Release of outer membrane vesicles by Gram-negative bacteria is a novel envelope stress response

Conditions that impair protein folding in the Gram-negative bacterial envelope cause stress. The destabilizing effects of stress in this compartment are recognized and countered by a number of signal transduction mechanisms. Data presented here reveal another facet of the complex bacterial stress response, release of outer membrane vesicles. Native vesicles are composed of outer membrane and periplasmic material, and they are released from the bacterial surface without loss of membrane integrity. Here we demonstrate that the quantity of vesicle release correlates directly with the level of protein accumulation in the cell envelope. Accumulation of material occurs under stress, and is exacerbated upon impairment of the normal housekeeping and stress-responsive mechanisms of the cell. Mutations that cause increased vesiculation enhance bacterial survival upon challenge with stressing agents or accumulation of toxic misfolded proteins. Preferential packaging of a misfolded protein mimic into vesicles for removal indicates that the vesiculation process can act to selectively eliminate unwanted material. Our results demonstrate that production of bacterial outer membrane vesicles is a fully independent, general envelope stress response. In addition to identifying a novel mechanism for alleviating stress, this work provides physiological relevance for vesicle production as a protective mechanism.     

Effects of cocultivation with transformed cells on surface proteins of normal cells.

Virally transformed fibroblasts do not have on their surface a major protein (large external transformation-sensitive, LETS) which is present in normal cells. Cocultivation of the transformed cells with normal cells whose surface proteins have been prelabelled induces an accelerated release of the LETS protein from the normal cells. We have investigated various conditions which affect this phenomenon. Our results show that alteration of cell surface proteins by cocultivation with the transformed cells is time and dose-dependent and requires cell contact. Serum was depleted at least 99% of plasminogen by affinity chromatography and used in the cocultivation experiments. It was found that activation of plasminogen was not required for the accelerated turnover of the LETS protein. Other diffusible proteases are also unlikely to be involved. The possibility that transformed cells have a membrane bound activity is discussed. The role of plasminogen activation was also tested for its relevance in transformation related proteolysis, growth and morphology of cells.     

Effects of cocultivation with transformed cells on surface proteins of normal cells

Virally transformed fibroblasts do not have on their surface a major protein (large external transformation-sensitive, LETS) which is present in normal cells. Cocultivation of the transformation cells with normal cells whose surface proteins have been prelabelled induces an accelerated release of the LETS protein from the normal cells. We have investigated various conditions which affect this phenomenon. Our results show that alteration of cell surface proteins by cocultivation with the transformed cells is time and dose-dependent and requires cell contact. Serum was depleted at least 99% of plasminogen by affinity chromatography and used in the cocultivation experiments. It was found that activation of plasminogen was not required for the accelerated turnover of the LETS protein. Other diffusible proteases are also unlikely to be involved. The possibility that transformed cells have a membrane bound activity is discussed. The role of plasminogen activation was also tested for its relevance in transformation related proteolysis, growth and morphology of cells.     

Cancer Cells Induced to Express Mesenchymal Phenotype Release Exosome-like Extracellular Vesicles Carrying Tissue Factor

Aggressive epithelial cancer cells frequently adopt mesenchymal characteristics and exhibit aberrant interactions with their surroundings, including the vasculature. Whether the release/uptake of extracellular vesicles (EVs) plays a role during these processes has not been studied. EVs are heterogeneous membrane structures that originate either at the surface (microparticles), or within (exosomes) activated or transformed cells, and are involved in intercellular trafficking of bioactive molecules. Here, we show that epithelial cancer cells (A431, DLD-1) adopt mesenchymal features (epithelial-to-mesenchymal transition-like state) upon activation of epidermal growth factor receptor (EGFR) coupled with blockade of E-cadherin. This treatment leads to a coordinated loss of EGFR and tissue factor (TF) from the plasma membrane and coincides with a surge in emission of small, exosome-like EVs containing both receptors. TF (but not EGFR) is selectively up-regulated in EVs produced by mesenchymal-like cancer cells and can be transferred to cultured endothelial cells rendering them highly procoagulant. We postulate that epithelial-to-mesenchymal transition-like changes may alter cancer cell interactions with the vascular systems through altered vesiculation and TF shedding.     

Differential Roles of C-terminal Eps15 Homology Domain Proteins as Vesiculators and Tubulators of Recycling Endosomes

Endocytic recycling involves the return of membranes and receptors to the plasma membrane following their internalization into the cell. Recycling generally occurs from a series of vesicular and tubular membranes localized to the perinuclear region, collectively known as the endocytic recycling compartment. Within this compartment, receptors are sorted into tubular extensions that later undergo vesiculation, allowing transport vesicles to move along microtubules and return to the cell surface where they ultimately undergo fusion with the plasma membrane. Recent studies have led to the hypothesis that the C-terminal Eps15 homology domain (EHD) ATPase proteins are involved in the vesiculation process. Here, we address the functional roles of the four EHD proteins. We developed a novel semipermeabilized cell system in which addition of purified EHD proteins to reconstitute vesiculation allows us to assess the ability of each protein to vesiculate MICAL-L1-decorated tubular recycling endosomes (TREs). Using this assay, we show that EHD1 vesiculates membranes, consistent with enhanced TRE generation observed upon EHD1 depletion. EHD4 serves a role similar to that of EHD1 in TRE vesiculation, whereas EHD2, despite being capable of vesiculating TREs in the semipermeabilized cells, fails to do so in vivo. Surprisingly, the addition of EHD3 causes tubulation of endocytic membranes in our semipermeabilized cell system, consistent with the lack of tubulation observed upon EHD3 depletion. Our novel vesiculation assay and in vitro electron microscopy analysis, combined with in vivo data, provide evidence that the functions of both EHD1 and EHD4 are primarily in TRE membrane vesiculation, whereas EHD3 is a membrane-tubulating protein.     

The effect of extracellular matrix interactions on morphologic transformation in vitro

There is emerging evidence that the structure and function of a cell is dependent in part on the contacts that cells make with the extracellular matrix. We report here the effect of extracellular matrices secreted from both normal and tumor cells have on the structure of normal rat kidney epithelial cells. Normal rat kidney cells plated on the basement membrane secreted by tumor cells adopt a morphology and phenotype which closely resembles a Kirsten-ras transformed normal rat kidney cell. This morphologic transformation was not observed for cells plated on individual extracellular matrix components or on basement membrane secreted by normal placenta cells. This suggests that tumor derived basement membrane has unique characteristics which may cause morphologic transformation of normal rat kidney cells.     

Molecular requirements for the mu-induced light chain gene rearrangement in pre-B cells.

During B cell differentiation rearrangement of immunoglobulin (Ig) genes is partially regulated by the Ig proteins. Rearrangement of heavy (H) chain genes is inhibited, whilst that of light (L) chain genes is induced by the membrane form of the mu H chain. In order to analyse additional structural requirements of mu induced L chain gene rearrangement we transfected wild-type mu and mutant mu constructs lacking functional exons encoding the first or second constant domains into Abelson murine leukemia virus (AMuLV) transformed pre-B cells. All mu chains are expressed on the surface of the pre-B cell and all associate with omega and iota, two proteins forming a surrogate light chain, necessary for mu membrane expression. Nevertheless, only wild-type mu and not the mutant mu proteins promote L gene rearrangement. A heterodimer of proteins with Mr of 33 kd and 36 kd was found associated with wild-type but not with the mutant mu proteins. Continuous presence of mu is required for L chain gene recombination since loss of mu stopped and readdition of mu started L gene rearrangement. We propose that the protein complex composed of mu and the 33 kd/36 kd protein heterodimer is responsible for the activation of the L chain gene locus and its rearrangement.     

Immunocytochemical localization in normal and transformed human cells in tissue culture using a monoclonal antibody to the src protein of the Harvey strain of murine sarcoma virus

Using a rat monoclonal antibody directed against the p21 src protein of the Harvey strain of Murine Sarcoma Virus (MSV), we have examined the reactivity of human cells in tissue culture using immunofluorescence and electron microscopy. Qualitative results indicated that untransformed mouse and human fibroblastic cells have undetectable amounts of p21; these levels were greatly increased after transformation with Harvey MSV. A group of human tumor cell lines adapted to tissue culture were examined and almost all of the epithelial tumor lines showed significant localization with this antibody. Notable exceptions were two melanoma cell lines which were negative for p21 by immunofluorescence. When normal human epithelial cells derived from esophageal or foreskin epithelium were examined, the antibody showed significant reactivity with subconfluent growing cells. After the normal cells were allowed to become quiescent, the reactivity with this antibody decreased. All of the localization seen by fluorescence was in a distribution consistent with the previously demonstrated location of p21 scr on the inner aspect of the plasma membrane. Electron microscope localization showed labeling for this antigen on the inner surface of the plasma membrane in both transformed mouse cells and in the human tumor cell lines MCF-7 and HTB-2 (RT4). These results suggest that the amounts of p21-like proteins detectable in human epithelial tumor cells do not necessarily reflect their malignant potential, but may be related to their epithelial nature. The loss of detectable localization at quiescence suggests that p21 levels decrease when these epithelial cells stop growing, and raises the possibility that an analog of p21 may be used by these human epithelial cells to regulate cell growth.     

Multilayering and loss of apical polarity in MDCK cells transformed with viral K-ras

The effects of viral Kirsten ras oncogene expression on the polarized phenotype of MDCK cells were investigated. Stable transformed MDCK cell lines expressing the v-K-ras oncogene were generated via infection with a helper-independent retroviral vector construct. When grown on plastic substrata, transformed cells formed continuous monolayers with epithelial-like morphology. However, on permeable filter supports where normal cells form highly polarized monolayers, transformed MDCK cells detached from the substratum and developed multilayers. Morphological analysis of the multilayers revealed that oncogene expression perturbed the polarized organization of MDCK cells such that the transformed cells lacked an apical--basal axis around which the cytoplasm is normally organized. Evidence for selective disruption of apical membrane polarity was provided by immunolocalization of membrane proteins; a normally apical 114-kD protein was randomly distributed on the cell surface in the transformed cell line, whereas normally basolateral proteins remained exclusively localized to areas of cell contact and did not appear on the free cell surface. The discrete distribution of the tight junction-associated ZO-1 protein as well as transepithelial resistance and flux measurements suggested that tight junctions were also assembled. These findings indicate that v-K-ras transformation alters cell-substratum and cell-cell interactions in MDCK cells. Furthermore, v-K-ras expression perturbs apical polarization but does not interfere with the development of a basolateral domain, suggesting that apical and basolateral polarity in epithelial cells may be regulated independently.     

Interactions between normal and transformed epithelial cells: Their contributions to tumourigenesis

During the initial stages of carcinogenesis, neoplastic transformation occurs in single epithelial cells and the transformed cells proliferate while being surrounded by normal epithelia. In Drosophila, normal and transformed epithelial cells compete with each other for survival, a process called cell competition. However, it was not known whether comparable phenomena also occur in mammals. Recently, several reports have shown that the interaction between normal and transformed epithelial cells causes various phenomena in mammals. For example, with elaborate cell culture systems that express oncoproteins or knockdown tumour suppressor proteins in an inducible manner, certain types of transformed cells have been shown to be apically eliminated from normal epithelial layers in an apoptosis-dependent or -independent manner. During the process of apical extrusion, various signalling pathways are modulated in transformed cells located within the normal epithelium, indicating that the presence of surrounding normal epithelial cells affects the behaviour and fate of transformed cells. Recent studies in mice have also shown that normal and transformed cells can compete with each other for survival during several processes such as liver regeneration. In this review, we will introduce these recent publications on interactions between normal and transformed mammalian epithelial cells. Furthermore, we will discuss how these studies can potentially lead to identification of biomarkers for precancerous cells and to invention of novel types of cancer prevention and treatment.     

Effect of sickling on dimyristoylphosphatidylcholine-induced vesiculation in sickle red blood cells

To study the effect of sickling on dimyristoylphosphatidylcholine (DMPC)-induced vesiculation, sickle (SS) red blood cells were incubated with sonicated suspensions of DMPC under either room air or nitrogen. Like normal red cells, when sickle cells were incubated with DMPC under oxygenated conditions, incorporation of DMPC into the erythrocyte membrane occurred, followed by echinocytic shape transformation and subsequent release of membrane vesicles. On the other hand, when SS cells were induced to sickle by deoxygenation, DMPC-induced vesiculation of these cells was dramatically reduced. However, upon reoxygenation, release of vesicles from these sickle erythrocytes occurred immediately. When SS cells were incubated under hypertonic (500 mosM) and deoxygenated conditions (where hemoglobin polymerization occurs but red cells do not show the typical sickle morphology), a similar decrease in the extent of vesiculation was observed. Experiments with radiolabelled lipid vesicles indicated that incorporation of DMPC into erythrocyte membranes occurred in all cases and therefore was not the limiting factor in the reduction of vesiculation in deoxygenated SS cells. Taken together, these results indicate that cellular viscosity and membrane rigidity, both of which are influenced by hemoglobin polymerization, are two important factors in process of vesicle release from sickle erythrocytes.     

Cell distribution and antigenic properties of mammalian sarcolectins

Sarcolectins are present in a great variety of tissues from mammalian origin. Such substances were observed to be secreted from cultures of human embryonic fibroblasts, human osteosarcoma and rat Rous sarcoma transformed cells and could be extracted from TG 180 Crocker Sarcoma or normal human placenta. All sarcolectins tested here, were comparable by their physicochemical properties to those previously reported in hamster or human sarcomas. Indeed, they are proteins or glycoproteins, resistant to pepsin and migrate in SDS-PAGE in the 65 kDa area. They agglutinate cells with an affinity for simple sugars and degrade previously established interferon-induced antiviral resistance. Considering the hamster sarcolectin as reference in this comparative study, both differences and similarities in the antigenic properties of mouse, rat and human sarcolectin variants were demonstrated. An indirect immunofluorescence assay showed that sarcolectins were specifically labelled on the cell surface but not detected in the cytoplasm after methanol or acetone permeabilization of the membrane. By electron microscopy, using immunoperoxidase labelling, sarcolectins can be localized on the surface of normal, transformed, human or rat cells. Only limited segments of normal cell membranes were labelled, while transformed cells were frequently stained on their whole surface. Other known extracellular proteins, such as fibronectin and collagen, did not share common antigenic determinants with sarcolectins.     

Surface alterations in transformed epithelial and fibroblastic cells in culture: A disturbance of membrane degradation versus biosynthesis?

Fibroblasts which were transformed with oncogenic DNA or RNA viruses as well as by chemicals or spontaneously have, with very few exceptions, been shown to possess increased agglutinabilities with various plant agglutinins. This surface membrane alteration is now shown to hold true also for transformed epithelial cells and for tumors of epithelial origin as monitored by wheat germ agglutinin, Concanavalin A, phytohemagglutinin, Ricinus communis and Great Northern bean (GNB) agglutinin. An agglutinin from lentils did the opposite. It permitted the agglutination of normal liver cells but not that of hepatoma cells which led to the suggestion that some membrane glycoproteins or glycolipids may possibly be turned inside out while others are turned outside in (during the transformation process. Inhibition of protein synthesis in normal cells brought about the agglutinable membrane state in non-growing or resting cells but not in actively growing cells. Since this agglutinability could be inhibited by protease inhibitors and since proteolytic enzymes could bring about the agglutinable membrane state in growing normal cells, it was suggested that growing cells have as active a biosynthesis of membrane proteins as do non growing cells but that non-growing cells are actively catabolizing their membranes with proteases, much more so than non growing, stationary cells.     

A cellular protein phosphorylated by the avian sarcoma virus transforming gene product is associated with ribonucleoprotein particles

In chick embryo fibroblasts transformed by Rous sarcoma virus (RSV) the tyrosine phosphorylation of a cellular protein of 34,000 daltons mol. wt. (34 kd) is greatly enhanced; this was shown to be catalyzed by the phosphotransferase activity of RSV transforming protein pp60src. We report here that in cytoplasmic extracts of both normal and transformed cells, in the presence of magnesium ions, the majority of the 34-kd protein is associated with large structures and that a fraction of 34 kd appears to be associated with ribonucleoprotein particles (RNPs). In addition, upon u.v. light cross-linking of RNA to protein in normal or transformed cells, an anti-34 kd serum immunoprecipitates RNA fragments of apparent low sequence complexity as detected by T1 fingerprint analysis. Our results indicate that the 34-kd protein may play a role in the cell at the level of RNPs.     

Lack of tropomyosin correlates with the absence of stress fibers in transformed rat kidney cells

We have utilized epithelial rat kidney cells and their Kirsten viral transformant (442) to examine the role of actin-binding proteins in cellular morphogenesis. Normal rat kidney cells are well spread while the transformed cells are more spherical, poorly adherent, and lack actin stress fibers (Rubin, R.W., Warren, R.H., Lukeman, D.S. and Clements, E. (1978) J. Cell Biol. 78, 28-35). By immunofluorescence, antitropomyosin prominently stains normal rat kidney cell stress fibers while only a weak, nonspecific fluorescence is observed in 442 cells. Using two-dimensional gel electrophoresis, tropomyosin can be detected in normal rat kidney cells homogenates. The tropomyosin subunits are enriched in Triton-extracted filamentous normal rat kidney cell models, and in extracts of normal rat kidney cell homogenate produced by using a rapid myosin affinity technique to isolate actin and actin-associated proteins. The identity of the tropomyosin subunits has been confirmed by electrophoretic mobility, lack of proline, and the peptide map generated by limited proteolysis. None of these techniques have detected tropomyosin in the corresponding 442 preparations. Our results suggest that the transformation of normal rat kidney cells has led to an overall reduction in tropomyosin content. This may be related to the inability of 442 cells to organize filamentous actin stress fibers.     

Release of host-derived membrane vesicles following pilus-mediated adhesion of Neisseria gonorrhoeae

Following attachment of Neisseria gonorrhoeae to human epithelial cell lines, the cellular pilus receptor CD46 is shed from the cell and accumulates in the media. In this report, we assess Neisseria-induced alterations in CD46 surface distribution and characterize this complement regulatory protein following its release from the infected cell. Within 3 h of attachment of gonococci to human epithelial cell lines, CD46 is enriched beneath sites of microcolony adhesion. By 6 h post infection, differential ultracentrifugation of culture media from ME-180 monolayers resulted in sedimentation of structurally and functionally intact CD46. Electron microscopy of these 100,000 g pellets revealed 30-200 nm vesicles. These vesicles likely originated from the host cell as they contained additional host cell surface proteins including CD55 and the epidermal growth factor receptor. Further, these vesicles were visualized by quick-freeze, deep-etch electron microscopy in association with the surface of infected ME-180 cells and with pili of adherent gonococci. Like CD46 shedding, CD46 redistribution and vesicle release were insensitive to colchicine and cytochalasin-D but dependent on expression of the pilus retraction protein PilT. This vesiculation may represent a host cell defence response in which surface proteins that are commonly exploited by pathogens, such as CD46, are removed from the cell.     

Coexpression of RTI40 with alveolar epithelial type II cell proteins in lungs following injury: identification of alveolar intermediate cell types

Injured alveolar epithelial type (AT) I cells are replaced following the proliferation and transformation of ATII cells to new ATI cells. RTI(40) is an ATI cell-specific protein required for normal lung development. We hypothesized that intermediate cell types in the ATII-to-ATI cell transformation would coexpress RTI(40) and ATII cell-selective proteins. To test this hypothesis, we used a rat model of Staphylococcus aureus-induced acute lung injury and a panel of ATI and ATII cell-specific and -selective antibodies. S. aureus induced an acute inflammatory reaction that was resolving by day 3 postinoculation. At day 3 postinoculation, the alveolar wall was thickened secondary to ATII cell hyperplasia. With the use of confocal microscopy, there was a fivefold increase in the fractional surface area of alveolar walls stained with ATII cell membrane proteins (RTII(70) and MMC4) and a decrease in the fractional surface area associated with RTI(40)-expressing cells. S. aureus-treated lungs also contained unique cell types that coexpressed the RTI(40) and ATII markers RTI(40)/MMC4/RTII(70)- and RTI(40)/MMC4-positive cells. These cells were not observed in control lungs. RTI(40)/MMC4-positive cells were also found in cultured ATII cells before they transformed to an ATI-like phenotype. Our data suggest that RTI(40)/MMC4/RTII(70)- and RTI(40)/MMC4-positive cells are intermediates in the ATII-to-ATI cell transformation. These data also suggest that the coexpression of RTI(40) with ATII cell proteins may be used to identify and investigate ATII cell transdifferentiation to ATI cells following injury.     

Effect of membrane cholesterol on dimyristoylphosphatidylcholine-induced vesiculation of human red blood cells

During incubation of intact human erythrocytes with sonicated dimyristoylphosphatidylcholine (DMPC) vesicles, the cells change their discoid morphology to form echinocytes and finally give rise to the release of membrane vesicles. In this process, the red cell membrane accumulates DMPC and loses up to 15% of its cholesterol. On the other hand, replacement of 25% of the endogenous phosphatidylcholine species by DMPC without affecting the cholesterol level of the erythrocytes can be achieved by incubation with DMPC/cholesterol (1:1, mol/mol) sonicated vesicles in the presence of the phosphatidylcholine-specific phospholipid-transfer protein from bovine liver. This replacement also gives rise to an echinocytic cell morphology, but no membrane vesiculation can be observed. However, the vesiculation process can as yet be initiated upon a subsequent decrease of the cholesterol level, by incubation of those modified cells in the presence of sonicated vesicles of pure egg phosphatidylcholine. Incubation of native erythrocytes with pure egg phosphatidylcholine vesicles, on the other hand, results in cholesterol depletion, but does neither induce the formation of echinocytes nor the release of membrane vesicles. Cellular ATP levels are not affected during these incubations. From these results, it can be concluded that a decrease in cholesterol content of the erythrocyte membrane is essential for the DMPC-induced vesiculation of those cells.     

Polarized endocytosis by Madin-Darby canine kidney cells transfected with functional chicken liver glycoprotein receptor

We have studied the expression of the chicken hepatic glycoprotein receptor (chicken hepatic lectin [CHL]) in Madin-Darby canine kidney (MDCK) cells, by transfection of its cDNA under the control of a retroviral promotor. Transfected cell lines stably express 87,000 surface receptors/cell with a kd = 13 nM. In confluent monolayers, approximately 40% of CHL is localized at the plasma membrane. 98% of the surface CHL is expressed at the basolateral surface where it performs polarized endocytosis and degradation of glycoproteins carrying terminal N-acetylglucosamine at a rate of 50,000 ligand molecules/h. Studies of the half-life of metabolically labeled receptor and of the stability of biotinylated cell surface receptor after internalization indicate that transfected CHL performs several rounds of uptake and recycling before it gets degraded. The successful expression of a functional basolateral receptor in MDCK cells opens the way for the characterization of the mechanisms that control targeting and recycling of proteins to the basolateral membrane of epithelial cells.     

Oligomerization state of dynamin 2 in cell membranes using TIRF and number and brightness analysis

Dynamin 2 is an ubiquitously expressed ∼100 kDa GTPase involved in receptor-mediated endocytosis, Golgi budding, and cytoskeletal reorganization. Dynamin molecules assemble around the necks of budding vesicles and constrict membranes in a GTP-dependent process, resulting in vesicle release. The oligomerization state of dynamin 2 in the membrane is still controversial. We investigated dynamin 2 within the plasma membrane of live cells using total internal reflection microscopy coupled with number and brightness analysis. Our results demonstrate that dynamin 2 is primarily tetrameric throughout the entire cell membrane, aside from punctate structures that may correspond to regions of membrane vesiculation.