Anna Marchetti

Mitofusin-2 Independent Juxtaposition of Endoplasmic Reticulum and Mitochondria: An Ultrastructural Study

Besides its role in controlling the morphology of mitochondria, mitofusin-2 has been proposed to tether mitochondria to the endoplasmic reticulum (ER), based largely on light microscopic analysis. In this study we have examined by electron microscopy the organization of ER and mitochondria in cells expressing or not mitofusin-2. Contrary to previous studies, we observed that loss of mitofusin-2 increased ER-mitochondria juxtaposition. These results suggest that mitofusin-2 does not play a critical role in the juxtapostion of ER and mitochondria, and highlight the essential role of ultrastructural analysis to visualize and measure contact between two intracellular compartments.

Specific host genes required for the killing of Klebsiella bacteria by phagocytes

The amoeba Dictyostelium discoideum shares many traits with mammalian macrophages, in particular the ability to phagocytose and kill bacteria. In response, pathogenic bacteria use conserved mechanisms to fight amoebae and mammalian phagocytes. Here we developed an assay using Dictyostelium to monitor phagocyte–bacteria interactions. Genetic analysis revealed that the virulence of Klebsiella pneumoniae measured by this test is very similar to that observed in a mouse pneumonia model. Using this assay, two new host resistance genes (PHG1 and KIL1) were identified and shown to be involved in intracellular killing of K. pneumoniae by phagocytes. Phg1 is a member of the 9TM family of proteins, and Kil1 is a sulphotransferase. The loss of PHG1 resulted in Dictyostelium susceptibility to a small subset of bacterial species including K. pneumoniae. Remarkably, Drosophila mutants deficient for PHG1 also exhibited a specific susceptibility to K. pneumoniae infections. Systematic analysis of several additional Dictyostelium mutants created a two‐dimensional virulence array, where the complex interactions between host and bacteria are visualized.

Dictyostelium discoideum : a model host to measure bacterial virulence

Dictyostelium amoebae have been used as a host model to measure virulence of a wide range of bacterial pathogens. The simple protocol described here takes advantage of the ability of Dictyostelium to grow and form plaques on a lawn of nonpathogenic bacteria but not on virulent bacteria. This assay can be modulated to measure the virulence of different bacterial pathogens. By adjusting various parameters such as cell numbers or media, a more quantitative measure of bacterial virulence can also be obtained. The entire procedure takes about 5 h to compete, and up to 2 further weeks may be required for plaques to form on the bacterial lawn.

A measure of endosomal pH by flow cytometry in Dictyostelium

BackgroundDictyostelium amoebae are frequently used to study the organization and function of the endocytic pathway, and specific protocols are essential to measure the dynamics of endocytic compartments and their internal pH.FindingsWe have revisited these classical protocols to measure more accurately endosomal pH, making use of a fluorescent probe (Oregon green) more adequate for very acidic pH values. This pH-sensitive probe was combined with a pH-insensitive marker, in order to visualize simultaneously endosome dynamics and pH changes. Finally, a flow cytometer was used to measure endosomal pH in individual cells.ConclusionUsing these simple protocols the endosomal pH of endocytic compartments can be assessed accurately, revealing the extreme acidity of Dictyostelium lysosomes (pH <3.5).

Role of magnesium and a phagosomal P‐type ATPase in intracellular bacterial killing

Bacterial ingestion and killing by phagocytic cells are essential processes to protect the human body from infectious microorganisms. However, only few proteins implicated in intracellular bacterial killing have been identified to date. We used Dictyostelium discoideum, a phagocytic bacterial predator, to study intracellular killing. In a random genetic screen we identified Kil2, a type V P‐ATPase as an essential element for efficient intracellular killing of Klebsiella pneumoniae bacteria. Interestingly, kil2 knockout cells still killed efficiently several other species of bacteria, and did not show enhanced susceptibility to Mycobacterium marinum intracellular replication. Kil2 is present in the phagosomal membrane, and its structure suggests that it pumps cations into the phagosomal lumen. The killing defect of kil2 knockout cells was rescued by the addition of magnesium ions, suggesting that Kil2 may function as a magnesium pump. In agreement with this, kil2 mutant cells exhibited a specific defect for growth at high concentrations of magnesium. Phagosomal protease activity was lower in kil2 mutant cells than in wild‐type cells, a phenotype reversed by the addition of magnesium to the medium. Kil2 may act as a magnesium pump maintaining magnesium concentration in phagosomes, thus ensuring optimal activity of phagosomal proteases and efficient killing of bacteria.

Evolution of Pseudomonas aeruginosa virulence in infected patients revealed in a Dictyostelium discoideum host model

Pseudomonas aeruginosa can cause acute lung infections in intubated patients or chronic infections in patients with cystic fibrosis (CF). In both situations, P. aeruginosa accumulates specific mutations, in particular in the lasR quorum-sensing regulator gene. Using a Dictyostelium discoideum amoeba model, we assessed whether these mutations affect bacterial virulence. Among a collection of clinical isolates from 16 CF patients, initial isolates were fully virulent in 15 patients, but for late isolates collected several years later, virulence was decreased in eight patients. No significant correlation between genetic inactivation of lasR and decreased virulence was observed. Among strains isolated from ten colonized intubated patients, all initial isolates were fully virulent. Despite the accumulation of lasR-inactivating mutations in strains collected over a 3-week period, no decrease in virulence was observed in eight of 10 patients. In one intubated patient, the virulent initial strain was replaced a few days later with a different, less virulent, strain. We observed a gradual decrease in bacterial virulence in only one intubated patient. We conclude that adaptation of P. aeruginosa to chronically infected CF patients can lead to a slow and gradual loss of virulence, as measured in a Dictyostelium model system. However, loss of virulence is not caused predominantly by mutations in lasR. During short-term colonization of intubated patients for up to 20 days, a decrease in virulence was exceptional, despite the accumulation of lasR mutations.

Formation of multivesicular endosomes in Dictyostelium.

Multivesicular endosomes are present in virtually every eucaryotic cell, where they arise by intra-endosomal budding of the limiting endosomal membrane. Some genetic diseases such as Chediak-Higashi syndrome are characterized by enlarged membrane-filled endosomes. The same altered endosomal morphology can be observed in cells exposed to certain drugs, for example U18666A. The mechanisms involved are still poorly characterized, partially because this atypical budding event is particularly difficult to observe in mammalian cells. Taking advantage of the simplicity of the endosomal structure in Dictyostelium discoideum, we could visualize intraendosomal budding at the ultrastructural level. In this model organism, the drug U18666A was shown to stimulate intra-endosomal budding, while an inhibitor of PI 3-kinase activity was found to have no effect on this process. Inactivation of a Dictyostelium gene with similarity to the gene responsible for Chediak-Higashi syndrome did not alter the intra-endosomal budding or the accumulation of intra-endosomal membranes. Thus, although treatment with U18666A and inactivation of the Chediak-Higashi gene cause similar morphological defects in mammalian cells, observations in a different model reveal that their respective modes of action are different.

STIM1L traps and gates Orai1 channels without remodeling the cortical ER.

STIM proteins populate and expand cortical endoplasmic reticulum (ER) sheets to mediate store‐operated Ca2+ entry (SOCE) by trapping and gating Orai channels in ER‐plasma membrane clusters. A longer splice variant, STIM1L, forms permanent ER‐plasma membrane clusters and mediates rapid Ca2+ influx in muscle. Here, we used electron microscopy, total internal reflection fluorescence (TIRF) microscopy and Ca2+ imaging to establish the trafficking and signaling properties of the two STIM1 isoforms in Stim1−/−/Stim2−/− fibroblasts. Unlike STIM1, STIM1L was poorly recruited into ER‐plasma membrane clusters and did not mediate store‐dependent expansion of cortical ER cisternae. Removal of the STIM1 lysine‐rich tail prevented store‐dependent cluster enlargement, whereas inhibition of cytosolic Ca2+ elevations or removal of the STIM1L actin‐binding domain had no impact on cluster expansion. Finally, STIM1L restored robust but not accelerated SOCE and clustered with Orai1 channels more slowly than STIM1 following store depletion. These results indicate that STIM1L does not mediate rapid SOCE but can trap and gate Orai1 channels efficiently without remodeling cortical ER cisternae. The ability of STIM proteins to induce cortical ER formation is dispensable for SOCE and requires the lysine‐rich tail of STIM1 involved in binding to phosphoinositides.

Phg1/TM9 Proteins Control Intracellular Killing of Bacteria by Determining Cellular Levels of the Kil1 Sulfotransferase in Dictyostelium

Dictyostelium discoideum has largely been used to study phagocytosis and intracellular killing of bacteria. Previous studies have shown that Phg1A, Kil1 and Kil2 proteins are necessary for efficient intracellular killing of Klebsiella bacteria. Here we show that in phg1a KO cells, cellular levels of lysosomal glycosidases and lysozyme are decreased, and lysosomal pH is increased. Surprisingly, overexpression of Kil1 restores efficient killing in phg1a KO cells without correcting these lysosomal anomalies. Conversely, kil1 KO cells are defective for killing, but their enzymatic content and lysosomal pH are indistinguishable from WT cells. The killing defect of phg1a KO cells can be accounted for by the observation that in these cells the stability and the cellular amount of Kil1 are markedly reduced. Since Kil1 is the only sulfotransferase characterized in Dictyostelium, an (unidentified) sulfated factor, defective in both phg1a and kil1 KO cells, may play a key role in intracellular killing of Klebsiella bacteria. In addition, Phg1B plays a redundant role with Phg1A in controlling cellular amounts of Kil1 and intracellular killing. Finally, cellular levels of Kil1 are unaffected in kil2 KO cells, and Kil1 overexpression does not correct the killing defect of kil2 KO cells, suggesting that Kil2 plays a distinct role in intracellular killing.

Transmembrane domains control exclusion of membrane proteins from clathrin-coated pits

Efficient sorting of proteins is essential to allow transport between intracellular compartments while maintaining their specific composition. During endocytosis, membrane proteins can be concentrated in endocytic vesicles by specific interactions between their cytoplasmic domains and cytosolic coat proteins. It is, however, unclear whether they can be excluded from transport vesicles and what the determinants for this sorting could be. Here, we show that in the absence of cytosolic sorting signals, transmembrane domains control the access of surface proteins to endosomal compartments. They act in particular by determining the degree of exclusion of membrane proteins from endocytic clathrin-coated vesicles. When cytosolic endocytosis signals are present, it is the combination of cytosolic and transmembrane determinants that ultimately controls the efficiency with which a given transmembrane protein is endocytosed.