Publisher Correction: Complement-dependent outer membrane perturbation sensitizes Gram-negative bacteria to Gram-positive specific antibiotics.

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Complement-dependent outer membrane perturbation sensitizes Gram-negative bacteria to Gram-positive specific antibiotics.

Gram-negative bacteria are refractory to the action of many antibiotics due to their impermeable outer membrane. An important player of the immune system is the complement system, a protein network in serum that directly kills Gram-negative bacteria through pore-formation by the Membrane Attack Complexes (MAC). We here show that the MAC rapidly perforates the outer membrane but that inner membrane damage, which is essential for killing, is relatively slow. Importantly, we demonstrate that MAC-induced outer membrane damage sensitizes Gram-negative bacteria to otherwise ineffective, Gram-positive-specific, antimicrobials. Synergy between serum and nisin was observed for 22 out of 53 tested Gram-negative clinical isolates and for multi-drug resistant (MDR) blood isolates. The in vivo relevance of this process is further highlighted by the fact that blood sensitizes a MDR K. pneumoniae strain to vancomycin. Altogether, these data imply that antibiotics that are considered ineffective to treat infections with Gram-negatives may have different functional outcomes in patients, due to the presence of the complement system.

Improved bovine embryo production in an oviduct-on-a-chip system: prevention of poly-spermic fertilization and parthenogenic activation.

The oviduct provides the natural micro-environment for gamete interaction, fertilization and early embryo development in mammals, such as the cow. In conventional culture systems, bovine oviduct epithelial cells (BOEC) undergo a rapid loss of essential differentiated cell properties; we aimed to develop a more physiological in vitro oviduct culture system capable of supporting fertilization. U-shaped chambers were produced using stereo-lithography and mounted with polycarbonate membranes, which were used as culture inserts for primary BOECs. Cells were grown to confluence and cultured at an air-liquid interface for 4 to 6 weeks and subsequently either fixed for immune staining, incubated with sperm cells for live-cell imaging, or used in an oocyte penetration study. Confluent BOEC cultures maintained polarization and differentiation status for at least 6 weeks. When sperm and oocytes were introduced into the system, the BOECs supported oocyte penetration in the absence of artificial sperm capacitation factors while also preventing polyspermy and parthenogenic activation, both of which occur in classical in vitro fertilization systems. Moreover, this "oviduct-on-a-chip" allowed live imaging of sperm-oviduct epithelium binding and release. Taken together, we describe for the first time the use of 3D-printing as a step further on bio-mimicking the oviduct, with polarized and differentiated BOECs in a tubular shape that can be perfused or manipulated, which is suitable for live imaging and supports in vitro fertilization.

The Rab7 effector protein RILP controls lysosomal transport by inducing the recruitment of dynein-dynactin motors.

Many intracellular compartments, including MHC class II-containing lysosomes, melanosomes, and phagosomes, move along microtubules in a bidirectional manner and in a stop-and-go fashion due to the alternating activities of a plus-end directed kinesin motor and a minus-end directed dynein-dynactin motor. It is largely unclear how motor proteins are targeted specifically to different compartments. Rab GTPases recruit and/or activate several proteins involved in membrane fusion and vesicular transport. They associate with specific compartments after activation, which makes Rab GTPases ideal candidates for controlling motor protein binding to specific membranes. We and others [7] have identified a protein, called RILP (for Rab7-interacting lysosomal protein), that interacts with active Rab7 on late endosomes and lysosomes. Here we show that RILP prevents further cycling of Rab7. RILP expression induces the recruitment of functional dynein-dynactin motor complexes to Rab7-containing late endosomes and lysosomes. Consequently, these compartments are transported by these motors toward the minus end of microtubules, effectively inhibiting their transport toward the cell periphery. This signaling cascade may be responsible for timed and selective dynein motor recruitment onto late endosomes and lysosomes.

Opposing motor activities of dynein and kinesin determine retention and transport of MHC class II-containing compartments.

MHC class II molecules exert their function at the cell surface by presenting to T cells antigenic fragments that are generated in the endosomal pathway. The class II molecules are targetted to early lysosomal structures, termed MIIC, where they interact with antigenic fragments and are subsequently transported to the cell surface. We previously visualised vesicular transport of MHC class II-containing early lysosomes from the microtubule organising centre (MTOC) region towards the cell surface in living cells. Here we show that the MIIC move bidirectionally in a 'stop-and-go' fashion. Overexpression of a motor head-deleted kinesin inhibited MIIC motility, showing that kinesin is the motor that drives its plus end transport towards the cell periphery. Cytoplasmic dynein mediates the return of vesicles to the MTOC area and effectively retains the vesicles at this location, as assessed by inactivation of dynein by overexpression of dynamitin. Our data suggest a retention mechanism that determines the perinuclear accumulation of MIIC, which is the result of dynein activity being superior over kinesin activity. The bidirectional nature of MIIC movement is the result of both kinesin and dynein acting reciprocally on the MIIC during its transport. The motors may be the ultimate targets of regulatory kinases since the protein kinase inhibitor staurosporine induces a massive release of lysosomal vesicles from the MTOC region that is morphologically similar to that observed after inactivation of the dynein motor.

Multivesicular body morphogenesis requires phosphatidyl-inositol 3-kinase activity.

Multivesicular bodies are endocytic compartments containing multiple small vesicles that originate from the invagination and 'pinching off' of the limiting membrane into the luminal space [1] [2] [3]. The molecular mechanisms responsible for the formation of these compartments are unknown. In the human melanoma cell line Mel JuSo, newly synthesised major histocompatibility complex (MHC) class II molecules accumulate in multivesicular early lysosomes [4]. The phosphatidylinositol (PI) 3-kinase inhibitor wortmannin induced the transient vacuolation of early MHC class II compartments, but also of early and late endosomes. We demonstrate that endocytic membrane influx is required for the wortmannin-induced swelling of vesicles. The wortmannin-induced vacuoles contained a reduced number of intraluminal vesicles that were linked to the limiting membrane by membraneous connections. These data suggest that wortmannin inhibits the invagination and/or pinching off of intraluminal vesicles and provide evidence of a role for PI 3-kinase in multivesicular body morphogenesis. We propose that the wortmannin-induced vacuolation occurs as a result of the inability of multivesicular bodies to store endocytosed membranes as intraluminal vesicles thereby causing the formation of large 'empty' vacuoles.

Intracellular transport and peptide loading of MHC class II molecules: regulation by chaperones and motors.

MHC class II molecules are important in the onset and modulation of cellular immune responses. Studies on the intracellular transport of these molecules has provided insight into the way pathogens are processed and presented at the cell surface and may result in future immunological intervention strategies. Recent reviews have extensively described structural properties and early events in the biosynthesis of MHC class II (1-3). In this review, the focus will be on the function of the dedicated chaperone proteins Ii, DM and DO in the class II assembly, transport and peptide loading as well on proteins involved in transport steps late in the intracellular transport of MHC class II.

Removal and degradation of the free MHC class II beta chain in the endoplasmic reticulum requires proteasomes and is accelerated by BFA.

We have studied the degradation of the free major histocompatibility complex (MHC) class II beta subunit in the ER. Domain swapping experiments demonstrate that both the intra- and extracellular domain determine the rate of degradation. Recently, it has been shown that some ER-retained proteins are exported from the ER by the translocon followed by deglycosylation and degradation in the cytosol by proteasomes. Degradation of the beta chain follows a different route. The proteasome is involved but inhibition of the proteasome by lactacystin does not result in deglycosylation and export to the cytosol. Instead, the beta chain is retained in the ER implying that extraction of the beta chain from the ER membrane requires proteasome activity. Surprisingly, brefeldin A accelerates the degradation of the beta chain by the proteasome. This suggests that various processes outside the ER are involved in ER-degradation. The ER is the site from where misfolded class II beta chains enter a proteasome-dependent degradation pathway.

MHC class II molecules: transport pathways for antigen presentation.

During biosynthesis, MHC class II molecules travel through the endocytic pathway and interact with antigenic peptides before their stable insertion in the plasma membrane. The process of class II association with these peptides and their final deposition at the cell surface are essential steps in boosting specific antibody responses. Therefore, the study of class II molecules is important in understanding how cell-biological events can direct an immune response.

Direct vesicular transport of MHC class II molecules from lysosomal structures to the cell surface.

Newly synthesized MHC class II molecules are sorted to lysosomal structures where peptide loading can occur. Beyond this point in biosynthesis, no MHC class II molecules have been detected at locations other than the cell surface. We studied this step in intracellular transport by visualizing MHC class II molecules in living cells. For this purpose we stably expressed a modified HLA-DR1 beta chain with the Green Fluorescent Protein (GFP) coupled to its cytoplasmic tail (beta-GFP) in class II-expressing Mel JuSo cells. This modification of the class II beta chain does not affect assembly, intracellular distribution, and peptide loading of the MHC class II complex. Transport of the class II/ beta-GFP chimera was studied in living cells at 37 degrees C. We visualize rapid movement of acidic class II/beta-GFP containing vesicles from lysosomal compartments to the plasma membrane and show that fusion of these vesicles with the plasma membrane occurs. Furthermore, we show that this transport route does not intersect the earlier endosomal pathway.

Allele-specific differences in the interaction of MHC class I molecules with transporters associated with antigen processing.

MHC class I molecules bind peptides that are translocated from the cytosol into the endoplasmic reticulum by the peptide transporter associated with antigen processing (TAP). Class I heterodimers have been shown to associate with TAP and are released when loaded with peptide. Here, we show the existence of two pools of class I heterodimers, one associated with TAP and one that is free. Whereas the free pool is recognized by the class I-specific Ab W6/32, the TAP-associated pool is not. Analysis of several class I alleles shows binding to TAP with different efficiencies, even at the earliest time points of MHC class I assembly. Most HLA-A and -C alleles tested interacted efficiently with TAP, whereas a considerable number of HLA-B alleles associated very inefficiently or not at all with TAP. This was also observed in cells with nonfunctional TAP. Sequence comparison of the different class I alleles allowed the definition of amino acids in the peptide binding groove that might be involved in TAP association. Binding of peptides to two different pools of class I heterodimers may ensure efficient peptide association in an environment where peptides have a short life span.

Cell surface expression of receptor protein tyrosine phosphatase RPTP mu is regulated by cell-cell contact.

RPTP mu is a transmembrane protein tyrosine phosphatase with an adhesion molecule-like ectodomain. It has recently been shown that RPTP mu mediates homophilic interactions when expressed in insect cells. In this study, we have examined how RPTP mu may function as a cell contact receptor in mink lung epithelial cells, which express RPTPmu endogenously, as well as in transfected 3T3 cells. We find that RPTP mu has a relatively short half-life (3-4 hours) and undergoes posttranslational cleavage into two noncovalently associated subunits, with both cleaved and uncleaved molecules being present on the cell surface (roughly at a 1:1 ratio); shedding of the ectodomain subunit is observed in exponentially growing cells. Immunofluorescence analysis reveals that surface expression of RPTPmu is restricted to regions of tight cell-cell contact. RPTPmu surface expression increases significantly with increasing cell density. This density-induced upregulation of RPTP mu is independent of its catalytic activity and is also observed when transcription is driven by a constitutive promoter, indicating that modulation of RPTPmu surface expression occurs posttranscriptionally. Based on our results, we propose the following model of RPTP mu function: In the absence of cell-cell contact, newly synthesized RPTP mu molecules are rapidly cleared from the cell surface. Cell-cell contact causes RPTPmu to be trapped at the surface through homophilic binding, resulting in accumulation of RPTP mu at intercellular contact regions. This contact-induced clustering of RPTPmu may then lead to tyrosine dephosphorylation of intracellular substrates at cell-cell contacts.

Major histocompatibility complex class II molecules induce the formation of endocytic MIIC-like structures.

During biosynthesis, major histochompatibility complex class II molecules are transported to the cell surface through a late endocytic multilaminar structure with lysosomal characteristics. This structure did not resemble any of the previously described endosomal compartments and was termed MIIC. We show here that continuous protein synthesis is required for the maintenance of MIIC in B cells. Transfection of class II molecules in human embryonal kidney cells induces the formation of multilaminar endocytic structures that are morphologically analogous to MIIC in B cells. Two lysosomal proteins (CD63 and lamp-1), which are expressed in MIIC of B cells, are also present in the structures induced by expression of major histocompatibility complex class II molecules. Moreover, endocytosed HRP enters the induced structures defining them as endocytic compartments. Exchanging the transmembrane and cytoplasmic tail of the class II alpha and beta chains for that of HLA-B27 does not result in the induction of multilaminar structures, and the chimeric class II molecules are now located in multivesicular structures. This suggests that expression of class II molecules is sufficient to induce the formation of characteristic MIIC-like multilaminar structures.

Cell-cell adhesion mediated by a receptor-like protein tyrosine phosphatase.

Receptor-like protein tyrosine phosphatases (receptor-PTPs) represent a novel family of transmembrane proteins that are thought to play important roles in cellular regulation. They consist of a cytoplasmic catalytic region, a single transmembrane segment and an extracellular, putative ligand-binding domain, but the nature of their physiological ligands is unknown. We have recently cloned a new receptor-PTP (RPTP mu), the ectodomain of which includes an Ig-like and four fibronectin type III-like domains, suggesting that RPTP mu may be involved in cell-cell or cell-matrix interactions. To test this hypothesis, we expressed RPTP mu in insect Sf9 cells using recombinant baculovirus. We demonstrate that RPTP mu dramatically promotes cell-to-cell adhesion in a homophilic, Ca(2+)-independent manner. No adhesion is observed in Sf9 cells expressing a chimeric RPTP mu molecule containing the extracellular domain of the epidermal growth factor receptor. Furthermore, cells expressing an enzymatically inactive, point-mutated RPTP mu or a truncated form of RPTP mu, lacking the entire catalytic region, show adhesive properties indistinguishable from those of wild-type RPTP mu, indicating that the catalytic domain is not essential for RPTP mu-mediated adhesion. These results assign a physiological role for RPTP mu in signaling cell-cell recognition.

Two dominant inhibitory mutants of p21ras interfere with insulin-induced gene expression.

Insulin induces a rapid activation of p21ras in NIH 3T3 and Chinese hamster ovary cells that overexpress the insulin receptor. Previously, we suggested that p21ras may mediate insulin-induced gene expression. To test such a function of p21ras more directly, we studied the effect of different dominant inhibitory mutants of p21ras on the induction of gene expression in response to insulin. We transfected a collagenase promoter-chloramphenicol acetyltransferase (CAT) gene or a fos promoter-luciferase gene into NIH 3T3 cells that overexpressed the insulin receptor. The activities of both promoters were strongly induced after treatment with insulin. This induction could be suppressed by cotransfection of two inhibitory mutant ras genes, H-ras(Asn-17) or H-ras(Leu-61,Ser-186). In particular, insulin-induced activation of the fos promoter was inhibited completely by H-ras(Asn-17). These results show that p21ras functions as an intermediate in the insulin signal transduction route leading to the induction of gene expression.