB.

Salmonella uses Type 3 secretion systems (T3SSs) to deliver virulence factors, called effectors, into host cells during infection. The T3SS effectors promote invasion into host cells and the generation of a replicative niche. SopB is a T3SS effector that plays an important role in Salmonella pathogenesis through its lipid phosphatase activity. Here, we show that SopB mediates the recruitment of Rho GTPases (RhoB, RhoD, RhoH, and RhoJ) to bacterial invasion sites. RhoJ contributes to Salmonella invasion, and RhoB and RhoH play an important role in Akt activation. R-Ras1 also contributes to SopB-dependent Akt activation by promoting the localised production of PI(3,4)P2 /PI(3,4,5)P3 . Our studies reveal new signalling factors involved in SopB-dependent Salmonella infection.

In vivo requirement for Atg5 in antigen presentation by dendritic cells.

Autophagy is known to be important in presentation of cytosolic antigens on MHC class II (MHC II). However, the role of autophagic process in antigen presentation in vivo is unclear. Mice with dendritic cell (DC)-conditional deletion in Atg5, a key autophagy gene, showed impaired CD4(+) T cell priming after herpes simplex virus infection and succumbed to rapid disease. The most pronounced defect of Atg5(-/-) DCs was the processing and presentation of phagocytosed antigens containing Toll-like receptor stimuli for MHC class II. In contrast, cross-presentation of peptides on MHC I was intact in the absence of Atg5. Although induction of metabolic autophagy did not enhance MHC II presentation, autophagic machinery was required for optimal phagosome-to-lysosome fusion and subsequent processing of antigen for MHC II loading. Thus, our study revealed that DCs utilize autophagic machinery to optimally process and present extracellular microbial antigens for MHC II presentation.

Structure of LIMP-2 provides functional insights with implications for SR-BI and CD36.

Members of the CD36 superfamily of scavenger receptor proteins are important regulators of lipid metabolism and innate immunity. They recognize normal and modified lipoproteins, as well as pathogen-associated molecular patterns. The family consists of three members: SR-BI (which delivers cholesterol to the liver and steroidogenic organs and is a co-receptor for hepatitis C virus), LIMP-2/LGP85 (which mediates lysosomal delivery of ß-glucocerebrosidase and serves as a receptor for enterovirus 71 and coxsackieviruses) and CD36 (a fatty-acid transporter and receptor for phagocytosis of effete cells and Plasmodium-infected erythrocytes). Notably, CD36 is also a receptor for modified lipoproteins and ß-amyloid, and has been implicated in the pathogenesis of atherosclerosis and of Alzheimer's disease. Despite their prominent roles in health and disease, understanding the function and abnormalities of the CD36 family members has been hampered by the paucity of information about their structure. Here we determine the crystal structure of LIMP-2 and infer, by homology modelling, the structure of SR-BI and CD36. LIMP-2 shows a helical bundle where ß-glucocerebrosidase binds, and where ligands are most likely to bind to SR-BI and CD36. Remarkably, the crystal structure also shows the existence of a large cavity that traverses the entire length of the molecule. Mutagenesis of SR-BI indicates that the cavity serves as a tunnel through which cholesterol(esters) are delivered from the bound lipoprotein to the outer leaflet of the plasma membrane. We provide evidence supporting a model whereby lipidic constituents of the ligands attached to the receptor surface are handed off to the membrane through the tunnel, accounting for the selective lipid transfer characteristic of SR-BI and CD36.

Dual loss of p110δ PI3-kinase and SKAP (KNSTRN) expression leads to combined immunodeficiency and multisystem syndromic features.

BACKGROUND: We previously reported a novel syndrome characterized by combined immunodeficiency associated with severe developmental defects-subsequently known as Roifman-Chitayat syndrome (RCS; OMIM 613328). Linkage analysis identified 2 disease-associated loci. OBJECTIVES: We sought to identify the genetic defect in these patients and characterize their immunologic cellular abnormalities. METHODS: Genetic, immunologic, protein, and cellular functional analyses were used to identify and characterize patient genetic deficiencies and aberrant patient cell behavior. RESULTS: Deleterious variants were found at both loci identified by linkage analysis: a homozygous stop codon in PI3-kinase p110δ (PIK3CD) and a homozygous frame shift mutation in SKAP (KNSTRN), both ablating protein expression. Patients with RCS display aberrant B-cell development, similar to p110δ-deficient mice, but also aberrant T-cell spreading, cell-cell interaction, and migration. Patients also display significant developmental abnormalities not seen in p110δ knockouts (eg, optic nerve atrophy and skeletal anomalies) that we ascribe to loss of SKAP. Aberrant SKAP expression can prolong anaphase and this may contribute to developmental defects. However, we also identified microtubule-associated protein 4 microtubule-binding protein as a novel SKAP-binding partner and show that it undergoes relocalization in patient T cells, with associated areas of aberrant microtubule hyperstabilization, likely contributing not only to the altered properties of RCS lymphoid cells but also to developmental defects. CONCLUSIONS: The complex RCS presentation, with combined developmental and immunologic defects, is associated with a combined deficiency of 2 genes products, PI3-kinase p110δ and SKAP, both of which appear to play a significant role in the disease.