Antigen-presenting cells contain a specialized late endocytic compartment, MIIC (major histocompatibility complex [MHC] class II-enriched compartment), that harbors newly synthesized MHC class II molecules in transit to the plasma membrane. MIICs have a limiting membrane enclosing characteristic internal membrane vesicles. Both the limiting membrane and the internal vesicles contain MHC class II. In this study on B lymphoblastoid cells, we demonstrate by immunoelectron microscopy that the limiting membrane of MIICs can fuse directly with the plasma membrane, resulting in release from the cells of internal MHC class II-containing vesicles. These secreted vesicles, named exosomes, were isolated from the cell culture media by differential centrifugation followed by flotation on sucrose density gradients. The overall surface protein composition of exosomes differed significantly from that of the plasma membrane. Exosome-bound MHC class II was in a compact, peptide-bound conformation. Metabolically labeled MHC class II was released into the extracellular medium with relatively slow kinetics, 10 +/- 4% in 24 h, indicating that direct fusion of MIICs with the plasma membrane is not the major pathway by which MHC class II reaches the plasma membrane. Exosomes derived from both human and murine B lymphocytes induced antigen-specific MHC class II-restricted T cell responses. These data suggest a role for exosomes in antigen presentation in vivo.

https://rupress.org/jem/article-pdf/183/3/1161/1107814/1161.pdf

B lymphocytes secrete antigen-presenting vesicles.

https://www.cell.com/cell/pdf/0092-8674(92)90189-J.pdf

Sorting of GPI-anchored proteins to glycolipid-enriched membrane subdomains during transport to the apical cell surface

Rab GTPases control intracellular vesicle traffic by acting as regulatable switches that recruit effector molecules when in their GTP-bound form. The functional coupling between multiple Rab GTPases ensures the spatiotemporally coordinated regulation of vesicle traffic. Membrane trafficking between organelles by vesiculotubular carriers is fundamental to the existence of eukaryotic cells. Central in ensuring that cargoes are delivered to their correct destinations are the Rab GTPases, a large family of small GTPases that control membrane identity and vesicle budding, uncoating, motility and fusion through the recruitment of effector proteins, such as sorting adaptors, tethering factors, kinases, phosphatases and motors. Crosstalk between multiple Rab GTPases through shared effectors, or through effectors that recruit selective Rab activators, ensures the spatiotemporal regulation of vesicle traffic. Functional impairments of Rab pathways are associated with diseases, such as immunodeficiencies, cancer and neurological disorders.

Rab GTPases as coordinators of vesicle traffic.

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

We have previously demonstrated that human peripheral blood low density mononuclear cells cultured in granulocyte/macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4 develop into dendritic cells (DCs) that are extremely efficient in presenting soluble antigens to T cells. To identify the mechanisms responsible for efficient antigen capture, we studied the endocytic capacity of DCs using fluorescein isothiocyanate-dextran, horseradish peroxidase, and lucifer yellow. We found that DCs use two distinct mechanisms for antigen capture. The first is a high level of fluid phase uptake via macropinocytosis. In contrast to what has been found with other cell types, macropinocytosis in DCs is constitutive and allows continuous internalization of large volumes of fluid. The second mechanism of capture is mediated via the mannose receptor (MR), which is expressed at high levels on DCs. At low ligand concentrations, the MR can deliver a large number of ligands to the cell in successive rounds. Thus, while macropinocytosis endows DCs with a high capacity, nonsaturable mechanism for capture of any soluble antigen, the MR gives an extra capacity for antigen capture with some degree of selectivity for non-self molecules. In addition to their high endocytic capacity, DCs from GM-CSF + IL-4-dependent cultures are characterized by the presence of a large intracellular compartment that contains high levels of class II molecules, cathepsin D, and lysosomal-associated membrane protein-1, and is rapidly accessible to endocytic markers. We investigated whether the capacity of DCs to capture and process antigen could be modulated by exogenous stimuli. We found that DCs respond to tumor necrosis factor alpha, CD40 ligand, IL-1, and lipopolysaccharide with a coordinate series of changes that include downregulation of macropinocytosis and Fc receptors, disappearance of the class II compartment, and upregulation of adhesion and costimulatory molecules. These changes occur within 1-2 d and are irreversible, since neither pinocytosis nor the class II compartment are recovered when the maturation-inducing stimulus is removed. The specificity of the MR and the capacity to respond to inflammatory stimuli maximize the capacity of DCs to present infectious non-self antigens to T cells.

https://rupress.org/jem/article-pdf/182/2/389/1107245/389.pdf

Dendritic cells use macropinocytosis and the mannose receptor to concentrate macromolecules in the major histocompatibility complex class II compartment: downregulation by cytokines and bacterial products.

Rab5 and rab7 proteins belong to a superfamily of small molecular weight GTPases known to be associated with early and late endosomes, respectively. The rab5 protein plays an important regulatory role in early endocytosis, yet the function of rab7 protein was previously uncharacterized. This question was addressed by comparing the kinetics of vesicular stomatitis virus (VSV) G protein internalization in baby hamster kidney cells overexpressing wild-type or dominant negative mutant forms of the rab7 protein (rab7N125I and rab7T22N). Overexpression of wild-type rab7 protein allowed normal transport to late endosomes (mannose 6-phosphate receptor positive), while the rab7N125I mutant caused the VSV G protein to accumulate specifically in early (transferrin receptor positive) endosomes. Horseradish peroxidase and paramyxovirus SV5 hemagglutinin-neuraminidase (HN) were used in quantitative biochemical assays to further demonstrate that rab7 function was not required for early internalization events, but was crucial in downstream degradative events. The characteristic cleavage of SV5 HN in the late endosome distinguishes internalization from transport to later stages of the endocytic pathway. Mutant rab7N125I or rab7T22N proteins had no effect on the internalization of either horseradish peroxidase or SV5 HN protein. In contrast, the mutant proteins markedly inhibited the subsequent cleavage of the SV5 HN protein. Taken together, these data support a key role for rab7, downstream of rab5, in regulating membrane transport leading from early to late endosomes. We compare our findings to those obtained for the yeast homologues Ypt51p, Ypt52p, Ypt53p, and Ypt7p.

/pdf/rab-7-an-important-regulator-of-late-endocytic-membrane-7y60uqxogw.pdf

Rab 7: an important regulator of late endocytic membrane traffic.

Polarized sorting in epithelia

Of the approximately 70 human Rab GTPases, nearly three-quarters are involved in endocytic trafficking. Significant plasticity in endosomal membrane transport pathways is closely coupled to receptor signaling and Rab GTPase-regulated scaffolds. Here we review current literature pertaining to endocytic Rab GTPase localizations, functions, and coordination with regulatory proteins and effectors. The roles of Rab GTPases in (1) compartmentalization of the endocytic pathway into early, recycling, late, and lysosomal routes; (2) coordination of individual transport steps from vesicle budding to fusion; (3) effector interactomes; and (4) integration of GTPase and signaling cascades are discussed.

/pdf/rab-proteins-and-the-compartmentalization-of-the-endosomal-20jfr0qn4v.pdf

Rab Proteins and the Compartmentalization of the Endosomal System

Endocytosis is characterized by vesicular transport along numerous pathways. Common steps in each pathway include membrane budding to form vesicles, transport to a particular destination, and ultimately docking and fusion with the target membrane. Specificity of vesicle targeting is rendered in part by associated Rab GTPases. This review summarizes current knowledge about Rab GTPase functions in the endocytic pathways and provides insight into the regulation of Rab GTPase activity and mechanisms of Rab protein function. Functional assays have identified some Rab proteins that operate on individual pathways, but Rab proteins in several pathways remain controversial or have not been identified. Control of Rab GTPase activity is exerted through multiple levels of regulation. Significant new information pertaining to Rab protein function in regulating transport has emerged. Remarkably, Rab5 GTPase links budding, cytoskeletal transport and docking/fusion activities. This paradigm will most likely be generally applicable to other Rab GTPase pathways. Together with the cross-talk between different Rab proteins and their effectors, this may provide an integrated system for the general coordination of endocytic pathways to maintain organelle homeostasis.

Rab GTPases coordinate endocytosis.

The rab11 GTPase has been localized to both the Golgi and recycling endosomes; however, its Golgi-associated function has remained obscure. In this study, rab11 function in exocytic transport was a...

Angela Wandinger-Ness

Papers

Rab 7: an important regulator of late endocytic membrane traffic.

Polarized sorting in epithelia

Rab Proteins and the Compartmentalization of the Endosomal System

Rab GTPases coordinate endocytosis.

Rab11 is required for trans-golgi network-to-plasma membrane transport and a preferential target for GDP dissociation inhibitor.