Jean-Pierre Paccaud

The Recycling of ERGIC-53 in the Early Secretory Pathway ERGIC-53 CARRIES A CYTOSOLIC ENDOPLASMIC RETICULUM-EXIT DETERMINANT INTERACTING WITH COPII

Further investigation of the targeting of the intracellular membrane lectin endoplasmic reticulum (ER)-Golgi intermediate compartment-53 (ERGIC-53) by site-directed mutagenesis revealed that its lumenal and transmembrane domains together confer ER retention. In addition we show that the cytoplasmic domain is required for exit from the ER indicating that ERGIC-53 carries an ER-exit determinant. Two phenylalanines at the C terminus are essential for ER-exit. Thus, ERGIC-53 contains determinants for ER retention as well as anterograde transport which, in conjunction with a dilysine ER retrieval signal, control the continuous recycling of ERGIC-53 in the early secretory pathway. In vitro binding studies revealed a specific phenylalanine-dependent interaction between an ERGIC-53 cytosolic tail peptide and the COPII coat component Sec23p. These results suggest that the ER-exit of ERGIC-53 is mediated by direct interaction of its cytosolic tail with the Sec23p·Sec24p complex of COPII and that protein sorting at the level of the ER occurs by a mechanism similar to receptor-mediated endocytosis or Golgi to ER retrograde transport.

PSA‐NCAM modulates BDNF‐dependent survival and differentiation of cortical neurons

We show that the loss or inactivation of the polysialic acid (PSA) tail of neural cell adhesion molecule (NCAM) on rat cortical neurons in culture leads to reduced differentiation and survival. The mechanism by which this negative effect is mediated appears to involve the neuronal response to brain‐derived neurotrophic factor (BDNF): (i) in the absence of PSA or in the presence of excess free PSA added to the culture medium, BDNF‐induced cell signalling is reduced; (ii) the addition of exogenous BDNF to the medium reverses the effect of PSA loss or inactivation. These data suggest that PSA‐NCAM, previously shown to modulate cell migration and plasticity, is needed for an adequate sensitivity of neurons to BDNF.

Role of Sec24 isoforms in selective export of membrane proteins from the endoplasmic reticulum

Sec24 of the COPII (coat protein complex II) vesicle coat mediates the selective export of membrane proteins from the endoplasmic reticulum (ER) in yeast. Human cells express four Sec24 isoforms, but their role is unknown. Here, we report the differential effects of Sec24 isoform‐specific silencing on the transport of the membrane reporter protein ERGIC‐53 (ER–Golgi intermediate compartment‐53) carrying the cytosolic ER export signals di‐phenylalanine, di‐tyrosine, di‐leucine, di‐isoleucine, di‐valine or terminal valine. Knockdown of single Sec24 isoforms showed dependence of di‐leucine‐mediated transport on Sec24A, but transport mediated by the other signals was not affected. By contrast, double knockdown of Sec24A with one of the other three Sec24 isoforms impaired all aromatic/hydrophobic signal‐dependent transport. Double knockdown of Sec24B/C or Sec24B/D preferentially affected di‐leucine‐mediated transport, whereas knockdown of Sec24C/D affected di‐isoleucine‐ and valine‐mediated transport. The isoform‐selective transport correlated with binding preferences of the signals for the corresponding isoforms in vitro. Thus, human Sec24 isoforms expand the repertoire of cargo for signal‐mediated ER export, but are in part functionally redundant.

Selective export of human GPI-anchored proteins from the endoplasmic reticulum

Selective export of transmembrane proteins from the endoplasmic reticulum (ER) relies on recognition of cytosolic-domain-localized transport signals by the Sec24 subunit of the COPII vesicle coat. Human cells express four Sec24 isoforms, termed Sec24A, Sec24B, Sec24C and Sec24D that are differentially required for selective, signal-mediated ER export of transmembrane proteins. By contrast, luminally exposed glycosylphosphatidylinositol (GPI)-anchored membrane proteins cannot bind directly to Sec24 and must either use membrane-spanning cargo receptors or alternative mechanisms for ER export. Little is known about the mechanism underlying export of GPI-anchored proteins from the ER in higher eukaryotes. Using siRNA-based silencing, we identified that ER-to-Golgi transport of the human GPI-anchored protein CD59 requires Sec24, with preference for the Sec24C and Sec24D isoforms, and the recycling transmembrane protein complex p24-p23 that exhibited the same Sec24C-Sec24D isoform preference for ER export. Co-immunoprecipitation indicated unprecedented physical interaction of CD59 as well as a GFP-folate-receptor-GPI-anchor hybrid with a p24-p23 complex. Density gradient centrifugation revealed co-partitioning of CD59 and p24-p23 into biosynthetically early lipid raft fractions, and CD59 transport to the Golgi was cholesterol dependent. The results suggest that the 24p-23p complex acts as a cargo receptor for GPI-anchored proteins by facilitating their export from the ER in a Sec24-isoform-selective manner involving lipid rafts as early sorting platforms.

Evidence for prebudding arrest of ER export in animal cell mitosis and its role in generating Golgi partitioning intermediates.

During mitosis the interconnected Golgi complex of animal cells breaks down to produce both finely dispersed elements and discrete vesiculotubular structures. The endoplasmic reticulum (ER) plays a controversial role in generating these partitioning intermediates and here we highlight the importance of mitotic ER export arrest in this process. We show that experimental inhibition of ER export (by microinjecting dominant negative Sar1 mutant proteins) is sufficient to induce and maintain transformation of Golgi cisternae to vesiculotubular remnants during interphase and telophase, respectively. We also show that buds on the ER, ER exit sites and COPII vesicles are markedly depleted in mitotic cells and COPII components Sec23p, Sec24p, Sec13p and Sec31p redistribute into the cytosol, indicating ER export is inhibited at an early stage. Finally, we find a markedly uneven distribution of Golgi residents over residual exit sites of metaphase cells, consistent with tubulovesicular Golgi remnants arising by fragmentation rather than redistribution via the ER. Together, these results suggest selective recycling of Golgi residents, combined with prebudding cessation of ER export, induces transformation of Golgi cisternae to vesiculotubular remnants in mitotic cells. The vesiculotubular Golgi remnants, containing populations of slow or nonrecycling Golgi components, arise by fragmentation of a depleted Golgi ribbon independently from the ER.

Inhibitors of bacterial virulence identified in a surrogate host model

Antibiotic resistance continues to reduce the number of available antibiotics, increasing the need for novel antibacterial drugs. Since the seminal work of Sir Alexander Fleming, antibiotic identification has been based exclusively on the inhibition of bacterial growth in vitro. Recently, inhibitors of bacterial virulence which interfere with bacterial pathogenesis mechanisms have been proposed as an alternative to antibiotics, and a few were discovered using assays targeting specific virulence mechanisms. Here we designed a simple surrogate host model for the measurement of virulence and systematic discovery of anti‐virulence molecules, based on the interaction of Tetrahymena pyriformis and Klebsiella pneumoniae cells. We screened a library of small molecules and identified several inhibitors of virulence. In a mouse pneumonia model we confirmed that an anti‐virulence molecule displayed antibacterial activity against Klebsiella pneumoniae and Pseudomonas aeruginosa, by reducing dramatically the bacterial load in the lungs. This molecule did not inhibit bacterial growth in vitro but prevented biosynthesis of the Klebsiella capsule and lipopolysaccharides, a key requirement for virulence. Our results demonstrate that anti‐virulence molecules represent an alternative to antibiotics and those can be discovered using non‐animal host models.

The N-formyl methionyl peptide, formyl-methionyl-leucyl phenylalanine (fMLF) increases the lateral diffusion of complement receptor 1 (CR1/CD35) in human neutrophils; a causative role for oxidative metabolites?

The effects of the N-formyl methionyl peptide, formyl-methionyl-leucyl phenylalanine (fMLF) on the lateral mobility of the complement receptor type 1 (CR1/CD35) in glass-adherent human neutrophils were investigated, using fluorescence recovery after photobleaching (FRAP) and confocal microscopy (CSLM). It was found that addition of 0.1–1 μM fMLF increased the diffusion constant (D) of CR1/CD35 to 167–278% of controls. No effect was observed on the receptor distribution or the mobile fraction of receptors. The effect of fMLF on the lateral diffusion of CR1/CD35 could be totally inhibited by addition of pertussis toxin (PT, 250 ng/ml) or of the free radical scavenger enzymes superoxide dismutase (SOD, 2000 U/ml) and catalase (CAT, 200 U/ml), added together the results show that oxidative metabolites produced by neutrophils in response to fMLF can modulate CR1/CD35 diffusion, and indicate a regulatory role for oxygen radicals in phagocytosis.

Illuminating cellular structure and function in the early secretory pathway by multispectral 3D imaging in living cells

Membrane traffic between the endoplasmic reticulum (ER) and the Golgi complex is regulated by two vesicular coat complexes, COPII and COPI. COPII has been implicated in selective packaging of anterograde cargo into coated transport vesicles budding from the ER. COPI-coated vesicles are proposed to mediate recycling of proteins from the Golgi complex to the ER. We have used multi spectral 3D imaging to visualize COPI and COPII behavior simultaneously with various GFP-tagged secretory markers in living cells. This shows that COPII and COPI act sequentially whereby COPI association with anterograde transport complexes is involved in microtubule-based transport and the en route segregation of ER recycling molecules from secretory cargo within TCS in transit to the Golgi complex. We have also investigated the possibility to discriminate spectrally GFP fusion proteins by fluorescence lifetime imaging. This shows that at least two, and possibly up to three GFP fusion proteins can be discriminated and localized in living cells using a single excitation wavelength and a single broad band emission filter.