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

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

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

STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Protease inhibitors rapidly modulate the repair of mechanical wounds.

<p>NRK (left columns) or HeLa (right columns) cells were wounded by scraping and PM repair assays were performed in the absence <b>(A, E)</b> or in the presence of the following protease inhibitors: <b>(B, F)</b> E64 (100 μM); <b>(C, G)</b> Pepstatin-A (PEP-A,100 μM); <b>(D, H)</b> AEBSF (100 μM). After a resealing period of 2 min at 37°C the cell population was stained with PI and analyzed by FACS. The dotted black lines in (A, E) show the Ca²⁺-free permeabilization controls, which determined the gating (dashed line). The data are representative of at least three independent experiments.</p

Active lysosomal proteases are released with differential kinetics during PM wounding and repair.

<p>NRK cells were pre-incubated on ice with SLO (100 ng/ml) followed by addition of Ca²⁺-containing media pre-warmed at 37°C to trigger pore-formation and lysosomal exocytosis. The supernatant was collected at the indicated periods of time and analyzed for activity of the cysteine proteases cathepsins B and L and the aspartyl protease cathepsin D. Results are expressed as percentage of the total activity present in the cells (determined by assaying enzymatic activity in whole cell lysates), and correspond to the mean +/- SD of three independent experiments.</p

PM repair is inhibited in cells deficient in the lysosomal cysteine proteases cathepsin B or cathepsin L.

<p>Time-lapse live imaging of FM1-43 influx was performed in HeLa cells exposed to SLO in the presence or absence of Ca²⁺, 48 h after transfection with control or cathepsin B siRNA <b>(A)</b> or 72 h after cathepsin L <b>(B)</b> siRNA. The insets show kinetic enzyme activity determinations using a specific fluorogenic substrate (left) or the protein levels of each enzyme detected by immunoblot with specific antibodies (right). Anti-tubulin antibodies were used in the same samples as loading controls. The data are representative of two independent experiments.</p

Plasma Membrane Repair Is Regulated Extracellularly by Proteases Released from Lysosomes

<div><p>Eukaryotic cells rapidly repair wounds on their plasma membrane. Resealing is Ca^2+-dependent, and involves exocytosis of lysosomes followed by massive endocytosis. Extracellular activity of the lysosomal enzyme acid sphingomyelinase was previously shown to promote endocytosis and wound removal. However, whether lysosomal proteases released during cell injury participate in resealing is unknown. Here we show that lysosomal proteases regulate plasma membrane repair. Extracellular proteolysis is detected shortly after cell wounding, and inhibition of this process blocks repair. Conversely, surface protein degradation facilitates plasma membrane resealing. The abundant lysosomal cysteine proteases cathepsin B and L, known to proteolytically remodel the extracellular matrix, are rapidly released upon cell injury and are required for efficient plasma membrane repair. In contrast, inhibition of aspartyl proteases or RNAi-mediated silencing of the lysosomal aspartyl protease cathepsin D enhances resealing, an effect associated with the accumulation of active acid sphingomyelinase on the cell surface. Thus, secreted lysosomal cysteine proteases may promote repair by facilitating membrane access of lysosomal acid sphingomyelinase, which promotes wound removal and is subsequently downregulated extracellularly by a process involving cathepsin D.</p></div

ASM secreted during cell injury associates with the PM and is proteolytically modulated.

<p><b>(A)</b> Effect of protease inhibitors on ASM activity released from wounded cells. NRK cells were permeabilized with SLO (100 ng/ml) in Ca²⁺ media containing (green) or not (blue) a protease inhibitor cocktail and incubated at 37°C for the indicated time. As controls, cells not treated with SLO (NT) were incubated with (purple) or without (red) inhibitors. Supernatant samples were placed on ice, protease inhibitors were added and ASM activity was assayed. Similar results were obtained with HeLa cells (not shown). <b>(B)</b> Effect of inhibitors on ASM activity released from wounded cells. NRK cells were permeabilized with SLO (100 ng/ml) in Ca²⁺-containing media with 100 μM E64, 100 μM pepstatin-A (PEP-A) or no inhibitors, and incubated at 37°C for 30 s. The data represent the mean +/- SD of triplicate assays. * P = 0.039, Student’s t test. <b>(C)</b> Detection of cell-associated ASM during the first seconds after SLO wounding. NRK cells were permeabilized with SLO (100 ng/ml), incubated at 37°C in Ca²⁺-containing media for the indicated time, washed and immunofluorescence was performed with rabbit anti-ASM antibodies, followed by imaging under identical settings. <b>(D)</b> Detection of the active 65 kDa form of ASM in supernatant and membrane-associated fractions of wounded cells. NRK cells were permeabilized or not with SLO (100 ng/ml) with or without 100 μM E64 or 100 μM pepstatin-A for the indicated periods of time, and samples of the supernatant or of material removed from the cell surface by an acid wash were analyzed by Western blot with rabbit anti-ASM antibodies. The data are representative of at least three independent experiments. <b>(E)</b> Detection of the 65 kDa ASM form in the supernatant and membrane-associated fractions of wounded cells depleted or not in cathepsin D (CATD). NRK cells were permeabilized or not with SLO and analyzed as described in (D). The data are representative of at least three independent experiments.</p