Phagocytosis of pathogens by macrophages initiates the innate immune response, which in turn orchestrates the adaptive response. In order to discriminate between infectious agents and self, macrophages have evolved a restricted number of phagocytic receptors, like the mannose receptor, that recognize conserved motifs on pathogens. Pathogens are also phagocytosed by complement receptors after relatively nonspecific opsonization with complement and by Fc receptors after specific opsonization with antibodies. All these receptors induce rearrangements in the actin cytoskeleton that lead to the internalization of the particle. However, important differences in the molecular mechanisms underlying phagocytosis by different receptors are now being appreciated. These include differences in the cytoskeletal elements that mediate ingestion, differences in vacuole maturation, and differences in inflammatory responses. Infectious agents, such as M. tuberculosis, Legionella pneumophila, and Salmonella typhimurium, enter macrophages via heterogeneous pathways and modify vacuolar maturation in a manner that favors their survival. Macrophages also play an important role in the recognition and clearance of apoptotic cells; a notable feature of this process is the absence of an inflammatory response.

Mechanisms of phagocytosis in macrophages

Endocytic mechanisms control the lipid and protein composition of the plasma membrane, thereby regulating how cells interact with their environments. Here, we review what is known about mammalian endocytic mechanisms, with focus on the cellular proteins that control these events. We discuss the well-studied clathrin-mediated endocytic mechanisms and dissect endocytic pathways that proceed independently of clathrin. These clathrin-independent pathways include the CLIC/GEEC endocytic pathway, arf6-dependent endocytosis, flotillin-dependent endocytosis, macropinocytosis, circular doral ruffles, phagocytosis, and trans-endocytosis. We also critically review the role of caveolae and caveolin1 in endocytosis. We highlight the roles of lipids, membrane curvature-modulating proteins, small G proteins, actin, and dynamin in endocytic pathways. We discuss the functional relevance of distinct endocytic pathways and emphasize the importance of studying these pathways to understand human disease processes.

Mechanisms of Endocytosis

Small GTP-binding proteins (G proteins) exist in eukaryotes from yeast to human and constitute a superfamily consisting of more than 100 members. This superfamily is structurally classified into at least five families: the Ras, Rho, Rab, Sar1/Arf, and Ran families. They regulate a wide variety of cell functions as biological timers (biotimers) that initiate and terminate specific cell functions and determine the periods of time for the continuation of the specific cell functions. They furthermore play key roles in not only temporal but also spatial determination of specific cell functions. The Ras family regulates gene expression, the Rho family regulates cytoskeletal reorganization and gene expression, the Rab and Sar1/Arf families regulate vesicle trafficking, and the Ran family regulates nucleocytoplasmic transport and microtubule organization. Many upstream regulators and downstream effectors of small G proteins have been isolated, and their modes of activation and action have gradually been elucidated. Cascades and cross-talks of small G proteins have also been clarified. In this review, functions of small G proteins and their modes of activation and action are described.

Small GTP-Binding Proteins

The Rho GTPases form a subgroup of the Ras superfamily of 20- to 30-kD GTP-binding proteins that have been shown to regulate a wide spectrum of cellular functions. These proteins are ubiquitously expressed across the species, from yeast to man. The mammalian Rho-like GTPases comprise at least 10 distinct proteins: RhoA, B, C, D, and E; Rac1 and 2; RacE; Cdc42Hs, and TC10. A comparison of the amino acid sequences of the Rho proteins from various species has revealed that they are conserved in primary structure and are 50%–55% homologous to each other. Like all members of the Ras superfamily, the Rho GTPases function as molecular switches, cycling between an inactive GDP-bound state and an active GTP-bound state. Until recently, members of the Rho subfamily were believed to be involved primarily in the regulation of cytoskeletal organization in response to extracellular growth factors. However, research from a number of laboratories over the past few years has revealed that the Rho GTPases play crucial roles in diverse cellular events such as membrane trafficking, transcriptional regulation, cell growth control, and development. Consequently, a major challenge has been to unravel the underlying molecular mechanisms by which the Rho GTPases mediate these various activities. Many targets of the Rho GTPases have now been identified and further characterization of some of them has provided major insights toward our understanding of Rho GTPase function at the molecular level. This review aims to summarize the general established principles about the Rho GTPases and some of the more recent exciting findings, hinting at novel, unanticipated functions of the Rho GTPases.

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Rho GTPases and signaling networks

Recent advances have uncovered the general protein apparatus used by all eukaryotes for intracellular transport, including secretion and endocytosis, and for triggered exocytosis of hormones and neurotransmitters. Membranes are shaped into vesicles by cytoplasmic coats which then dissociate upon GTP hydrolysis. Both vesicles and their acceptor membranes carry targeting proteins which interact specifically to initiate docking. A general apparatus then assembles at the docking site and fuses the vesicle with its target.

Mechanisms of intracellular protein transport

THE definition of cellular organelles has evolved over the last hundred years largely driven by morphologic observations, but more recently has been supplemented and complemented by functional and biochemical studies (Palade, 1975) . Thus, organelles are now identified both by their morphology and by the set ofcomponents that comprise them . Determining how organelle identity is established and maintained and how newly synthesized protein and membrane are sorted to different organelles are the central issues of organellogenesis . Essential to the many cellular functions that take place within the central vacuolar system (which consists ofthe ER, Golgi apparatus, secretory vesicles, endosomes, and lysosomes) is membrane traffic which mediates the exchange of components between different organelles . There are two critical characteristics of membrane traffic . First, only certain sets oforganelles exchange membrane and the patterns of this exchange define what are called membrane pathways . Second, multiple pathways intersect at specific points within the central vacuolar system . For specific components to "choose" the correct pathway at such points of crossing, mechanisms exist to impose choices on specific molecules . This process is called sorting . The characteristicsofeachorganelle within the central vacuolar system are likely to be intimately tied to the properties ofmembrane traffic . An imbalance in the magnitude ofmembrane input into and egress from an organelle would have profound effects on the size ofthat compartment . In addition, failures in sorting or aberrations in targeting pathways would be expected to profoundly affect the identity of individual organelles . Recently, the relationship between the control of membrane traffic and the maintenance of organelle structure has been investigated with the use ofa remarkable drug, brefeldin A (BFA).' In this review we will summarize recent findings with BFA and propose some speculative models concerning the mechanism and regulation ofmembrane traffic within the central vacuolar system .

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Brefeldin A: insights into the control of membrane traffic and organelle structure.

Endocytic recycling is coordinated with endocytic uptake to control the composition of the plasma membrane. Although much of our understanding of endocytic recycling has come from studies on the transferrin receptor, a protein internalized through clathrin-dependent endocytosis, increased interest in clathrin-independent endocytosis has led to the discovery of new endocytic recycling systems. Recent insights into the regulatory mechanisms that control endocytic recycling have focused on recycling through tubular carriers and the return to the cell surface of cargo that enters cells through clathrin-independent mechanisms. Recent work emphasizes the importance of regulated recycling in such diverse processes as cytokinesis, cell adhesion and morphogenesis, cell fusion, and learning and memory.

Pathways and mechanisms of endocytic recycling.

Microtubule-dependent retrograde transport of proteins into the ER in the presence of brefeldin A suggests an ER recycling pathway.

Members of the ADP-ribosylation factor (ARF) family of guanine-nucleotide-binding (G) proteins, including the ARF-like (ARL) proteins and SAR1, regulate membrane traffic and organelle structure by recruiting cargo-sorting coat proteins, modulating membrane lipid composition, and interacting with regulators of other G proteins. New roles of ARF and ARL proteins are emerging, including novel functions at the Golgi complex and in cilia formation. Their function is under tight spatial control, which is mediated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) that catalyse GTP exchange and hydrolysis, respectively. Important advances are being gained in our understanding of the functional networks that are formed not only by the GEFs and GAPs themselves but also by the inactive forms of the ARF proteins.

ARF family G proteins and their regulators: roles in membrane transport, development and disease

THE fungal metabolite brefeldin A is a powerful tool for investigating membrane traffic in eukaryotic cells1. The effects of brefeldin A on traffic are partly explained by its ability to prevent binding of cytosolic coat proteins onto membranes2–5. The non-clathrin coatomer complex6,7 binds reversibly to Golgi membranes in a GTP-controlled cycle8–10. The low-molecular-mass GTP-binding protein ADP-ribosylation factor (ARF), which also associates reversibly with Golgi membranes11,12, is required for coatomer binding13 and probably accounts for the control by guanine nucleotide of the coatomer–membrane interaction. Brefeldin A prevents the assembly of coatomer onto the membrane by inhibiting the GTP-dependent interaction of ARF with the Golgi membrane13, but the nature of this interaction has not been established. Here we demonstrate that Golgi membranes can specifically catalyse the exchange of GTP onto ARF and that brefeldin A prevents this function.

Julie G. Donaldson

Papers

Brefeldin A: insights into the control of membrane traffic and organelle structure.

Pathways and mechanisms of endocytic recycling.

Microtubule-dependent retrograde transport of proteins into the ER in the presence of brefeldin A suggests an ER recycling pathway.

ARF family G proteins and their regulators: roles in membrane transport, development and disease

Brefeldin A inhibits Golgi membrane-catalysed exchange of guanine nucleotide onto ARF protein