Sitting in the driver's seat: Manipulation of mammalian cell Rab GTPase functions by apicomplexan parasites

Many intracellular microbial pathogens subvert, disrupt or otherwise modulate host membrane trafficking pathways to establish a successful infection. Among them, bacteria that are trapped in a phagosome during mammalian cell invasion, disengage the programmed degradation process by altering the identity of their replicative niche through the exclusion or recruitment of specific Rab GTPases to their vacuole. Many viruses co-opt essential cellular trafficking pathways to perform key steps in their lifecycles. Among protozoan parasites, Apicomplexa are obligate intracellular microbes that invade mammalian cells by creating a unique, nonfusogenic membrane-bound compartment that protects the parasites straightaway from lysosomal degradation. Recent compelling evidence demonstrates that apicomplexan parasites are master manipulators of mammalian Rab GTPase proteins, and benefit or antagonise Rab functions for development within host cells. This review covers the exploitation of mammalian Rab proteins and vesicles by Apicomplexa, focusing on Toxoplasma, Neospora, Plasmodium and Theileria parasites.     

Proteomic identification of intracellular vesicle trafficking and protein glycosylation requirements for lumen inflation in Ciona notochord

Lumen formation and inflation are crucial steps for tubular organ morphogenesis, yet the underling mechanism remains largely unrevealed. Here, we applied 4D proteomics to screen the lumenogenesis-related proteins and revealed the biological pathways potentially that are involved in lumen inflation during notochord lumen formation in the ascidian Ciona savignyi. In total, 910 differentiated expressed proteins (DEPs) were identified before and after notochord lumen formation utilizing Mfuzz analysis. Those DEPs were grouped into four upregulated clusters based on their quantitative expression patterns; the functions of these proteins were enriched in protein metabolic and biosynthetic process, the establishment of localization, and vesicle-mediated transport. We analyzed the vesicle trafficking cluster and focused on several vesicle transport hub proteins. In vivo function-deficient experiments showed that mutation of vesicle transport proteins resulted in an abnormal lumen in notochord development, demonstrating the crucial role of intracellular trafficking for lumen formation. Moreover, abundant extracellular matrix proteins were identified, the majority of which were predicted to be glycosylated proteins. Inhibition of glycosylation markedly reduced the lumen expansion rate in notochord cells, suggesting that protein glycosylation is essential for lumenogenesis. Overall, our study provides an invaluable resource and reveals the crucial mechanisms in lumen formation and expansion.     

Probing intracellular vesicle trafficking and membrane remodelling by cryo-EM

Protein transport between the membranous compartments of the eukaryotic cells is mediated by the constant fission and fusion of the membrane-bounded vesicles from a donor to an acceptor membrane. While there are many membrane remodelling complexes in eukaryotes, COPII, COPI, and clathrin-coated vesicles are the three principal classes of coat protein complexes that participate in vesicle trafficking in the endocytic and secretory pathways. These vesicle-coat proteins perform two key functions: deforming lipid bilayers into vesicles and encasing selective cargoes. The three trafficking complexes share some commonalities in their structural features but differ in their coat structures, mechanisms of cargo sorting, vesicle formation, and scission. While the structures of many of the proteins involved in vesicle formation have been determined in isolation by X-ray crystallography, elucidating the proteins' structures together with the membrane is better suited for cryogenic electron microscopy (cryo-EM). In recent years, advances in cryo-EM have led to solving the structures and mechanisms of several vesicle trafficking complexes and associated proteins.     

Components of the endocytic and recycling trafficking pathways interfere with the integrity of the Legionella‐containing vacuole

Legionella pneumophila requires the Dot/Icm translocation system to replicate in a vacuolar compartment within host cells. Strains lacking the translocated substrate SdhA form a permeable vacuole during residence in the host cell, exposing bacteria to the host cytoplasm. In primary macrophages, mutants are defective for intracellular growth, with a pyroptotic cell death response mounted due to bacterial exposure to the cytosol. To understand how SdhA maintains vacuole integrity during intracellular growth, we performed high‐throughput RNAi screens against host membrane trafficking genes to identify factors that antagonise vacuole integrity in the absence of SdhA. Depletion of host proteins involved in endocytic uptake and recycling resulted in enhanced intracellular growth and lower levels of permeable vacuoles surrounding the ΔsdhA mutant. Of interest were three different Rab GTPases involved in these processes: Rab11b, Rab8b and Rab5 isoforms, that when depleted resulted in enhanced vacuole integrity surrounding the sdhA mutant. Proteins regulated by these Rabs are responsible for interfering with proper vacuole membrane maintenance, as depletion of the downstream effectors EEA1, Rab11FIP1, or VAMP3 rescued vacuole integrity and intracellular growth of the sdhA mutant. To test the model that specific vesicular components associated with these effectors could act to destabilise the replication vacuole, EEA1 and Rab11FIP1 showed increased density about the sdhA mutant vacuole compared with the wild type (WT) vacuole. Depletion of Rab5 isoforms or Rab11b reduced this aberrant redistribution. These findings are consistent with SdhA interfering with both endocytic and recycling membrane trafficking events that act to destabilise vacuole integrity during infection.     

腺病毒感染及胞内运输途径的研究进展

目前关于腺病毒感染及胞内运输的分子机制研究主要来源于C亚群腺病毒在肿瘤细胞系中的研究结果。腺病毒对靶细胞的感染及胞内运输大致分为几步:病毒与细胞表面受体的特异结合,胞吞介导的病毒内化,病毒逃脱胞内体进入细胞质,病毒沿着微管运输至核孔,病毒基因组入核。病毒胞内运输效率极高,感染后1 h,80%以上的病毒基因组被送至核内。但是腺病毒胞内的运输方式会因以下几个因素变化而产生差异:靶细胞类型,细胞生理状态,病毒血清型。文中对腺病毒感染靶细胞及胞内运输的已有分子机制进行综述,为临床基因治疗用途的病毒载体研发提供思路。     

Toward the systems biology of vesicle transport

Systems biology aims to study complex biological processes, such as intracellular traffic, as a whole. Systematic genome-wide assays have the potential to identify the transport machinery, delineate pathways and uncover the molecular components of physiological processes that influence trafficking. A goal of this approach is to create predictive models of intracellular trafficking pathways that reflect these relationships. In this review, we highlight current genome-wide technologies of particular relevance to vesicle transport and describe recent applications of these technologies in the framework of systems biology. Systems approaches hold great promise for placing trafficking pathways in their cellular contexts.     

Visualizing of the cellular uptake and intracellular trafficking of exosomes by live‐cell microscopy

Cells release exosomes to transfer various molecules to other cells. Exosomes are involved in a number of physiological and pathological processes. They are emerging great potential utility for diseases diagnosis and treatment recently. However, the internalization and intracellular trafficking of exosomes have not been described clearly. In this work, exosomes were isolated from the culture medium of PC12 cells, labeled by lipophilic dye and amino‐reactive fluorophore, incubated with resting PC12 cells. The results of live‐cell microscopy indicated that exosomes were internalized through endocytosis pathway, trapped in vesicles, and transported to perinuclear region. Particle tracking fluorescent vesicles suggested that the active transport of exosomes may be mediated by cytoskeleton. The proteins on exosome membrane were found to be released from exosomes and trapped in lysosome. The inverted transport of lipophilic dye from perinuclear region to cell peripheries was revealed, possibly caused by recycling of the exosome lipids. This study provides new sight into the mechanisms of exosome uptake and intracellular fate. J. Cell. Biochem. 111: 488–496, 2010. © 2010 Wiley‐Liss, Inc.     

Endocytosis and intracellular trafficking contribute to necrotic neurodegeneration in C. elegans

Unlike apoptosis, necrotic cell death is characterized by marked loss of plasma membrane integrity. Leakage of cytoplasmic material to the extracellular space contributes to cell demise, and is the cause of acute inflammatory responses, which typically accompany necrosis. The mechanisms underlying plasma membrane damage during necrotic cell death are not well understood. We report that endocytosis is critically required for the execution of necrosis. Depletion of the key endocytic machinery components dynamin, synaptotagmin and endophilin suppresses necrotic neurodegeneration induced by diverse genetic and environmental insults in C. elegans. We used genetically encoded fluorescent markers to monitor the formation and fate of specific types of endosomes during cell death in vivo. Strikingly, we find that the number of early and recycling endosomes increases sharply and transiently upon initiation of necrosis. Endosomes subsequently coalesce around the nucleus and disintegrate during the final stage of necrosis. Interfering with kinesin-mediated endosome trafficking impedes cell death. Endocytosis synergizes with autophagy and lysosomal proteolytic mechanisms to facilitate necrotic neurodegeneration. These findings demonstrate a prominent role for endocytosis in cellular destruction during neurodegeneration, which is likely conserved in metazoans.     

Glucose phosphotransferase and intracelular trafficking

Glycoproteins containing phosphodiester-linked glucose residues have recently been described. The synthesis of this structure occurs due to the intact transfer of glucose-1-phosphate from UDP-glucose and is catalyzed by the enzyme glucose phosphotransferase (GlcPTase). The endogenous acceptors for GlcPTase have been characterized as to molecular weight following incubation of selected homogenates with (³²P)UDP-glucose. These glycoproteins are distinct from the lysosomal hydrolases recognized by the GlcNAc phosphotransferase. The transfer of ³²P from (³²P)UDP-Glc can also be detected when the nucleotide sugar is microinjected into the cytoplasm of individual neurons in Aplysia. The phosphorylated acceptors in this system seem to be predominantly two glycoproteins that are subjected to rapid axoplasmic transport. The possible role of this post-translational modification in the intracellular trafficking of a subset of newly synthesized glycoproteins is discussed.     

The integration of autophagy and cellular trafficking pathways via RAB GAPs

Macroautophagy is a conserved degradative pathway in which a double-membrane compartment sequesters cytoplasmic cargo and delivers the contents to lysosomes for degradation. Efficient formation and maturation of autophagic vesicles, so-called phagophores that are precursors to autophagosomes, and their subsequent trafficking to lysosomes relies on the activity of small RAB GTPases, which are essential factors of cellular vesicle transport systems. The activity of RAB GTPases is coordinated by upstream factors, which include guanine nucleotide exchange factors (RAB GEFs) and RAB GTPase activating proteins (RAB GAPs). A role in macroautophagy regulation for different TRE2-BUB2-CDC16 (TBC) domain-containing RAB GAPs has been established. Recently, however, a positive modulation of macroautophagy has also been demonstrated for the TBC domain-free RAB3GAP1/2, adding to the family of RAB GAPs that coordinate macroautophagy and additional cellular trafficking pathways.     

Regulation of developmental intercellular signalling by intracellular trafficking

Universal trafficking components within the cell can be recruited to coordinate and regulate the developmental signalling cascades. We will present ways in which the intracellular trafficking machinery is used to affect and modulate the outcome of signal transduction in developmental contexts, thus regulating multicellular development. Each of the signalling components must reach its proper intracellular destination, in a form that is properly folded and modified. In many instances, the ability to bring components together or segregate them into distinct compartments within the cell actually provides the switch mechanism to turn developmental signalling pathways on or off. The review will begin with a focus on the signal-sending cells, and the ways in which ligand trafficking can impinge on the signalling outcome, via processing, endocytosis and recycling. We will then turn to the signal-receiving cell, and discuss mechanisms by which endocytosis can affect the spatial features of the signal, and the compartmentalization of components downstream to the receptor.     

Peculiarities of host cholesterol transport to the unique intracellular vacuole containing Toxoplasma

The intracellular protozoan Toxoplasma gondii is auxotrophic for low-density lipoprotein (LDL)-derived cholesterol (C). We previously showed that T. gondii scavenges this essential lipid from host endolysosomal compartments and that C delivery to the parasitophorous vacuole (PV) does not require transit through host Golgi or endoplasmic reticulum. In this study, we explore the itinerary of C from the host endolysosomes to the PV. Labeled C incorporated into LDL is rapidly detected in intravacuolar parasites and partially esterified by the parasites. In contrast to diverse mammalian organelles, the post-endolysosomal transfer of C to the PV does not involve the host plasma membrane as an intermediate. Nevertheless, the PV membrane is accessible to extracellular sterol acceptors, suggesting C trafficking from intracellular parasites to host plasma membrane. C movement to the PV requires temperatures permissive for vesicular transport, metabolic energy and functional microtubules. Host caveolae vesicles and the sterol carrier protein-2 do not participate in this process. Proteolytic treatment of purified PV or free parasites abolishes C acquisition by the parasites. Altogether, these results support a vesicular transport system from host endolysosomes to the PV, and a requirement for PV membrane and parasite plasma membrane proteins in C delivery to T. gondii.     

From transporter to transceptor: signaling from transporters provokes re-evaluation of complex trafficking and regulatory controls: endocytic internalization and intracellular trafficking of nutrient transceptors may, at least in part, be governed by their signaling function

When cells are starved of their substrate, many nutrient transporters are induced. These undergo rapid endocytosis and redirection of their intracellular trafficking when their substrate becomes available again. The discovery that some of these transporters also act as receptors, or transceptors, suggests that at least part of the sophisticated controls governing the trafficking of these proteins has to do with their signaling function rather than with control of transport. In yeast, the general amino acid permease Gap1 mediates signaling to the protein kinase A pathway. Its endocytic internalization and intracellular trafficking are subject to amino acid control. Other nutrient transceptors controlling this signal transduction pathway appear to be subject to similar trafficking regulation. Transporters with complex regulatory control have also been suggested to function as transceptors in other organisms. Hence, precise regulation of intracellular trafficking in nutrient transporters may be related to the need for tight control of nutrient-induced signaling.     

Determinants of AQP6 trafficking to intracellular sites versus the plasma membrane in transfected mammalian cells

BACKGROUND INFORMATION: Most AQPs (aquaporins) function at the plasma membrane, however AQP6 is exclusively localized to membranes of intracellular vesicles in acid-secreting type-A intercalated cells of renal collecting ducts. The intracellular distribution indicates that AQP6 has a function distinct from trans-epithelial water movement. RESULTS: We show by mutational analyses and immunofluorescence that the N-terminus of AQP6 is a determinant for its intracellular localization. Presence or absence at the plasma membrane of AQP6 constructs was confirmed by electrophysiological methods. Addition of a GFP (green fluorescent protein) or a HA (haemagglutinin) epitope tag (GFP-AQP6 or HA-AQP6) to the N-terminus of AQP6, directed AQP6 to the plasma membranes of transfected Madin-Darby canine kidney cells. In contrast, addition of a GFP tag to the C-terminus (AQP6-GFP) caused the protein to remain intracellular, similar to untagged wild-type AQP6. Replacement of the N-terminus of AQP6 by that of AQP1 also directed AQP6 to the plasma membranes, whereas the N-terminus of AQP6 retained AQP1 in cytosolic sites. CONCLUSION: Our results suggest that the N-terminus of AQP6 is critical for trafficking of the protein to the intracellular sites. Moreover, our studies provide an approach for future identification of proteins involved in vesicle sorting in the acid-secreting type-A intercalated cells.     

The level of HER2 expression is a predictor of antibody-HER2 trafficking behavior in cancer cells

The receptor tyrosine kinase HER2 is known to play a central role in mitogenic signaling, motivating the development of targeted, HER2-specific therapies. However, despite the longstanding use of antibodies to target HER2, controversies remain concerning antibody/HER2 trafficking behavior in cancer cells. Understanding this behavior has direct relevance to the mechanism of action and effective design of such antibodies. In the current study, we analyzed the intracellular dynamics of trastuzumab, a marketed HER2-targeting antibody, in a panel of breast and prostate cancer cell lines that have a wide range of HER2 expression levels. Our results reveal distinct post-endocytic trafficking behavior of antibody-HER2 complexes in cells with different HER2 expression levels. In particular, HER2-overexpressing cells exhibit efficient HER2 recycling and limited reductions in HER2 levels upon antibody treatment, and consequently display a high level of antibody persistence on their plasma membrane. By contrast, in cells with low HER2 expression, trastuzumab treatment results in rapid antibody clearance from the plasma membrane combined with substantial decreases in HER2 levels and undetectable levels of recycling. A cell line with intermediate levels of HER2 expression exhibits both antibody recycling and clearance from the cell surface. Significantly, these analyses demonstrate that HER2 expression levels, rather than cell origin (breast or prostate), is a determinant of subcellular trafficking properties. Such studies have relevance to optimizing the design of antibodies to target HER2.     

The costs of sex due to deleterious intracellular parasites

A major problem in evolutionary theory is to explain the widespread occurrence of sexual recombination. This is particularly difficult in anisogamous species where the familiar ‘two-fold cost of sex’ is encountered. Another cost has recently been identified: that fusion of gametes allows intracellular parasites or deleterious ‘selfish’ genomes to invade a population. These costs of anisogamy and the ability of cytoplasmic agents to invade a sexual population are quantified, allowing the costs and consequences of different modes of reproduction to be compared. It is found that the costs of selfish elements are likely to be very high and, in particular, that isogamous sexual reproduction (the putative ‘primitive’ form) is not cost-free, but incurs a fitness reduction of the order of 90%; thus a large selective disadvantage occurs in the initial evolution of sex which is ignored in standard analysis. Even once anisogamy has evolved, the low levels of ‘paternal leakage’ observed in many extant organisms may allow selfish cytoplasmic elements to spread, resulting in moderate to large decreases in host population fitness. However, much of the cost of selfish elements is avoided in sexual lifecycles with a large number of asexual cellular divisions between sexual reproduction: this greatly impedes the spread of selfish agents and reduces the fitness loss attributable to selfish elements.     

Secretion of proteins into host cells by Apicomplexan parasites

The phylum Apicomplexa consists of a diverse group of obligate, intracellular parasites. The distinct evolutionary pressures on these protozoans as they have adapted to their respective niches have resulted in a variety of methods that they use to interact with and modify their hosts. One of these is the secretion and trafficking of parasite proteins into the host cell. We review this process for Theileria , Toxoplasma and Plasmodium . We also present what is known about the mechanisms by which parasite proteins are exported into the host cell, as well as information on their known and putative functions once they have reached their final destination.