Sergio Grinstein

How nascent phagosomes mature to become phagolysosomes

Phagocytosis mediates the clearance of apoptotic bodies and also the elimination of microbial pathogens. The nascent phagocytic vacuole formed upon particle engulfment lacks microbicidal and degradative activity. These capabilities are acquired as the phagosome undergoes maturation; a progressive remodeling of its membrane and contents that culminates in the formation of phagolysosomes. Maturation entails orderly sequential fusion of the phagosomal vacuole with specialized endocytic and secretory compartments. Concomitantly, the phagosomal membrane undergoes both inward and outward vesiculation and tubulation followed by fission, thereby recycling components and maintaining its overall size. Here, we summarize what is known about the molecular machinery that governs this complex metamorphosis of phagosome maturation.

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.

Subversion of Phagocytosis for Pathogen Survival

Professional phagocytes, such as neutrophils and macrophages, effectively engulf and eliminate invading microorganisms. To survive this onslaught, pathogens have developed an astounding array of countermeasures aimed at avoiding detection, impairing signaling, or paralyzing the machinery that underlies phagocytosis. On the other hand, certain pathogens benefit from attaching to, entering, or traversing host cells to establish and spread infection. This is accomplished by yet other types of effectors that either co-opt or mimic host cell phagocytic components. Here, we briefly summarize the basic features of the phagocytic process and proceed to describe the types of strategies deployed by pathogens to either impair phagocytosis or to gain entry into cells where they can establish a safe survival niche.

Regulation from within: the cytoskeleton in transmembrane signaling.

There is mounting evidence that the plasma membrane is highly dynamic and organized in a complex manner. The cortical cytoskeleton is proving to be a particularly important regulator of plasmalemmal organization, modulating the mobility of proteins and lipids in the membrane, facilitating their segregation, and influencing their clustering. This organization plays a critical role in receptor-mediated signaling, especially in the case of immunoreceptors, which require lateral clustering for their activation. Based on recent developments, we discuss the structures and mechanisms whereby the cortical cytoskeleton regulates membrane dynamics and organization, and how the nonuniform distribution of immunoreceptors and their self-association may affect activation and signaling.

Phosphoinositides in phagocytosis and macropinocytosis.

Professional phagocytes provide immunoprotection and aid in the maintenance of tissue homeostasis. They perform these tasks by recognizing, engulfing and eliminating pathogens and endogenous cell debris. Here, we examine the paramount role played by phosphoinositides in phagocytosis and macropinocytosis, two major endocytic routes that mediate the uptake of particulate and fluid matter, respectively. We analyze accumulating literature describing the molecular mechanisms whereby phosphoinositides translate environmental cues into the complex, sophisticated responses that underlie the phagocytic and macropinocytic responses. In addition, we exemplify virulence strategies involving modulation of host cell phosphoinositide signaling that are employed by bacteria to undermine immunity. This article is part of a Special Issue entitled Phosphoinositides.

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.

The vacuolar-type H⁺-ATPase at a glance - more than a proton pump.

ABSTRACT The vacuolar H+-ATPase (V-ATPase) has long been appreciated to function as an electrogenic H+ pump. By altering the pH of intracellular compartments, the V-ATPase dictates enzyme activity, governs the dissociation of ligands from receptors and promotes the coupled transport of substrates across membranes, a role often aided by the generation of a transmembrane electrical potential. In tissues where the V-ATPase is expressed at the plasma membrane, it can serve to acidify the extracellular microenvironment. More recently, however, the V-ATPase has been implicated in a bewildering variety of additional roles that seem independent of its ability to translocate H+. These non-canonical functions, which include fusogenicity, cytoskeletal tethering and metabolic sensing, are described in this Cell Science at a Glance article and accompanying poster, together with a brief overview of the conventional functions of the V-ATPase.

Phosphatidic acid is required for the constitutive ruffling and macropinocytosis of phagocytes

Phagocytes spontaneously ruffle as they probe their environment and take up antigens. These cells are uniquely enriched in phosphatidic acid, which is necessary for ruffling and macropinocytosis.

Contrasting phagosome pH regulation and maturation in human M1 and M2 macrophages.

Phagosomal pH is regulated in diametrically opposed ways in M1 and M2 macrophages. M2 phagosomes acidify rapidly and monotonically, whereas M1 phagosomes undergo cyclic alkaline oscillations caused by proton consumption upon dismutation of superoxide, followed by activation of HV1 channels.

Actin Cytoskeleton Reorganization by Syk Regulates Fcγ Receptor Responsiveness by Increasing Its Lateral Mobility and Clustering

Clustering of immunoreceptors upon association with multivalent ligands triggers important responses including phagocytosis, secretion of cytokines, and production of immunoglobulins. We applied single-molecule detection and tracking methods to study the factors that control the mobility and clustering of phagocytic Fcγ receptors (FcγR). While the receptors exist as monomers in resting macrophages, two distinct populations were discernible based on their mobility: some diffuse by apparent free motion, while others are confined within submicron boundaries that reduce the frequency of spontaneous collisions. Src-family and Syk kinases determine the structure of the actin cytoskeleton, which is fenestrated, accounting for the heterogeneous diffusion of the FcγR. Stimulation of these kinases during phagocytosis induces reorganization of the cytoskeleton both locally and distally in a manner that alters receptor mobility and clustering, generating a feedback loop that facilitates engagement of FcγR at the tip of pseudopods, directing the progression of phagocytosis.