The exocyst is required for trypanosome invasion and the repair of mechanical plasma membrane wounds

The process of host cell invasion by Trypanosoma cruzi shares mechanistic elements with plasma membrane injury and repair. Both processes require Ca2+-triggered exocytosis of lysosomes, exocytosis of acid sphingomyelinase and formation of ceramide-enriched endocytic compartments. T. cruzi invades at peripheral sites, suggesting a need for spatial regulation of membrane traffic. Here, we show that Exo70 and Sec8 (also known as EXOC7 and EXOC4, respectively), components of the exocyst complex, accumulate in nascent T. cruzi vacuoles and at sites of mechanical wounding. Exo70 or Sec8 depletion inhibits T. cruzi invasion and Ca2+-dependent resealing of mechanical wounds, but does not affect the repair of smaller lesions caused by pore-forming toxins. Thus, T. cruzi invasion and mechanical lesion repair share a unique requirement for the exocyst, consistent with a dependence on targetedmembrane delivery.The process of host cell invasion by Trypanosoma cruzi shares mechanistic elements with plasma membrane injury and repair. Both processes require Ca2+-triggered exocytosis of lysosomes, exocytosis of acid sphingomyelinase and formation of ceramide-enriche12812732sem informaçãosem informaçãoWe thank Dr D. Toomre (Yale University) for the VSVG construct, Dr W. Guo (University of Pennsylvania) for antibodies and A. Beaven and K. Class (University of Maryland) for assistance with confocal microscopy and flow cytometry, respectivel

Caveolae protect endothelial cells from membrane rupture during increased cardiac output.

Caveolae are strikingly abundant in endothelial cells, yet the physiological functions of caveolae in endothelium and other tissues remain incompletely understood. Previous studies suggest a mechanoprotective role, but whether this is relevant under the mechanical forces experienced by endothelial cells in vivo is unclear. In this study we have sought to determine whether endothelial caveolae disassemble under increased hemodynamic forces, and whether caveolae help prevent acute rupture of the plasma membrane under these conditions. Experiments in cultured cells established biochemical assays for disassembly of caveolar protein complexes, and assays for acute loss of plasma membrane integrity. In vivo, we demonstrate that caveolae in endothelial cells of the lung and cardiac muscle disassemble in response to acute increases in cardiac output. Electron microscopy and two-photon imaging reveal that the plasma membrane of microvascular endothelial cells in caveolin 1(-/-) mice is much more susceptible to acute rupture when cardiac output is increased. These data imply that mechanoprotection through disassembly of caveolae is important for endothelial function in vivo

The delivery of personalised, precision medicines via synthetic proteins

Introduction: The design of advanced drug delivery systems based on synthetic and su-pramolecular chemistry has been very successful. Liposomal doxorubicin (Caelyx®), and liposomal daunorubicin (DaunoXome®), estradiol topical emulsion (EstrasorbTM) as well as soluble or erodible polymer systems such as pegaspargase (Oncaspar®) or goserelin acetate (Zoladex®) represent considerable achievements. The Problem: As deliverables have evolved from low molecular weight drugs to biologics (currently representing approximately 30% of the market), so too have the demands made of advanced drug delivery technology. In parallel, the field of membrane trafficking (and endocytosis) has also matured. The trafficking of specific receptors i.e. material to be recycled or destroyed, as well as the trafficking of protein toxins has been well characterized. This, in conjunction with an ability to engineer synthetic, recombinant proteins provides several possibilities. The Solution: The first is using recombinant proteins as drugs i.e. denileukin diftitox (Ontak®) or agalsidase beta (Fabrazyme®). The second is the opportunity to use protein toxin architecture to reach targets that are not normally accessible. This may be achieved by grafting regulatory domains from multiple species to form synthetic proteins, engineered to do multiple jobs. Examples include access to the nucleocytosolic compartment. Herein the use of synthetic proteins for drug delivery has been reviewed

β-1,3-Glucan-Induced Host Phospholipase D Activation Is Involved in Aspergillus fumigatus Internalization into Type II Human Pneumocyte A549 Cells

The internalization of Aspergillus fumigatus into lung epithelial cells is a process that depends on host cell actin dynamics. The host membrane phosphatidylcholine cleavage driven by phospholipase D (PLD) is closely related to cellular actin dynamics. However, little is known about the impact of PLD on A. fumigatus internalization into lung epithelial cells. Here, we report that once germinated, A. fumigatus conidia were able to stimulate host PLD activity and internalize more efficiently in A549 cells without altering PLD expression. The internalization of A. fumigatus in A549 cells was suppressed by the downregulation of host cell PLD using chemical inhibitors or siRNA interference. The heat-killed swollen conidia, but not the resting conidia, were able to activate host PLD. Further, β-1,3-glucan, the core component of the conidial cell wall, stimulated host PLD activity. This PLD activation and conidia internalization were inhibited by anti-dectin-1 antibody. Indeed, dectin-1, a β-1,3-glucan receptor, was expressed in A549 cells, and its expression profile was not altered by conidial stimulation. Finally, host cell PLD1 and PLD2 accompanied A. fumigatus conidia during internalization. Our data indicate that host cell PLD activity induced by β-1,3-glucan on the surface of germinated conidia is important for the efficient internalization of A. fumigatus into A549 lung epithelial cells

Phospholipase D signaling: orchestration by PIP2 and small GTPases

Hydrolysis of phosphatidylcholine by phospholipase D (PLD) leads to the generation of the versatile lipid second messenger, phosphatidic acid (PA), which is involved in fundamental cellular processes, including membrane trafficking, actin cytoskeleton remodeling, cell proliferation and cell survival. PLD activity can be dramatically stimulated by a large number of cell surface receptors and is elaborately regulated by intracellular factors, including protein kinase C isoforms, small GTPases of the ARF, Rho and Ras families and, particularly, by the phosphoinositide, phosphatidylinositol 4,5-bisphosphate (PIP2). PIP2 is well known as substrate for the generation of second messengers by phospholipase C, but is now also understood to recruit and/or activate a variety of actin regulatory proteins, ion channels and other signaling proteins, including PLD, by direct interaction. The synthesis of PIP2 by phosphoinositide 5-kinase (PIP5K) isoforms is tightly regulated by small GTPases and, interestingly, by PA as well, and the concerted formation of PIP2 and PA has been shown to mediate receptor-regulated cellular events. This review highlights the regulation of PLD by membrane receptors, and describes how the close encounter of PLD and PIP5K isoforms with small GTPases permits the execution of specific cellular functions

Dynamic caveolae exclude bulk membrane proteins and are required for sorting of excess glycosphingolipids

Caveolae have long been implicated in endocytosis. Recent data question this link, and in the absence of specific cargoes the potential cellular function of caveolar endocytosis remains unclear. Here we develop new tools, including doubly genome-edited cell lines, to assay the subcellular dynamics of caveolae using tagged proteins expressed at endogenous levels. We find that around 5% of the cellular pool of caveolae is present on dynamic endosomes, and is delivered to endosomes in a clathrin-independent manner. Furthermore, we show that caveolae are indeed likely to bud directly from the plasma membrane. Using a genetically encoded tag for electron microscopy and ratiometric light microscopy, we go on to show that bulk membrane proteins are depleted within caveolae. Although caveolae are likely to account for only a small proportion of total endocytosis, cells lacking caveolae show fundamentally altered patterns of membrane traffic when loaded with excess glycosphingolipid. Altogether, these observations support the hypothesis that caveolar endocytosis is specialized for transport of membrane lipid

Le trafic membranaire au cours de la phagocytose : régulation par la phospholipase D et les GTPases Ral

La phagocytose est un mécanisme d’internalisation de particules > 0,5µm conservé au cours de l’évolution et jouant un rôle dans le développement, l’homéostasie des tissus et l’immunité. Ce processus nécessite l’extension de pseudopodes autour de la particule grâce à la force motrice du cytosquelette d’actine et à l’insertion de membrane intracellulaire afin de maintenir la surface cellulaire après fermeture et internalisation du phagosome. Le but de ma thèse a été l’étude de la régulation de la phagocytose par la phospholipase D (PLD), une enzyme produisant l’acide phosphatidique (PA), un lipide second messager impliqué dans le réarrangement de l’actine et le trafic membranaire. Nous avons également examiné les GTPases Ral, potentiellement impliquées dans la régulation de la PLD au cours de la phagocytose. Dans un premier temps, nos résultats ont montré que la PLD1 et la PLD2 sont impliquées dans la régulation de la phagocytose par l’intermédiaire du PA et sont toutes deux recrutées au niveau du phagosome naissant. La PLD1, localisée sur des compartiments vésiculaires, pourrait jouer un rôle dans le trafic membranaire tandis que la PLD2, localisée à la membrane plasmique est susceptible de participer au recrutement et à l’activation de protéines nécessaires au réarrangement de l’actine ou au trafic membranaire. Dans un second temps, l’étude des GTPases Ral montre que RalA joue un rôle positif dans la phagocytose tandis que RalB joue un rôle négatif. Ces deux protéines sont localisées au niveau de la membrane plasmique et de vésicules intracellulaires de la voie endosomale et recrutées au niveau du phagosome pendant sa formation. Nous suggérons que pendant la phagocytose, RalA est susceptible de réguler la PLD ou l’exocyste, un complexe impliqué dans le trafic membranaire. RalB pourrait activer la RalBP1, un régulateur du cytosquelette d’actine, bien que des expériences complémentaires soient nécessaires pour l’impliquer dans la phagocytose. L’ensemble de ces résultats pourrait permettre de définir une voie de signalisation à l’interface entre le réarrangement de l’actine et le trafic membranaires, deux phénomènes nécessaires à la formation du phagosome.Phagocytosis is an evolutionary conserved process for internalizing particles > 0,5µm and plays a role during development, tissue homeostasis and immunity. Phagosome formation involves pseudopod extension which requires actin polymerization as a driving force and membrane insertion to maintain the cell surface constant after phagosome internalization. The aim of my PhD project was to investigate the role of phospholipase D (PLD) and its product phosphatidic acid (PA) in this process. Ral GTPases, potential regulators of PLD and other effectors during phagocytosis, were also studied. Our results show that endogenous PLD1 and PLD2 are necessary for efficient phagocytosis. Both PLDs are recruited to nascent phagosomes where PA is produced. PLD1, localized on a vesicular compartment, is likely to play a role in membrane trafficking, whereas PLD2, localized at the plasma membrane, may play a role in early signaling events leading to actin rearrangements and vesicle trafficking. The results concerning the implication of Ral GTPases indicated that RalA plays a positive role during phagocytosis and RalB a negative role. Both proteins are observed on nascent phagosomes. Ral GTPases are distributed at the plasma membrane and on intracellular vesicles of the endosomal pathway. We hypothesize that during phagocytosis, RalA may regulate PLD activity and the formation of the exocyst, a complex implicated in membrane trafficking. On the other hand, the negative effects of RalB may be due to the activation of RalBP1, a known regulator of the actin cytoskeleton. Complementary experiments are necessary to confirm these possibilities. Taken together, our results indicate a signaling pathway at the interface of actin rearrangements and membrane trafficking, during phagosomal formation

Membrane trafficking during phagocytosis,regulation by phospholipase D and Ral GTPases

La phagocytose est un mécanisme d'internalisation de particules > 0,5 m conservé au cours de l'évolution et jouant un rôle dans le développement, l'homéostasie des tissus et l'immunité. Ce processus nécessite l'extension de pseudopodes autour de la particule grâce à la force motrice du cytosquelette d'actine et à l'insertion de membrane intracellulaire afin de maintenir la surface cellulaire après fermeture et internalisation du phagosome. Le but de ma thèse a été l'étude de la régulation de la phagocytose par la phospholipase D (PLD), une enzyme produisant l'acide phosphatidique (PA), un lipide second messager impliqué dans le réarrangement de l'actine et le trafic membranaire. Nous avons également examiné les GTPases Ral, potentiellement impliquées dans la régulation de la PLD au cours de la phagocytose. Dans un premier temps, nos résultats ont montré que la PLD1 et la PLD2 sont impliquées dans la régulation de la phagocytose par l'intermédiaire du PA et sont toutes deux recrutées au niveau du phagosome naissant. La PLD1, localisée sur des compartiments vésiculaires, pourrait jouer un rôle dans le trafic membranaire tandis que la PLD2, localisée à la membrane plasmique est susceptible de participer au recrutement et à l'activation de protéines nécessaires au réarrangement de l'actine ou au trafic membranaire. Dans un second temps, l'étude des GTPases Ral montre que RalA joue un rôle positif dans la phagocytose tandis que RalB joue un rôle négatif. Ces deux protéines sont localisées au niveau de la membrane plasmique et de vésicules intracellulaires de la voie endosomale et recrutées au niveau du phagosome pendant sa formation. Nous suggérons que pendant la phagocytose, RalA est susceptible de réguler la PLD ou l'exocyste, un complexe impliqué dans le trafic membranaire. RalB pourrait activer la RalBP1, un régulateur du cytosquelette d'actine, bien que des expériences complémentaires soient nécessaires pour l'impliquer dans la phagocytose. L'ensemble de ces résultats pourrait permettre de définir une voie de signalisation à l'interface entre le réarrangement de l'actine et le trafic membranaires, deux phénomènes nécessaires à la formation du phagosome.Phagocytosis is an evolutionary conserved process for internalizing particles > 0,5 m and plays a role during development, tissue homeostasis and immunity. Phagosome formation involves pseudopod extension which requires actin polymerization as a driving force and membrane insertion to maintain the cell surface constant after phagosome internalization. The aim of my PhD project was to investigate the role of phospholipase D (PLD) and its product phosphatidic acid (PA) in this process. Ral GTPases, potential regulators of PLD and other effectors during phagocytosis, were also studied. Our results show that endogenous PLD1 and PLD2 are necessary for efficient phagocytosis. Both PLDs are recruited to nascent phagosomes where PA is produced. PLD1, localized on a vesicular compartment, is likely to play a role in membrane trafficking, whereas PLD2, localized at the plasma membrane, may play a role in early signaling events leading to actin rearrangements and vesicle trafficking. The results concerning the implication of Ral GTPases indicated that RalA plays a positive role during phagocytosis and RalB a negative role. Both proteins are observed on nascent phagosomes. Ral GTPases are distributed at the plasma membrane and on intracellular vesicles of the endosomal pathway. We hypothesize that during phagocytosis, RalA may regulate PLD activity and the formation of the exocyst, a complex implicated in membrane trafficking. On the other hand, the negative effects of RalB may be due to the activation of RalBP1, a known regulator of the actin cytoskeleton. Complementary experiments are necessary to confirm these possibilities. Taken together, our results indicate a signaling pathway at the interface of actin rearrangements and membrane trafficking, during phagosomal formation

Membrane trafficking during phagocytosis,regulation by phospholipase D and Ral GTPases

La phagocytose est un mécanisme d'internalisation de particules > 0,5 m conservé au cours de l'évolution et jouant un rôle dans le développement, l'homéostasie des tissus et l'immunité. Ce processus nécessite l'extension de pseudopodes autour de la particPhagocytosis is an evolutionary conserved process for internalizing particles > 0,5 m and plays a role during development, tissue homeostasis and immunity. Phagosome formation involves pseudopod extension which requires actin polymerization as a driving