Diverse and essential roles of mammalian vacuolar-type proton pump ATPase: toward the physiological understanding of inside acidic compartments

The vacuolar-type H(+)-ATPases (V-ATPase) are a family of multi-subunit ATP-dependent proton pumps involved in a wide variety of physiological processes. They are present in endomembrane organelles such as vacuoles, lysosomes, endosomes, the Golgi apparatus, chromaffin granules and coated vesicles, and acidify the luminal pH of these intracellular compartments. They also pump protons across the plasma membranes of specialized cells including osteoclasts and epithelial cells in kidneys and male genital tracts. Here, we briefly summarize our recent studies on the diverse and essential roles of mammalian V-ATPase.     

Lysosome-related organelles.

Lysosomes are membrane-bound cytoplasmic organelles involved in intracellular protein degradation. They contain an assortment of soluble acid-dependent hydrolases and a set of highly glycosylated integral membrane proteins. Most of the properties of lysosomes are shared with a group of cell type-specific compartments referred to as 'lysosome-related organelles', which include melanosomes, lytic granules, MHC class II compartments, platelet-dense granules, basophil granules, azurophil granules, and Drosophila pigment granules. In addition to lysosomal proteins, these organelles contain cell type-specific components that are responsible for their specialized functions. Abnormalities in both lysosomes and lysosome-related organelles have been observed in human genetic diseases such as the Chediak-Higashi and Hermansky-Pudlak syndromes, further demonstrating the close relationship between these organelles. Identification of genes mutated in these human diseases, as well as in mouse and Drosophila: pigmentation mutants, is beginning to shed light on the molecular machinery involved in the biogenesis of lysosomes and lysosome-related organelles.     

Inhibitory effect of intracellular lipid load on macroautophagy

Degradation of intracellular components via macroautophagy is a complex multi-step process that starts with the sequestration of cytosolic cargo in a de novo formed double-membrane vesicle or autophagosome. This compartment acquires the hydrolases required for cargo digestion by fusion with lysosomes. In contrast to the detailed molecular dissection of the components that participate in the induction, regulation and execution of the early steps in macroautophagy, through the engulfment of cargo in autophagosomes, the mechanisms involved in the lysosomal clearance of autophagosomes have been poorly characterized in mammals. One of the major limitations in this respect has been the fact that autophagosome-lysosome fusion in intact cells involves several independent steps, namely binding of the molecular motors associated to the surface of the vesicles with the cytoskeletal network, directional vesicular trafficking and fusion between the two vesicular compartments. Furthermore, both lysosomes and autophagosomes are very dynamic organelles that can fuse with different vesicular structures involved in macroautophagy, but also along the endocytic and phagocytic pathways. To resolve these limitations and directly analyze the fusion step between autophagosomes and different compartments of the endocytic-lysosomal pathway, we have recently developed an in vitro fusion assay with autophagosomes, lysosomes and endosomes isolated from cells or tissues. Fluorescent labeling of these compartments allows for the tracking of fusion events by fluorescence microscopy or by fluorescence activated cell sorting (FACS). Labeling of either membrane proteins on the surface of the organelles or dye-loading of the vesicles permits the monitoring of hemi-membrane fusion and complete vesicular fusion (cargo mixing).     

Vacuolar H+ Pumping ATPases in Luminal Acidic Organelles and Extracellular Compartments: Common Rotational Mechanism and Diverse Physiological Roles

Cytoplasmic organelles with an acidic luminal pH include vacuoles, coated vesicles, lysosomes, the Golgi apparatus, and synaptic vesicles. Acidic compartments are also known outside specialized cells such as osteoclasts. The unique acidic pH is formed by V-ATPase (Vacuolar type ATPase), other ion transporters, and the buffering action of proteins inside the organelles. V-ATPase hydrolyzes ATP and transports protons inside an organelle or extracellular compartment. We have summarized recent progress on mouse V-ATPases and their varying localizations together with their mechanism emphasizing similarities with F-type ATPases.     

Four Na+/H+ exchanger isoforms are distributed to Golgi and post-Golgi compartments and are involved in organelle pH regulation

Four isoforms of the Na+/H+ exchanger (NHE6-NHE9) are distributed to intracellular compartments in human cells. They are localized to Golgi and post-Golgi endocytic compartments as follows: mid- to trans-Golgi, NHE8; trans-Golgi network, NHE7; early recycling endosomes, NHE6; and late recycling endosomes, NHE9. No significant localization of these NHEs was observed in lysosomes. The distribution of these NHEs is not discrete in the cells, and there is partial overlap with other isoforms, suggesting that the intracellular localization of the NHEs is established by the balance of transport in and out of the post-Golgi compartments as the dynamic membrane trafficking. The overexpression of NHE isoforms increased the luminal pH of the compartments in which the protein resided from the mildly acidic pH to the cytosolic pH, suggesting that their in vivo function is to regulate the pH and monovalent cation concentration in these organelles. We propose that the specific NHE isoforms contribute to the maintenance of the unique acidic pH values of the Golgi and post-Golgi compartments in the cell.     

Accumulation of sialic acid in endocytic compartments interferes with the formation of mature lysosomes. Impaired proteolytic processing of cathepsin B in fibroblasts of patients with lysosomal sialic acid storage disease.

The impact of an altered endocytic environment on the biogenesis of lysosomes was studied in fibroblasts of patients suffering from sialic acid storage disease (SASD). This inherited disorder is characterized by the accumulation of acidic monosaccharides in lysosomal compartments and a concomitant decrease of their buoyant density. We demonstrate that C-terminal trimming of the lysosomal cysteine proteinase cathepsin B is inhibited in SASD fibroblasts. This late event in the biosynthesis of cathepsin B normally takes place in mature lysosomes, suggesting an impaired biogenesis of these organelles in SASD cells. When normal fibroblasts are loaded with sucrose, which inhibits transport from late endosomes to lysosomes, C-terminal cathepsin B processing is prevented to the same extent. Further characterization of the terminal endocytic compartments of SASD cells revealed properties usually associated with late endosomes/prelysosomes. In addition to a decreased buoyant density, SASD "lysosomes" show a reduced acidification capacity and appear smaller than their normal counterparts. We conclude that the accumulation of small non-diffusible compounds within endocytic compartments interferes with the formation of mature lysosomes and that the acidic environment of the latter organelles is a prerequisite for C-terminal processing of lysosomal hydrolases.     

Lysosome-related organelles: a view from immunity and pigmentation

Lysosomes are ubiquitous organelles that carry out essential household functions. Certain cell types, however, contain lysosome-related organelles with specialized functions. Their specialized functions are usually reflected by specific morphological and compositional features. A number of diseases that develop due to genetic mutations, pathogen exposure or cell transformation are characterized by dysfunctional lysosomes and/or lysosome-related organelles. In this review we highlight adaptations and malfunction of the endosomal/lysosomal system in normal and pathological situations with special focus on MHC class II compartments in antigen presenting cells and melanosomes in pigment cells.     

The Arf-like GTPase Arl8 Mediates Delivery of Endocytosed Macromolecules to Lysosomes in Caenorhabditis elegans

Late endocytic organelles including lysosomes are highly dynamic acidic organelles. Late endosomes and lysosomes directly fuse for content mixing to form hybrid organelles, from which lysosomes are reformed. It is not fully understood how these processes are regulated and maintained. Here we show that the Caenorhabditis elegans ARL-8 GTPase is localized primarily to lysosomes and involved in late endosome-lysosome fusion in the macrophage-like coelomocytes. Loss of arl-8 results in an increase in the number of late endosomal/lysosomal compartments, which are smaller than wild type. In arl-8 mutants, late endosomal compartments containing endocytosed macromolecules fail to fuse with lysosomal compartments enriched in the aspartic protease ASP-1. Furthermore, loss of arl-8 strongly suppresses formation of enlarged late endosome-lysosome hybrid organelles caused by mutations of cup-5, which is the orthologue of human mucolipin-1. These findings suggest that ARL-8 mediates delivery of endocytosed macromolecules to lysosomes by facilitating late endosome-lysosome fusion.     

Hyperacidification of cellubrevin endocytic compartments and defective endosomal recycling in cystic fibrosis respiratory epithelial cells.

The cystic fibrosis transmembrane conductance regulator (CFTR), which is aberrant in patients with cystic fibrosis, normally functions both as a chloride channel and as a pleiotropic regulator of other ion transporters. Here we show, by ratiometric imaging with luminally exposed pH-sensitive green fluorescent protein, that CFTR affects the pH of cellubrevin-labeled endosomal organelles resulting in hyperacidification of these compartments in cystic fibrosis lung epithelial cells. The excessive acidification of intracellular organelles was corrected with low concentrations of weak base. Studies with proton ATPase and sodium channel inhibitors showed that the increased acidification was dependent on proton pump activity and sodium transport. These observations implicate sodium efflux in the pH homeostasis of a subset of endocytic organelles and indicate that a dysfunctional CFTR in cystic fibrosis leads to organellar hyperacidification in lung epithelial cells because of a loss of CFTR inhibitory effects on sodium transport. Furthermore, recycling of transferrin receptor was altered in CFTR mutant cells, suggesting a previously unrecognized cellular defect in cystic fibrosis, which may have functional consequences for the receptors on the plasma membrane or within endosomal compartments.     

A toolbox for the immunomagnetic purification of signaling organelles

Homeostasis and the complex functions of organisms and cells rely on the sophisticated spatial and temporal regulation of signaling in different intra‐ and extracellular compartments and via different mediators. We here present a set of fast and easy to use protocols for the target‐specific immunomagnetic enrichment of receptor containing endosomes (receptosomes), plasma membranes, lysosomes and exosomes. Isolation of subcellular organelles and exosomes is prerequisite for and will advance their detailed subsequent biochemical and functional analysis. Sequential application of the different subprotocols allows isolation of morphological and functional intact organelles from one pool of cells. The enrichment is based on a selective labelling using receptor ligands or antibodies together with superparamagnetic microbeads followed by separation in a patented matrix‐free high‐gradient magnetic purification device. This unique magnetic chamber is based on a focusing system outside of the empty separation column, generating an up to 3 T high‐gradient magnetic field focused at the wall of the column.      

溶酶体途径在细胞自噬过程中的功能意义

溶酶体具有高度保守的异质性,是细胞自噬的关键细胞器。细胞质中的蛋白质和细胞器最终在溶酶体降解,故溶酶体在维持细胞结构和功能的平衡方面起着重要生理作用。通过自噬溶酶体途径,细胞可清除某些病原体并参与抗原呈递。细胞自噬与异噬经溶酶体密切联系。自噬过程中溶酶体功能障碍与某些疾病和衰老等相关。对细胞自噬的溶酶体途径及其功能意义作了概述。     

Endocytic delivery to lysosomes mediated by concurrent fusion and kissing events in living cells

In mammalian cells, macromolecules internalized by endocytosis are transported via endosomes for digestion by lysosomal acid hydrolases . The mechanism by which endosomes and lysosomes exchange content remains equivocal . However, lysosomes are reusable organelles because they remain accessible to endocytic enzyme replacement therapies and undergo content mixing with late endosomes . The maturation model, which proposes that endosomes mature into lysosomes , cannot explain these observations. Three mechanisms for content mixing have been proposed. The first is vesicular transport, best supported by a yeast cell-free assay . The second suggests that endosomes and lysosomes engage in repeated transient fusions termed "kiss-and-run" . The third is that endosomes and lysosomes fuse completely, yielding hybrid compartments from which lysosomes reform , termed "fusion-fission" . We utilized time-lapse confocal microscopy to test these hypotheses in living cells. Lysosomes were loaded with rhodamine dextran by pulse-chase, and subsequently late endosomes were loaded with Oregon green 488 dextran. Direct fusions were observed between endosomes and lysosomes, and one such event was captured by correlative electron microscopy. Fluorescence intensity analyses of endosomes that encountered lysosomes revealed a gradual accumulation of lysosomal content. Our data are compatible with a requirement for direct contact between organelles before content is exchanged.     

Lysosome changes in exponentially growing, synchronized and differentiating L-cell cultures

Using supravital fluorescent staining of lysosomes with Euchrysine 3R, the morphology of these organelles was studied in L cells examined from cultures being at different growth phases in the course of cell cycle and after adipocyte conversion of L cells due to the 60% bovine serum administration. As cells were passing from the lag-phase to the stationary phase of culture growth, the number of lysosomes was seen to increase. The appearance of large lysosomes is characteristic of cells in confluent and senescent cultures. During G1-period, lysosomes are often confined to the perinuclear area of L-cells, to be extended later during S and G2-periods. In dividing cells, these are commonly seen scattered throughout the cell periphery, around the mitotic spindle. In cells undergoing differentiation, within 4-7 days the seeding in the medium supplemented with 60% bovine serum, the number of lysosomes became augmented to be gradually reduced during the next 10-15 days, concomittantly with the accumulation of lipid drops in the cell cytoplasm. The activity of the Golgi complex and the intensity of autophagy are discussed as possible regulation points of lysosome formation during the cell growth.     

Cytoskeletal rearrangement during migration and activation of T lymphocytes.

T lymphocytes have an inherent ability to migrate along a chemotactic gradient, which enables them to exit the bloodstream and reach different tissues. Motile T cells display a polarized morphology with two distinct cell compartments: the leading edge and the uropod. During cell polarization, chemoattractant receptors, cell-adhesion molecules and cytoskeletal proteins are redistributed within these cellular compartments. The polarity of T lymphocytes changes during the establishment of antigen-specific cell-cell interactions, and this involves rearrangement of cytoskeletal proteins. This article discusses the regulation of these cytoskeletal rearrangements, and their role in the activation, migration and effector function of T cells.     

Mature and juvenile tissue models of regeneration in small fish species

The multitude of cells constituting organisms are fragile and easily damaged day by day. Therefore, maintenance of tissue morphology and function is fundamental for multicellular organisms to attain long life. For proper maintenance of tissue integrity, organisms must have mechanisms that detect the loss of tissue mass, activate the de novo production of cells, and organize those cells into functional tissues. However, these processes are only poorly understood. Here we give an overview of adult and juvenile tissue regeneration models in small fish species, such as zebrafish and medaka, and highlight recent advances at the molecular level. From these advances, we have come to realize that the epidermal and mesenchymal parts of the regenerating fish fin-that is, the wound epidermis and blastema, respectively-comprise heterogeneous populations of cells with different molecular identities that can be termed "compartments." These compartments and their mutual interactions are thought to play important roles in promoting the proper progression of tissue regeneration. We further describe the current understanding of these compartments and discuss the possible approaches to affording a better understanding of their roles and interactions during regeneration.     

Ultrastructure of intermediate stages in polarity reversal of thyroid epithelium in follicles in suspension culture

Separated thyroid follicles can be maintained in suspension culture in Coon's modified F-12 medium in 0.5% calf serum. If the serum concentration is raised to 5%, the follicles undergo inversion in 3-5 d. During the process of inversion, epithelial cells can be observed in intermediate stages of polarity reversal. The earliest ultrastructural changes recognized are surface changes in which tight junctions and microvilli appear at the lateral margins of the cell near the medium. Later, changes in the distribution of intracellular organelles occur. The Golgi apparatus shifts towards the end of the cell facing the medium, and lysosomes shift toward the luminal end of the cell. The right junctions and microvilli at the luminal end of the cell disappear sometime after the cytoplasmic organelles rearrange. The luminal colloid disappears only after the surface changes (loss of tight junctions and microvilli) occur at the luminal end of the cell. There appears to be some regulation of the order in which changes occur during polarity reversal of the thyroid epithelial cell.     

Intracellular traffic and fate of protein transduction domains HIV-1 TAT peptide and octaarginine. Implications for their utilization as drug delivery vectors

Transduction domains such as those derived from the HIV-TAT protein are candidate vectors for intracellular delivery of therapeutic macromolecules such as DNA and proteins. The mechanism by which they enter cells is controversial, and very little spatial information regarding the downstream fate of these peptides from the plasma membrane is available. We studied endocytic traffic of fluorescent conjugates of HIV-TAT peptide and octaarginine in human hematopoietic cell lines K562 (CD34-) and KG1a (CD34+) and substantiated our findings in epithelia cells. Both peptides were efficiently internalized to endocytic pathways of both hematopoietic cell lines; however, comparative analysis of the intracellular location of the peptides with endocytic probes revealed major differences in spatial organization of their endocytic organelles and their interaction with the peptides at low temperatures. Double labeling confocal microscopy demonstrates that prelabeled lysosomes of all the tested cells are accessible to internalized peptides within 60 min of endocytic uptake. Incubation of cells with nocodazole and cytochalasin D inhibited peptide traffic from early to late endosomal structures, demonstrating a cytoskeletal requirement for lysosomal delivery. Disruption of Golgi and endoplasmic reticulum dynamics was without effect on peptide localization, suggesting that endosomes and lysosomes rather than these organelles are the major acceptor compartments for these molecules.     

Selective perturbation of early endosome and/or trans-Golgi network pH but not lysosome pH by dose-dependent expression of influenza M2 protein

Many sorting stations along the biosynthetic and endocytic pathways are acidified, suggesting a role for pH regulation in protein traffic. However, the function of acidification in individual compartments has been difficult to examine because global pH perturbants affect all acidified organelles in the cell and also have numerous side effects. To circumvent this problem, we have developed a method to selectively perturb the pH of a subset of acidified compartments. We infected HeLa cells with a recombinant adenovirus encoding influenza virus M2 protein (an acid-activated ion channel that dissipates proton gradients across membranes) and measured the effects on various steps in protein transport. At low multiplicity of infection (m.o.i.), delivery of influenza hemagglutinin from the trans-Golgi network to the cell surface was blocked, but there was almost no effect on the rate of recycling of internalized transferrin. At higher m.o.i., transferrin recycling was inhibited, suggesting increased accumulation of M2 in endosomes. Interestingly, even at the higher m.o.i., M2 expression had no effect on lysosome morphology or on EGF degradation, suggesting that lysosomal pH was not compromised by M2 expression. However, delivery of newly synthesized cathepsin D to lysosomes was slowed in cells expressing active M2, suggesting that acidification of the TGN and endosomes is important for efficient delivery of lysosomal hydrolases. Fluorescence labeling using a pH-sensitive dye confirmed the reversible effect of M2 on the pH of a subset of acidified compartments in the cell. The ability to dissect the role of acidification in individual steps of a complex pathway should be useful for numerous other studies on protein processing and transport.     

The yeast lysosome-like vacuole: endpoint and crossroads

Fungal vacuoles are acidic organelles with degradative and storage capabilities that have many similarities to mammalian lysosomes and plant vacuoles. In the past several years, well-developed genetic, genomic, biochemical and cell biological tools in S. cerevisiae have provided fresh insights into vacuolar protein sorting, organelle acidification, ion homeostasis, autophagy, and stress-related functions of the vacuole, and these insights have often found parallels in mammalian lysosomes. This review provides a broad overview of the defining features and functions of S. cerevisiae vacuoles and compares these features to mammalian lysosomes. Recent research challenges the traditional view of vacuoles and lysosomes as simply the terminal compartment of biosynthetic and endocytic pathways (i.e. the "garbage dump" of the cell), and suggests instead that these compartments are unexpectedly dynamic and highly regulated.     

Toward unraveling membrane biogenesis in mammalian autophagy

Autophagy is a unique form of membrane trafficking, which delivers macromolecules and organelles from the cytoplasm to lysosomes for degradation. This fundamental and ubiquitous process in eukaryotic cells is mediated by the double-membrane-bound structures called autophagosomes, which transiently emerge in the cytoplasm. The recent remarkable explosion of knowledge of autophagy has revealed its multiple roles, especially in mammals; in addition to its basic role in turnover and reuse of cellular constituents, the process unexpectedly functions in elimination of invading bacteria and antigen presentation. Analysis of mammalian homologs of the autophagy-related (Atg) proteins identified in yeast has shed light on not only the common molecular mechanisms underlying autophagosome formation, but also specialized mechanisms that are related to the diverse functions and complex regulation of autophagy in higher organisms.