Cameron C. Scott

LAMP proteins are required for fusion of lysosomes with phagosomes

Lysosome‐associated membrane proteins 1 and 2 (LAMP‐1 and LAMP‐2) are delivered to phagosomes during the maturation process. We used cells from LAMP‐deficient mice to analyze the role of these proteins in phagosome maturation. Macrophages from LAMP‐1‐ or LAMP‐2‐deficient mice displayed normal fusion of lysosomes with phagosomes. Because ablation of both the lamp‐1 and lamp‐2 genes yields an embryonic‐lethal phenotype, we were unable to study macrophages from double knockouts. Instead, we reconstituted phagocytosis in murine embryonic fibroblasts (MEFs) by transfection of FcγIIA receptors. Phagosomes formed by FcγIIA‐transfected MEFs obtained from LAMP‐1‐ or LAMP‐2‐ deficient mice acquired lysosomal markers. Remarkably, although FcγIIA‐transfected MEFs from double‐deficient mice ingested particles normally, phagosomal maturation was arrested. LAMP‐1 and LAMP‐2 double‐deficient phagosomes acquired Rab5 and accumulated phosphatidylinositol 3‐phosphate, but failed to recruit Rab7 and did not fuse with lysosomes. We attribute the deficiency to impaired organellar motility along microtubules. Time‐lapse cinematography revealed that late endosomes/lysosomes as well as phagosomes lacking LAMP‐1 and LAMP‐2 had reduced ability to move toward the microtubule‐organizing center, likely precluding their interaction with each other.

Amiloride inhibits macropinocytosis by lowering submembranous pH and preventing Rac1 and Cdc42 signaling.

Inhibitors of Na+/H+ exchange proteins block macropinocytosis by lowering the pH near the plasma membrane, which in turn inhibits actin remodeling by Rho family GTPases.

Phosphatidylinositol-4,5-bisphosphate hydrolysis directs actin remodeling during phagocytosis

The Rho GTPases play a critical role in initiating actin polymerization during phagocytosis. In contrast, the factors directing the disassembly of F-actin required for fission of the phagocytic vacuole are ill defined. We used fluorescent chimeric proteins to monitor the dynamics of association of actin and active Cdc42 and Rac1 with the forming phagosome. Although actin was found to disappear from the base of the forming phagosome before sealing was complete, Rac1/Cdc42 activity persisted, suggesting that termination of GTPase activity is not the main determinant of actin disassembly. Furthermore, fully internalized phagosomes engineered to associate constitutively with active Rac1 showed little associated F-actin. The disappearance of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) from the phagosomal membrane closely paralleled the course of actin disassembly. Furthermore, inhibition of PI(4,5)P2 hydrolysis or increased PI(4,5)P2 generation by overexpression of phosphatidylinositol phosphate kinase I prevented the actin disassembly necessary for the completion of phagocytosis. These observations suggest that hydrolysis of PI(4,5)P2 dictates the remodeling of actin necessary for completion of phagocytosis.

Phagosome Maturation: A Few Bugs in the System

Cells of the innate immune system ingest and destroy invading microorganisms by initially engulfing them into a specialized vacuole, known as the phagosome. The membrane of the forming phagosome is similar to the plasmalemma and its contents resemble the extracellular milieu. As such, the nascent phagosome is not competent to kill and eliminate the ingested microorganisms. However, shortly after sealing, the phagosome undergoes a series of rapid and extensive changes in its composition, the result of a sophisticated sequence of membrane fusion and fission reactions. Understanding the molecular basis of these events is of particular importance, since they are often the target of disruption by intracellular parasites such as Mycobacterium, Salmonella and Legionella. The objective of this review is to summarize the current knowledge of the molecular mechanisms underlying phagosomal maturation and its subversion by parasitic microorganisms.

Endosome maturation, transport and functions.

Efficient sorting of the material internalized by endocytosis is essential for key cellular functions and represents a, if not the, major trafficking pathway in mammalian cells. Incoming material - solutes, receptors and cargos, lipids and even pathogenic agents - are routed to various destinations within mammalian cells at two major sorting stations: the early and late endosome. The early endosome receives all manner of incoming material from the plasma membrane, as well as from the Golgi, and serves as an initial sorting nexus routing molecules back to the cell surface through recycling endosomes, to the trans-Golgi network by retrograde transport, or on to the late endosome/lysosome. The early endosome also regulates cell signaling, through the downregulation of internalized receptors, which are packaged into intralumenal vesicles that arise from inward invaginations of the limiting membrane. These multivesicular regions detach or mature from early endosomes and become free endocytic carrier vesicle/multivesicular body, which transports cargoes to late endosomes. The late endosome provides a central hub for incoming traffic from the endocytic, biosynthetic and autophagic pathways and outgoing traffic to the lysosomes, the Golgi complex or the plasma membrane. They also function as a key sensing/signaling platform that inform the cell about the nutrient situation. Herein we summarize the current understanding of the organization and functions of the endocytic pathway, differences across species, and the process of endosome maturation.

Ion flux and the function of endosomes and lysosomes: pH is just the start The flux of ions across endosomal membranes influences endosome function not only through regulation of the luminal pH

The ionic nature of endosomes varies considerably in character along the endocytic pathway. Counter‐ion flux across the limiting membrane of endosomes has long been considered essential for full acidification and normal endosome/lysosomal function. The proximal functions of luminal ions, however, have been difficult to assess. The recent development of transgenic mice carrying mutations in the intracellular chloride channels (ClCs) has provided a tool to uncouple Cl− influx from endosomal acidification. Intriguingly, many of the defects of the endo‐lysomal system attributed to aberrant pH persist in the Cl−‐deficient mice implying a direct regulatory role for Cl− influx in endosome function. These observations may begin to explain the abundance of endosomal ion transporters, including ClCs, sodium‐proton exchangers, two‐pore channels and mucolipins, that have been localized to endo‐lysosomes, and the extensive changes in luminal ion composition therein. In this review, we summarize what is known regarding the mediators of endosomal ion flux, and discuss the implications of changing ionic content on endo‐lysosomal function.

Single-cell analysis of population context advances RNAi screening at multiple levels

Isogenic cells in culture show strong variability, which arises from dynamic adaptations to the microenvironment of individual cells. Here we study the influence of the cell population context, which determines a single cells microenvironment, in image‐based RNAi screens. We developed a comprehensive computational approach that employs Bayesian and multivariate methods at the single‐cell level. We applied these methods to 45 RNA interference screens of various sizes, including 7 druggable genome and 2 genome‐wide screens, analysing 17 different mammalian virus infections and four related cell physiological processes. Analysing cell‐based screens at this depth reveals widespread RNAi‐induced changes in the population context of individual cells leading to indirect RNAi effects, as well as perturbations of cell‐to‐cell variability regulators. We find that accounting for indirect effects improves the consistency between siRNAs targeted against the same gene, and between replicate RNAi screens performed in different cell lines, in different labs, and with different siRNA libraries. In an era where large‐scale RNAi screens are increasingly performed to reach a systems‐level understanding of cellular processes, we show that this is often improved by analyses that account for and incorporate the single‐cell microenvironment.

Acquisition of Hrs, an Essential Component of Phagosomal Maturation, Is Impaired by Mycobacteria

ABSTRACT Pathogenic mycobacteria survive within macrophages by precluding the fusion of phagosomes with late endosomes or lysosomes. Because the molecular determinants of normal phagolysosome formation are poorly understood, the sites targeted by mycobacteria remain unidentified. We found that Hrs, an adaptor molecule involved in protein sorting, associates with phagosomes prior to their fusion with late endosomes or lysosomes. Recruitment of Hrs required the interaction of its FYVE domain with phagosomal phosphatidylinositol 3-phosphate, but two other attachment sites were additionally involved. Depletion of Hrs by use of small interfering RNA impaired phagosomal maturation, preventing the acquisition of lysobisphosphatidic acid and reducing luminal acidification. As a result, the maturation of phagosomes formed in Hrs-depleted cells was arrested at an early stage, characterized by the acquisition and retention of sorting endosomal markers. This phenotype is strikingly similar to that reported to occur in phagosomes of cells infected by mycobacteria. We therefore tested whether Hrs is recruited to phagosomes containing mycobacteria. Hrs associated readily with phagosomes containing inert particles but poorly with mycobacterial phagosomes. Moreover, Hrs was found more frequently in phagosomes containing avirulent Mycobacterium smegmatis than in phagosomes with the more virulent Mycobacterium marinum. These findings suggest that the inability to recruit Hrs contributes to the arrest of phagosomal maturation induced by pathogenic mycobacteria.

In Vitro Budding of Intralumenal Vesicles into Late Endosomes Is Regulated by Alix and Tsg101

Endosomes along the degradation pathway leading to lysosomes accumulate membranes in their lumen and thus exhibit a characteristic multivesicular appearance. These lumenal membranes typically incorporate down-regulated EGF receptor destined for degradation, but the mechanisms that control their formation remain poorly characterized. Here, we describe a novel quantitative biochemical assay that reconstitutes the formation of lumenal vesicles within late endosomes in vitro. Vesicle budding into the endosome lumen was time-, temperature-, pH-, and energy-dependent and required cytosolic factors and endosome membrane components. Our light and electron microscopy analysis showed that the compartment supporting the budding process was accessible to endocytosed bulk tracers and EGF receptor. We also found that the EGF receptor became protected against trypsin in our assay, indicating that it was sorted into the intraendosomal vesicles that were formed in vitro. Our data show that the formation of intralumenal vesicles is ESCRT-dependent, because the process was inhibited by the K173Q dominant negative mutant of hVps4. Moreover, we find that the ESCRT-I subunit Tsg101 and its partner Alix control intralumenal vesicle formation, by acting as positive and negative regulators, respectively. We conclude that budding of the limiting membrane toward the late endosome lumen, which leads to the formation of intraendosomal vesicles, is controlled by the positive and negative functions of Tsg101 and Alix, respectively.

Phosphoinositide Involvement in Phagocytosis and Phagosome Maturation

Cells of the innate immune system engulf invading microorganisms into plasma membrane-derived vacuoles called phagosomes. Newly formed phagosomes gradually acquire microbicidal properties by a maturation process which involves sequential and coordinated rounds of fusion with endomembranes and concomitant fission. Some pathogens interfere with this maturation sequence and thereby evade killing by the immune cells, managing to survive intracellularly as parasites. Phosphoinositides seem to be intimately involved in the processes of phagosome formation and maturation, and initial observations suggest that the ability of some microorganisms to survive intracellularly is associated with alterations in phosphoinositide metabolism. This chapter presents a brief overview of phosphoinositides in cells of the immune system, their metabolism in the context of phagocytosis and phagosome maturation and their possible derangements during infectious pathogenosis.