SnapShot: Enveloped Virus Entry.

In order to initiate successful infection, viruses have to transmit and deliver their genome from one host cell or organism to another. To achieve this, enveloped viruses must first fuse their membrane with those of the target host cell. Here, we describe the sequence of events leading to the entry of representative enveloped viruses, highlighting the strategies they use to gain access to the host cell cytosol.

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.

Two-pore channels regulate endomembrane tension to enable remodeling and resolution of phagolysosomes.

Phagocytes promptly resolve ingested targets to replenish lysosomes and maintain their responsiveness. The resolution process requires that degradative hydrolases, solute transporters, and proteins involved in lipid traffic are delivered and made active in phagolysosomes. It also involves extensive membrane remodeling. We report that cation channels that localize to phagolysosomes were essential for resolution. Specifically, the conductance of Na+ by two-pore channels (TPCs) and the presence of a Na+ gradient between the phagolysosome lumen and the cytosol were critical for the controlled release of membrane tension that permits deformation of the limiting phagolysosome membrane. In turn, membrane deformation was a necessary step to efficiently transport the cholesterol extracted from cellular targets, permeabilizing them to hydrolases. These results place TPCs as regulators of endomembrane remodeling events that precede target degradation in cases when the target is bound by a cholesterol-containing membrane. The findings may help to explain lipid metabolism dysfunction and autophagic flux impairment reported in TPC KO mice and establish stepwise regulation to the resolution process that begins with lysis of the target.

The resolution of phagosomes.

Phagocytosis is a fundamental immunobiological process responsible for the removal of harmful particulates. While the number of phagocytic events achieved by a single phagocyte can be remarkable, exceeding hundreds per day, the same phagocytic cells are relatively long-lived. It should therefore be obvious that phagocytic meals must be resolved in order to maintain the responsiveness of the phagocyte and to avoid storage defects. In this article, we discuss the mechanisms involved in the resolution process, including solute transport pathways and membrane traffic. We describe how products liberated in phagolysosomes support phagocyte metabolism and the immune response. We also speculate on mechanisms involved in the redistribution of phagosomal metabolites back to circulation. Finally, we highlight the pathologies owed to impaired phagosome resolution, which range from storage disorders to neurodegenerative diseases.

Interleukin-1 mediated hyperinflammation in XIAP-deficiency is associated with defective autophagy.

Deficiency of X-linked Inhibitor of Apoptosis Protein (XIAP) is a rare genetic condition that can present with recurrent episodes of hemophagocytic lymphohistiocytosis (HLH), though the exact mechanisms leading to this hyperinflammatory disorder are unclear. Understanding its biology is critical to developing targeted therapies for this potentially fatal disease. Here we report on a novel multi-exonic intragenic duplication leading to XIAP deficiency with recurrent HLH that demonstrated complete response to interleukin (IL)-1b blockade. We further demonstrate using both primary patient cells and genetically modified THP-1 monocyte cell lines that, contrary to what has previously been shown in mouse cells, XIAP-deficient human macrophages do not produce excess IL-1b when stimulated under standard conditions. Instead, NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome-mediated hyperproduction of IL-1b is observed only when the XIAP-deficient cells are stimulated under autophagy-promoting conditions and this correlates with defective autophagic flux as measured by decreased accumulation of the early autophagy marker LC3-II. This work therefore highlights IL-1b blockade as a therapeutic option for patients with XIAP deficiency experiencing recurrent HLH and identifies a critical role for XIAP in promoting autophagy as a means of limiting IL-1b-mediated hyperinflammation during periods of cellular stress.

Solute Transport Controls Membrane Tension and Organellar Volume.

The regulation of cellular volume in response to osmotic change has largely been studied at the whole cell level. Such regulation occurs by the inhibition or activation of ionic and organic solute transport pathways at the cell surface and is coincident with remodelling of the plasma membrane. However, it is only in rare instances that osmotic insults are experienced by cells and tissues. By contrast, the relatively minute luminal volumes of membrane-bound organelles are constantly subject to shifts in their solute concentrations as exemplified in the endocytic pathway where these evolve alongside with maturation. In this review, we summarize recent evidence that suggests trafficking events are in fact orchestrated by the solute fluxes of organelles that briefly impose osmotic gradients. We first describe how hydrostatic pressure and the resultant tension on endomembranes can be readily dissipated by controlled solute efflux since water is obliged to exit. In such cases, the relief of tension on the limiting membrane of the organelle can promote its remodelling by coat proteins, ESCRT machinery, and motors. Second, and reciprocally, we propose that osmotic gradients between organellar lumens and the cytosol may persist or be created. Such gradients impose osmotic pressure and tension on the endomembrane that prevent its remodelling. The control of endomembrane tension is dysregulated in lysosomal storage disorders and can be usurped by pathogens in endolysosomes. Since trafficking and signaling pathways conceivably sense and respond to endomembrane tension, we anticipate that understanding how cells control organellar volumes and the movement of endocytic fluid in particular will be an exciting new area of research.

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.

The Rap1-cofilin-1 pathway coordinates actin reorganization and MTOC polarization at the B cell immune synapse.

B cells that bind antigens displayed on antigen-presenting cells (APCs) form an immune synapse, a polarized cellular structure that optimizes the dual functions of the B cell receptor (BCR), signal transduction and antigen internalization. Immune synapse formation involves polarization of the microtubule-organizing center (MTOC) towards the APC. We now show that BCR-induced MTOC polarization requires the Rap1 GTPase (which has two isoforms, Rap1a and Rap1b), an evolutionarily conserved regulator of cell polarity, as well as cofilin-1, an actin-severing protein that is regulated by Rap1. MTOC reorientation towards the antigen contact site correlated strongly with cofilin-1-dependent actin reorganization and cell spreading. We also show that BCR-induced MTOC polarization requires the dynein motor protein as well as IQGAP1, a scaffolding protein that can link the actin and microtubule cytoskeletons. At the periphery of the immune synapse, IQGAP1 associates closely with F-actin structures and with the microtubule plus-end-binding protein CLIP-170 (also known as CLIP1). Moreover, the accumulation of IQGAP1 at the antigen contact site depends on F-actin reorganization that is controlled by Rap1 and cofilin-1. Thus the Rap1-cofilin-1 pathway coordinates actin and microtubule organization at the immune synapse.

Applied stretch initiates directional invasion through the action of Rap1 GTPase as a tension sensor.

Although it is known that a stiffening of the stroma and the rearrangement of collagen fibers within the extracellular matrix facilitate the movement of tumor cells away from the primary lesion, the underlying mechanisms responsible are not fully understood. We now show that this invasion, which can be initiated by applying tensional loads to a three-dimensional collagen gel matrix in culture, is dependent on the Rap1 GTPases (Rap1a and Rap1b, referred to collectively as Rap1). Under these conditions Rap1 activity stimulates the formation of focal adhesion structures that align with the tensional axis as single tumor cells move into the matrix. These effects are mediated by the ability of Rap1 to induce the polarized polymerization and retrograde flow of actin, which stabilizes integrins and recruits vinculin to preformed adhesions, particularly those near the leading edge of invasive cells. Rap1 activity also contributes to the tension-induced collective invasive elongation of tumor cell clusters and it enhances tumor cell growth in vivo Thus, Rap1 mediates the effects of increased extracellular tension in multiple ways that are capable of contributing to tumor progression when dysregulated.

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.

Multistep Track Segmentation and Motion Classification for Transient Mobility Analysis.

Molecular interactions are often transient and might change within the window of observation, leading to changes in molecule movement. Therefore, accurate motion analysis often requires transient motion classification. Here we present an accurate and computationally efficient transient mobility analysis framework, termed "divide-and-conquer moment scaling spectrum" (DC-MSS). DC-MSS works in a multistep fashion: 1) it utilizes a local movement descriptor throughout a track to divide it into initial segments of putatively different motion classes; 2) it classifies these segments via moment scaling spectrum (MSS) analysis of molecule displacements; and 3) it uses the MSS analysis results to refine the track segmentation. This strategy uncouples the initial identification of motion switches from motion classification, allowing DC-MSS to circumvent the sensitivity-accuracy tradeoff of classic rolling window approaches for transient motion analysis, while at the same time harnessing the classification power of MSS analysis. Testing of DC-MSS demonstrates that it detects switches among free diffusion, confined diffusion, directed diffusion, and immobility with great sensitivity. To illustrate the utility of DC-MSS, we have applied it to single-particle tracks of the transmembrane protein CD44 on the surface of macrophages, revealing actin cortex-dependent transient mobility changes.

Phagocytosis: receptors, signal integration, and the cytoskeleton.

Phagocytosis is a remarkably complex and versatile process: it contributes to innate immunity through the ingestion and elimination of pathogens, while also being central to tissue homeostasis and remodeling by clearing effete cells. The ability of phagocytes to perform such diverse functions rests, in large part, on their vast repertoire of receptors. In this review, we address the various receptor types, their mobility in the plane of the membrane, and two modes of receptor crosstalk: priming and synergy. A major section is devoted to the actin cytoskeleton, which not only governs receptor mobility and clustering but also is instrumental in particle engulfment. Four stages of the actin remodeling process are identified and discussed: (i) the 'resting' stage that precedes receptor engagement, (ii) the disruption of the cortical actin prior to formation of the phagocytic cup, (iii) the actin polymerization that propels pseudopod extension, and (iv) the termination of polymerization and removal of preassembled actin that are required for focal delivery of endomembranes and phagosomal sealing. These topics are viewed in the larger context of the differentiation and polarization of the phagocytic cells.

(R)-PFI-2 is a potent and selective inhibitor of SETD7 methyltransferase activity in cells.

SET domain containing (lysine methyltransferase) 7 (SETD7) is implicated in multiple signaling and disease related pathways with a broad diversity of reported substrates. Here, we report the discovery of (R)-PFI-2-a first-in-class, potent (Ki (app) = 0.33 nM), selective, and cell-active inhibitor of the methyltransferase activity of human SETD7-and its 500-fold less active enantiomer, (S)-PFI-2. (R)-PFI-2 exhibits an unusual cofactor-dependent and substrate-competitive inhibitory mechanism by occupying the substrate peptide binding groove of SETD7, including the catalytic lysine-binding channel, and by making direct contact with the donor methyl group of the cofactor, S-adenosylmethionine. Chemoproteomics experiments using a biotinylated derivative of (R)-PFI-2 demonstrated dose-dependent competition for binding to endogenous SETD7 in MCF7 cells pretreated with (R)-PFI-2. In murine embryonic fibroblasts, (R)-PFI-2 treatment phenocopied the effects of Setd7 deficiency on Hippo pathway signaling, via modulation of the transcriptional coactivator Yes-associated protein (YAP) and regulation of YAP target genes. In confluent MCF7 cells, (R)-PFI-2 rapidly altered YAP localization, suggesting continuous and dynamic regulation of YAP by the methyltransferase activity of SETD7. These data establish (R)-PFI-2 and related compounds as a valuable tool-kit for the study of the diverse roles of SETD7 in cells and further validate protein methyltransferases as a druggable target class.

Target lysis by cholesterol extraction is a rate limiting step in the resolution of phagolysosomes.

The ongoing phagocytic activity of macrophages necessitates an extraordinary capacity to digest and resolve incoming material. While the initial steps leading to the formation of a terminal phagolysosome are well studied, much less is known about the later stages of this process, namely the degradation and resolution of the phagolysosomal contents. We report that the degradation of targets such as splenocytes and erythrocytes by phagolysosomes occurs in a stepwise fashion, requiring lysis of their plasmalemmal bilayer as an essential initial step. This is achieved by the direct extraction of cholesterol facilitated by Niemann-Pick protein type C2 (NPC2), which in turn hands off cholesterol to NPC1 for export from the phagolysosome. The removal of cholesterol ulimately destabilizes and permeabilizes the membrane of the phagocytic target, allowing access of hydrolases to its internal compartments. In contrast, we found that saposins, which activate the hydrolysis of sphingolipids, are required for lysosomal tubulation, yet are dispensable for the resolution of targets by macrophages. The extraction of cholesterol by NPC2 is therefore envisaged as rate-limiting in the clearance of membrane-bound targets such as apoptotic cells. Selective cholesterol removal appears to be a primary mechanism that enables professional phagocytes to distinguish the target membrane from the phagolysosomal membrane and may be conserved in the resolution of autolysosomes.

Redistribution of the glycocalyx exposes phagocytic determinants on apoptotic cells.

Phagocytes remove dead and dying cells by engaging "eat-me" ligands such as phosphatidylserine (PtdSer) on the surface of apoptotic targets. However, PtdSer is obscured by the bulky exofacial glycocalyx, which also exposes ligands that activate "don't-eat-me" receptors such as Siglecs. Clearly, unshielding the juxtamembrane "eat-me" ligands is required for the successful engulfment of apoptotic cells, but the mechanisms underlying this process have not been described. Using human and murine cells, we find that apoptosis-induced retraction and weakening of the cytoskeleton that anchors transmembrane proteins cause an inhomogeneous redistribution of the glycocalyx: actin-depleted blebs emerge, lacking the glycocalyx, while the rest of the apoptotic cell body retains sufficient actin to tether the glycocalyx in place. Thus, apoptotic blebs can be engaged by phagocytes and are targeted for engulfment. Therefore, in cells with an elaborate glycocalyx, such as mucinous cancer cells, this "don't-come-close-to-me" barrier must be removed to enable clearance by phagocytosis.