Emmanuel Boucrot

Salmonella maintains the integrity of its intracellular vacuole through the action of SifA

A method based on the Competitive Index was used to identify Salmonella typhimurium virulence gene interactions during systemic infections of mice. Analysis of mixed infections involving single and double mutant strains showed that OmpR, the type III secretion system of Salmonella pathogenicity island 2 (SPI‐2) and SifA [required for the formation in epithelial cells of lysosomal glycoprotein (lgp)‐containing structures, termed Sifs] are all involved in the same virulence function. sifA gene expression was induced after Salmonella entry into host cells and was dependent on the SPI‐2 regulator ssrA. A sifA− mutant strain had a replication defect in macrophages, similar to that of SPI‐2 and ompR− mutant strains. Whereas wild‐type and SPI‐2 mutant strains reside in vacuoles that progressively acquire lgps and the vacuolar ATPase, the majority of sifA− bacteria lost their vacuolar membrane and were released into the host cell cytosol. We propose that the wild‐type strain, through the action of SPI‐2 effectors (including SpiC), diverts the Salmonella‐containing vacuole from the endocytic pathway, and subsequent recruitment and maintenance of vacuolar ATPase/lgp‐containing membranes that enclose replicating bacteria is mediated by translocation of SifA.

Endosomal recycling controls plasma membrane area during mitosis

The shape and total surface of a cell and its daughters change during mitosis. Many cells round up during prophase and metaphase and reacquire their extended and flattened shape during cytokinesis. How does the total area of plasma membrane change to accommodate these morphological changes and by what mechanism is control of total membrane area achieved? Using single-cell imaging methods, we have found that the amount of plasma membrane in attached cells in culture decreases at the beginning of mitosis and recovers rapidly by the end. Clathrin-based endocytosis is normal throughout all phases of cell division, whereas recycling of internalized membranes back to the cell surface slows considerably during the rounding up period and resumes at the time at which recovery of cell membrane begins. Interference with either one of these processes by genetic or chemical means impairs cell division. The total cell-membrane area recovers even in the absence of a functional Golgi apparatus, which would be needed for export of newly synthesized membrane lipids and proteins. We propose a mechanism by which modulation of endosomal recycling controls cell area and surface expression of membrane-bound proteins during cell division.

Perforin pores in the endosomal membrane trigger the release of endocytosed granzyme B into the cytosol of target cells.

How the pore-forming protein perforin delivers apoptosis-inducing granzymes to the cytosol of target cells is uncertain. Perforin induces a transient Ca2+ flux in the target cell, which triggers a process to repair the damaged cell membrane. As a consequence, both perforin and granzymes are endocytosed into enlarged endosomes called gigantosomes. Here we show that perforin formed pores in the gigantosome membrane, allowing endosomal cargo, including granzymes, to be gradually released. After about 15 min, gigantosomes ruptured, releasing their remaining content. Thus, perforin delivers granzymes by a two-step process that involves first transient pores in the cell membrane that trigger the endocytosis of granzyme and perforin and then pore formation in endosomes to trigger cytosolic release.

The Salmonella effector protein PipB2 is a linker for kinesin-1

Understanding the mechanisms of Salmonella virulence is an important challenge. The capacity of this intracellular bacterial pathogen to cause diseases depends on the expression of virulence factors including the second type III secretion system (TTSS-2), which is used to translocate into the eukaryotic cytosol a set of effector proteins that divert the biology of the host cell and shape the bacterial replicative niche. Yet little is known about the eukaryotic functions affected by individual Salmonella effectors. Here we report that the TTSS-2 effector PipB2 interacts with the kinesin light chain, a subunit of the kinesin-1 motor complex that drives anterograde transport along microtubules. Translocation of PipB2 is both necessary and sufficient for the recruitment of kinesin-1 to the membrane of the Salmonella-containing vacuole. In vivo, PipB2 contributes to the attenuation of Salmonella mutant strains in mice. Taken together, our data indicate that the TTSS-2-mediated fine-tuning of kinesin-1 activity associated with the bacterial vacuole is crucial for the virulence of Salmonella.

The Translocated Salmonella Effector Proteins SseF and SseG Interact and Are Required To Establish an Intracellular Replication Niche

ABSTRACT The facultative intracellular pathogen Salmonella enterica causes a variety of diseases, including gastroenteritis and typhoid fever. Inside epithelial cells, Salmonella replicates in vacuoles, which localize in the perinuclear area in close proximity to the Golgi apparatus. Among the effector proteins translocated by the Salmonella pathogenicity island 2-encoded type III secretion system, SifA and SseG have been shown necessary but not sufficient to ensure the intracellular positioning of Salmonella vacuoles. Hence, we have investigated the involvement of other secreted effector proteins in this process. Here we show that SseF interacts functionally and physically with SseG but not SifA and is also required for the perinuclear localization of Salmonella vacuoles. The observations show that the intracellular positioning of Salmonella vacuoles is a complex phenomenon resulting from the combined action of several effector proteins.

Roles of AP-2 in Clathrin-Mediated Endocytosis

Background The notion that AP-2 clathrin adaptor is an essential component of an endocytic clathrin coat appears to conflict with recent observations that substantial AP-2 depletion, using RNA interference with synthesis of AP-2 subunits, fails to block uptake of certain ligands known to internalize through a clathrin-based pathway. Methodology/Principal Findings We report here the use of in vivo imaging data obtained by spinning-disk confocal microscopy to study the formation of clathrin-coated structures at the plasma membranes of BSC1 and HeLa cells depleted by RNAi of the clathrin adaptor, AP-2. Very few clathrin coats continue to assemble after AP-2 knockdown. Moreover, there is a total absence of clathrin-containing structures completely lacking AP-2 while all the remaining coats still contain a small amount of AP-2. These observations suggest that AP-2 is essential for endocytic coated-pit and coated-vesicle formation. We also find that AP-2 knockdown strongly inhibits light-density lipoprotein (LDL) receptor-mediated endocytosis, as long as cells are maintained in complete serum and at 37°C. If cells are first incubated with LDL at 4°C, followed by warming, there is little or no decrease in LDL uptake with respect to control cells. LDL uptake at 37°C is also not affected in AP-2 depleted cells first deprived of LDL by incubation with either serum-starved or LDL-starved cells for 24 hr. The LDL-deprived cells display a significant increase in endocytic structures enriched on deeply invaginated tubes that contain LDL and we suggest that under this condition of stress, LDL might enter through this alternative pathway. Conclusions/Significance These results suggest that AP-2 is essential for endocytic clathrin coated-pit and coated-vesicle formation. They also indicate that under normal conditions, functional endocytic clathrin coated pits are required for LDL internalization. We also show that under certain conditions of stress, cells can upregulate alternative endocytic structures with the potential to provide compensatory trafficking pathways.

Perforin activates clathrin- and dynamin-dependent endocytosis, which is required for plasma membrane repair and delivery of granzyme B for granzyme-mediated apoptosis

Cytotoxic T lymphocytes and natural killer cells destroy target cells via the polarized exocytosis of lytic effector proteins, perforin and granzymes, into the immunologic synapse. How these molecules enter target cells is not fully understood. It is debated whether granzymes enter via perforin pores formed at the plasma membrane or whether perforin and granzymes are first endocytosed and granzymes are then released from endosomes into the cytoplasm. We previously showed that perforin disruption of the plasma membrane induces a transient Ca(2+) flux into the target cell that triggers a wounded membrane repair response in which lysosomes and endosomes donate their membranes to reseal the damaged membrane. Here we show that perforin activates clathrin- and dynamin-dependent endocytosis, which removes perforin and granzymes from the plasma membrane to early endosomes, preserving outer membrane integrity. Inhibiting clathrin- or dynamin-dependent endocytosis shifts death by perforin and granzyme B from apoptosis to necrosis. Thus by activating endocytosis to preserve membrane integrity, perforin facilitates granzyme uptake and avoids the proinflammatory necrotic death of a membrane-damaged cell.

Targeting of AMSH to Endosomes Is Required for Epidermal Growth Factor Receptor Degradation

To reach the lysosomes, down-regulated receptors such as the epidermal growth factor receptor must first be sorted into internal vesicles of late endosomes (multivesicular bodies), a ubiquitin-dependent event that requires the coordinated function of the endosome sorting complex required for transport (ESCRT) proteins. Here we report that CHMP3, an ESCRT-III complex component, and associated molecule of SH3 domain of STAM (AMSH), a deubiquitinating enzyme, interact with each other in cells. A dominant-negative version of CHMP3, which specifically prevents targeting of AMSH to endosomes, inhibits degradation but not internalization of EGFR, suggesting that endosomal AMSH is a functional component of the multivesicular body pathway.

SKIP, the host target of the Salmonella virulence factor SifA, promotes kinesin-1-dependent vacuolar membrane exchanges.

In Salmonella‐infected cells, the bacterial effector SifA forms a functional complex with the eukaryotic protein SKIP (SifA and kinesin‐interacting protein). The lack of either partner has important consequences on the intracellular fate and on the virulence of this pathogen. In addition to SifA, SKIP binds the microtubule‐based motor kinesin‐1. Yet the absence of SifA or SKIP results in an unusual accumulation of kinesin‐1 on the bacterial vacuolar membrane. To understand this apparent contradiction, we investigated the interaction between SKIP and kinesin‐1 and the function of this complex. We show that the C‐terminal RUN (RPIP8, UNC‐14 and NESCA) domain of SKIP interacted specifically with the tetratricopeptide repeat (TPR) domain of the kinesin light chain. Overexpression of SKIP induced a microtubule‐ and kinesin‐1‐dependent anterograde movement of late endosomal/lysosomal compartments. In infected cells, SifA contributed to the fission of vesicles from the bacterial vacuole and the SifA/SKIP complex was required for the formation and/or the anterograde transport of kinesin‐1‐enriched vesicles. These observations reflect the role of SKIP as a linker and/or an activator for kinesin‐1.

Mammalian cells change volume during mitosis.

Using single cell-imaging methods we have found that the volume of adherent cells grown in culture decreases as the cells rounds when it enters mitosis. A minimal volume is reached at metaphase. Rapid volume recovery initiates before abscission as cells make the transition from metaphase to cytokinesis. These volume changes are simultaneous with the rapid surface area decrease and recovery observed in mitotic cells [1].