Chloroquine and ammonium chloride prevent terminal glycosylation of immunoglobulins in plasma cells without affecting secretion

The generation of an acidic pH in intracellular organelles is required for several membrane and protein recycling processes. For instance, the internalization of ligands by receptor-mediated endocytosis is followed by the development of an acidic pH inside endosomes; this allows dissociation of the ligand, which is then transported to the lysosomes, from the receptor, which is recycled to the cell surface. There is evidence that part of this recycling process involves the distal region of the Golgi complex, where terminal glycosylation occurs: when the plasma membrane transferrin receptor is desialylated by neuraminidase treatment, it acquires new sialic acid molecules after endocytosis and before cell-surface re-expression. Golgi membranes have been shown to contain a proton pump and the distal Golgi cisternae appear to have an acidic content. Here, we have studied the effects of chloroquine and ammonium chloride, which raise the pH of acidic intracellular compartments, on the processing and secretion of immunoglobulins by plasma cells. Sialic acid transfer to terminal galactose residues, a reaction known to occur in the distal Golgi shortly before secretion, is completely and rapidly inhibited in the presence of these drugs, without significant modification of the secretion rate. This effect is accompanied by a dilatation of the Golgi cisternae and is not rapidly reversible.     

Dendritic cell maturation triggers retrograde MHC class II transport from lysosomes to the plasma membrane

Central to the initiation of immune responses is recognition of peptide antigen by T lymphocytes. The cell biology of dendritic cells makes them ideally suited for the essential process of antigen presentation. Their life cycle includes several stages characterized by distinct functions and mechanisms of regulation. Immature dendritic cells synthesize large amounts of major histocompatibility complex class II molecules (MHC II), but the alpha beta-dimers are targeted to late endosomes and lysosomes (often referred to as MHC class II compartments) where they reside unproductively with internalized antigens. After exposure to microbial products or inflammatory mediators, endocytosis is downregulated, the expression of co-stimulatory molecules is enhanced, and newly formed immunogenic MHC II-peptide complexes are transported to the cell surface. That these MHC II molecules reach the surface is surprising, as the lysosomes comprise the terminal degradative compartment of the endocytic pathway from which exogenous components generally cannot be recovered intact. Here we have visualized this pathway in live dendritic cells by video microscopy, using cells expressing MHC II tagged with green fluorescent protein (GFP). We show that on stimulation, dendritic cells generate tubules from lysosomal compartments that go on to fuse directly with the plasma membrane.     

Targeting of cell-surface beta-amyloid precursor protein to lysosomes: alternative processing into amyloid-bearing fragments.

Progressive cerebral deposition of the amyloid beta-peptide is an early and invariant feature of Alzheimer's disease. The beta-peptide is released by proteolytic cleavages from the beta-amyloid precursor protein (beta APP), a membrane-spanning glycoprotein expressed in most mammalian cells. Normal secretion of beta APP involves a cleavage in the beta-peptide region, releasing the soluble extramembranous portion and retaining a 10K C-terminal fragment in the membrane. Because this secretory pathway precludes beta-amyloid formation, we searched for an alternative proteolytic processing pathway that can generate beta-peptide-bearing fragments from full-length beta APP. Incubation of living human endothelial cells with a beta APP antibody revealed reinternalization of mature beta APP from the cell surface and its targeting to endosomes/lysosomes. After cell-surface biotinylation, full-length biotinylated beta APP was recovered inside the cells. Purification of lysosomes directly demonstrated the presence of mature beta APP and an extensive array of beta-peptide-containing proteolytic products. Our results define a second processing pathway for beta APP and suggest that it may be responsible for generating amyloid-bearing fragments in Alzheimer's disease.     

Receptor-rich intracellular membrane vesicles transporting asialotransferrin and insulin in liver

A wide range of receptors are located at the blood sinusoidal aspect of the hepatocyte plasma membrane. Many circulating ligands that bind to receptors on the cell surfaces are interiorized along two pathways. Asialoglycoproteins are transferred from the plasma membrane to lysosomes and degraded, whereas immunoglobulin A and bile acids are transported across the hepatocyte interior and released into bile. Asialotransferrin type 3 (ref. 6) follows a further pathway termed diacytosis. After binding to the asialoglycoprotein receptor, asialotransferrin is endocytosed and then returned to blood with a proportion of its carbohydrate side chains resialylated. We now describe in liver the properties of intracellular asialotransferrin-enclosing vesicles (diacytosomes) and show that they differ from Golgi, lysosome and plasma membrane fractions. Furthermore, we show that the asialoglycoprotein binding sites are located on the cytoplasmic (outer) surface of diacytosomes.     

Exposure of an antigen of chromaffin granules on cell surface during exocytosis

The synthesis rate of the membrane proteins of the catecholamine-storing vesicles (chromaffin granules) of the adrenal medulla is lower than that of the secretory proteins of the contents. Based on these results we proposed that after exocytosis the membranes of chromaffin granules are retrieved and are re-used for several secretion cycles (see also ref. 4). This concept of re-use of granule membranes has been further strengthened by the finding that exogenous markers which are taken up by secretory cells during stimulation can be traced to the Golgi region and to immature secretory organelles. However, one basic question remains: are the membranes of secretory organelles specifically and completely removed from the plasma membrane and if so, how fast is this process? By using an antiserum against a membrane glycoprotein of chromaffin granules we have now obtained quantitative data which demonstrate that during exocytosis this antigen becomes exposed on the cell surface and disappears again to a large degree within 30 min.     

Preparation and cellular uptake of PLGA particles loaded with lamivudine

Poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles loaded with lamivudine and coated with bovine serum albumin (BSA) were prepared via a double emulsion method. The influences of experiments parameters such as volume of inner aqueous phase, concentration of organic phase and ultrasonication time on the particle size and drug entrapment efficiency were investigated, obtaining PLGA particles with a diameter of ~260 nm and drug entrapment efficiency of ~35%. The particles were observed by scanning electron microscopy and transmittance electron microscopy, showing a core-shell structure. BCA assay found that 58 mg BSA was present on/in 1 g LPB particles. The loaded lamivudine showed a burst release at beginning and sustained release until 24 h in physiological conditions. Low pH could accelerate the release of lamivudine from PLGA particles, making the PLGA particles potential intelligent intracellular drug carriers. The PLGA particles were readily internalized into the human liver cells within a short time and increased gradually with the prolongation of incubation time regardless of the loading of lamivudine. The particles either resided within lysosomes or transferred to cytoplasm, but could not enter into the cell nucleus. The cell viability was not significantly influenced in the presence of the particles regardless of lamivudine encapsulation, suggesting that this kind of particles may be a good candidate for the intracellular anti-hepatitis B drug delivery.     

Ebola virus entry requires the cholesterol transporter Niemann-Pick C1

Infections by the Ebola and Marburg filoviruses cause a rapidly fatal haemorrhagic fever in humans for which no approved antivirals are available. Filovirus entry is mediated by the viral spike glycoprotein (GP), which attaches viral particles to the cell surface, delivers them to endosomes and catalyses fusion between viral and endosomal membranes. Additional host factors in the endosomal compartment are probably required for viral membrane fusion; however, despite considerable efforts, these critical host factors have defied molecular identification. Here we describe a genome-wide haploid genetic screen in human cells to identify host factors required for Ebola virus entry. Our screen uncovered 67 mutations disrupting all six members of the homotypic fusion and vacuole protein-sorting (HOPS) multisubunit tethering complex, which is involved in the fusion of endosomes to lysosomes, and 39 independent mutations that disrupt the endo/lysosomal cholesterol transporter protein Niemann-Pick C1 (NPC1). Cells defective for the HOPS complex or NPC1 function, including primary fibroblasts derived from human Niemann-Pick type C1 disease patients, are resistant to infection by Ebola virus and Marburg virus, but remain fully susceptible to a suite of unrelated viruses. We show that membrane fusion mediated by filovirus glycoproteins and viral escape from the vesicular compartment require the NPC1 protein, independent of its known function in cholesterol transport. Our findings uncover unique features of the entry pathway used by filoviruses and indicate potential antiviral strategies to combat these deadly agents.     

A kinase-regulated PDZ-domain interaction controls endocytic sorting of the beta2-adrenergic receptor.

A fundamental question in cell biology is how membrane proteins are sorted in the endocytic pathway. The sorting of internalized beta2-adrenergic receptors between recycling endosomes and lysosomes is responsible for opposite effects on signal transduction and is regulated by physiological stimuli. Here we describe a mechanism that controls this sorting operation, which is mediated by a family of conserved protein-interaction modules called PDZ domains. The phosphoprotein EBP50 (for ezrinradixin-moesin(ERM)-binding phosphoprotein-50) binds to the cytoplasmic tail of the beta2-adrenergic receptor through a PDZ domain and to the cortical actin cytoskeleton through an ERM-binding domain. Disrupting the interaction of EBP50 with either domain or depolymerization of the actin cytoskeleton itself causes missorting of endocytosed beta2-adrenergic receptors but does not affect the recycling of transferrin receptors. A serine residue at position 411 in the tail of the beta2-adrenergic receptor is a substrate for phosphorylation by GRK-5 (for G-protein-coupled-receptor kinase-5) and is required for interaction with EBP50 and for proper recycling of the receptor. Our results identify a new role for PDZ-domain-mediated protein interactions and for the actin cytoskeleton in endocytic sorting, and suggest a mechanism by which GRK-mediated phosphorylation could regulate membrane trafficking of G-protein-coupled receptors after endocytosis.     

高尔基体蛋白功能研究进展

高尔基体蛋白是一类定位在高尔基体表面上的螺旋卷曲蛋白家族.目前研究最为广泛的有11种高尔基体蛋白,它们分别定位在高尔基体堆栈不同的部位上.其中有3种定位在高尔基体正面膜囊表面上,高尔基体的反面膜囊表面和高尔基体膜囊边缘表面各定位4种高尔基体蛋白,它们以各自肽链羧基端或以跨膜结构域或与小的GTPase结合锚定到高尔基体膜表面.目前它们比较明确的主要功能是参与维持高尔基体结构的稳定和胞质内的膜运输,还有很多重要的功能在持续地研究发掘中.该文综述了这11种高尔基体蛋白功能的最新研究进展,以期为研究人员在该领域中更深入地进行研究提供参考.     

Cell-surface labelling reveals no evidence for membrane assembly and disassembly during fibroblast locomotion.

As a cultured fibroblast moves, particles or pieces of debris attached to its surface move backwards with respect to both the cell and the substrate1,2, as also do concanavalin A (Con A) receptors in the membrane of the leading lamella3. One suggested explanation is that membrane components from the cytoplasm are assembled and introduced into the surface membrane of the moving cell close to its leading edge and flow backwards to a 'sink'; there the membrane is disassembled, and its components enter the cytoplasm and flow forward within the cell, to be re-introduced later into the surface membrane1,4. However, because cell-surface receptors can be redistributed as a result of cross-linking by external ligands5,6 it has been proposed that the backward flow of Con A receptors and particles may result from such a cross-linking rather than from a flow of intact cell membrane7,8. To investigate these alternatives, I have studied moving fibroblasts by means of a cell membrane label that does not induce the redistribution of its receptors. My results do not seem compatible with the membrane flow model.     

Bacillus subtilis expressing a haemolysin gene from Listeria monocytogenes can grow in mammalian cells

Intracellular parasites can be classified into those that reside within a host vacuole and those which grow directly in the host cytoplasm. Members of the latter group, which includes Rickettsia, Shigellae, Trypanosoma cruzi, and Listeria monocytogenes, possess haemolytic activity associated with the ability to enter the host cytoplasm. Therefore mutants of L. monocytogenes lacking a pore-forming haemolysin, listeriolysin O, do not escape from the endosomal compartment and consequently fail to become established in the cytoplasm. To examine the role of listeriolysin O, we cloned the structural gene for the L. monocytogenes haemolysin, hlyA, into an asporogenic mutant of Bacillus subtilis under the control of an IPTG-inducible promoter. After being internalized by the macrophage-like cell line J774, haemolytic B. subtilis disrupted the phagosomal membrane and grew rapidly within the macrophage cytoplasm. These results show that a single gene product is sufficient to convert a common soil bacterium into a parasite that can grow in the cytoplasm of a mammalian cell.     

Signal sequence directs localized secretion of bacterial surface proteins

All living cells require specific mechanisms that target proteins to the cell surface. In eukaryotes, the first part of this process involves recognition in the endoplasmic reticulum of amino-terminal signal sequences and translocation through Sec translocons, whereas subsequent targeting to different surface locations is promoted by internal sorting signals. In bacteria, N-terminal signal sequences promote translocation across the cytoplasmic membrane, which surrounds the entire cell, but some proteins are nevertheless secreted in one part of the cell by poorly understood mechanisms. Here we analyse localized secretion in the Gram-positive pathogen Streptococcus pyogenes, and show that the signal sequences of two surface proteins, M protein and protein F (PrtF), direct secretion to different subcellular regions. The signal sequence of M protein promotes secretion at the division septum, whereas that of PrtF preferentially promotes secretion at the old pole. Our work therefore shows that a signal sequence may contain information that directs the secretion of a protein to one subcellular region, in addition to its classical role in promoting secretion. This finding identifies a new level of complexity in protein translocation and emphasizes the potential of bacterial systems for the analysis of fundamental cell-biological problems.     

Retrograde transport of endocytosed Shiga toxin to the endoplasmic reticulum.

Shiga toxin and some other protein toxins that act on targets in the cytosol have previously been shown to enter the trans-Golgi network. Transport by this route may be necessary for translocation of the toxin to the cytosol and for intoxication, but it is not known whether the enzymatically active part of the toxins actually enters the cytosol from the trans-Golgi network. It has been suggested that such toxins are transported in a retrograde manner to the endoplasmic reticulum and that translocation occurs in this organelle, but retrograde transport of endocytosed material beyond the trans-Golgi network has never been demonstrated. Here we show that in butyric acid-treated A431 cells endocytosed Shiga toxin is not only transported to the trans-Golgi network, but also to all Golgi stacks, to the endoplasmic reticulum and to the nuclear envelope. Furthermore, butyric acid sensitizes the cells to Shiga toxin, which is consistent with the possibility that retrograde transport is required for translocation of the toxin to the cytosol.     

The type IV mucolipidosis-associated protein TRPML1 is an endolysosomal iron release channel

TRPML1 (mucolipin 1, also known as MCOLN1) is predicted to be an intracellular late endosomal and lysosomal ion channel protein that belongs to the mucolipin subfamily of transient receptor potential (TRP) proteins. Mutations in the human TRPML1 gene cause mucolipidosis type IV disease (ML4). ML4 patients have motor impairment, mental retardation, retinal degeneration and iron-deficiency anaemia. Because aberrant iron metabolism may cause neural and retinal degeneration, it may be a primary cause of ML4 phenotypes. In most mammalian cells, release of iron from endosomes and lysosomes after iron uptake by endocytosis of Fe(3+)-bound transferrin receptors, or after lysosomal degradation of ferritin-iron complexes and autophagic ingestion of iron-containing macromolecules, is the chief source of cellular iron. The divalent metal transporter protein DMT1 (also known as SLC11A2) is the only endosomal Fe(2+) transporter known at present and it is highly expressed in erythroid precursors. Genetic studies, however, suggest the existence of a DMT1-independent endosomal and lysosomal Fe(2+) transport protein. By measuring radiolabelled iron uptake, by monitoring the levels of cytosolic and intralysosomal iron and by directly patch-clamping the late endosomal and lysosomal membrane, here we show that TRPML1 functions as a Fe(2+) permeable channel in late endosomes and lysosomes. ML4 mutations are shown to impair the ability of TRPML1 to permeate Fe(2+) at varying degrees, which correlate well with the disease severity. A comparison of TRPML1(-/- )ML4 and control human skin fibroblasts showed a reduction in cytosolic Fe(2+) levels, an increase in intralysosomal Fe(2+) levels and an accumulation of lipofuscin-like molecules in TRPML1(-/-) cells. We propose that TRPML1 mediates a mechanism by which Fe(2+) is released from late endosomes and lysosomes. Our results indicate that impaired iron transport may contribute to both haematological and degenerative symptoms of ML4 patients.     

Magnetic particles in the liver: a probe for intracellular movement

Previous studies have used magnetic particles to estimate the viscosity of cell cytoplasm in vitro 1-4. Here we describe how magnetic Fe2O3 particles can be used to estimate non-invasively the motion of organelles in hepatic macrophages in intact animals. We report that when these particles are injected intravenously (i.v.), most are phagocytosed by hepatic macrophages (Fig. 1)5. When an external magnetic field is applied to the rabbit, these particles become magnetized and aligned. After removal of the field, the particles collectively produce a remanent magnetic field which can be measured at the body surface. This field decreases with time due to particle rotation (relaxation) 6,7. As the particles are contained in phagosomes or secondary lysosomes, we conclude that motions of these organelles are responsible for the particle rotation and relaxation.     

Rab5 is necessary for the biogenesis of the endolysosomal system in vivo

An outstanding question is how cells control the number and size of membrane organelles. The small GTPase Rab5 has been proposed to be a master regulator of endosome biogenesis. Here, to test this hypothesis, we developed a mathematical model of endosome dependency on Rab5 and validated it by titrating down all three Rab5 isoforms in adult mouse liver using state-of-the-art RNA interference technology. Unexpectedly, the endocytic system was resilient to depletion of Rab5 and collapsed only when Rab5 decreased to a critical level. Loss of Rab5 below this threshold caused a marked reduction in the number of early endosomes, late endosomes and lysosomes, associated with a block of low-density lipoprotein endocytosis. Loss of endosomes caused failure to deliver apical proteins to the bile canaliculi, suggesting a requirement for polarized cargo sorting. Our results demonstrate for the first time, to our knowledge, the role of Rab5 as an endosome organizer in vivo and reveal the resilience mechanisms of the endocytic system.     

Structural characterization of the molecular platform for type III secretion system assembly

Type III secretion systems (TTSSs) are multi-protein macromolecular 'machines' that have a central function in the virulence of many Gram-negative pathogens by directly mediating the secretion and translocation of bacterial proteins (termed effectors) into the cytoplasm of eukaryotic cells. Most of the 20 unique structural components constituting this secretion apparatus are highly conserved among animal and plant pathogens and are also evolutionarily related to proteins in the flagellar-specific export system. Recent electron microscopy experiments have revealed the gross 'needle-shaped' morphology of the TTSS, yet a detailed understanding of the structural characteristics and organization of these protein components within the bacterial membranes is lacking. Here we report the 1.8-A crystal structure of EscJ from enteropathogenic Escherichia coli (EPEC), a member of the YscJ/PrgK family whose oligomerization represents one of the earliest events in TTSS assembly. Crystal packing analysis and molecular modelling indicate that EscJ could form a large 24-subunit 'ring' superstructure with extensive grooves, ridges and electrostatic features. Electron microscopy, labelling and mass spectrometry studies on the orthologous Salmonella typhimurium PrgK within the context of the assembled TTSS support the stoichiometry, membrane association and surface accessibility of the modelled ring. We propose that the YscJ/PrgK protein family functions as an essential molecular platform for TTSS assembly.     

金纳米颗粒的摄入对细胞光学性质影响的数值仿真

采用时域有限差分法(FDTD)方法计算了细胞摄入金纳米颗粒后的消光光谱,并对金纳米颗粒在细胞中3种不同的分布做了比较:金纳米颗粒均匀分布细胞膜上;金纳米颗粒在细胞质内均匀分布,不存在于细胞核中;金纳米颗粒在整个细胞中均匀分布.分析结果表明,由于金纳米颗粒的等离子共振效应,细胞在可见光区域的散射截面产生增强,并且其消光光谱的峰形和消光峰的位置也发生相应的变化,这种变化依赖于金纳米颗粒在细胞内的分布情况.当金纳米颗粒在细胞膜上均匀分布的时候,细胞的消光光谱的峰值最大,而另外2种情况则形成较宽的消光峰.计算结果有助进一步理解纳米颗粒在细胞内的扩散或生长的过程.     

The condensing vacuole of exocrine cells is more acidic than the mature secretory vesicle

A number of intracellular, membrane-bound compartments in both the endocytic and exocytic pathways of eukaryotic cells have an acidic internal pH. In endocrine cells, the mature secretory vesicle has an acidic pH; secretory vesicles isolated from exocrine cells, however, appear to have a neutral pH. Recently we have used a newly developed immunocytochemical technique to map low-pH compartments in insulin-secreting islet cells with the electron microscope and find that during the maturation of the secretory vesicle there is a progressive acidification of these vesicles that begins as soon as the trans Golgi condensing vacuoles form. Now we have used this technique to examine two exocrine cells: the pancreatic acinar cell and the parotid serous cell. In both cell types, the trans Golgi condensing vacuoles are acidic and accumulate the low-pH probe to the same extent as condensing vacuoles of insulin-secreting islet cells. Unlike insulin-secreting cells, however, maturation of the granules is accompanied by a return of luminal pH to near neutrality. Therefore, although the pH of storage granules in exocrine and endocrine cells is different, the pH of the condensing vacuoles in both cells is acidic.