微生物细胞表面工程研究进展*

微生物细胞表面工程是近年来发展起来的,它利用细胞表面展示技术使外源蛋白固定化于细胞表面,从而生产微生物细胞表面蛋白。微生物细胞表面工程可用于细胞催化剂、细胞吸附剂、活疫苗、生物传感器的开发等。微生物细胞表面工程具有广阔的应用前景,但是国内对这一领域的研究尚刚起步。在介绍了细胞表面工程的基础上,对微生物细胞表面工程技术进展进行了综述,并对该技术的发展给予展望。     

微生物细胞表面呈现技术研究进展

微生物细胞表面工程是近年来发展起来的,它利用细胞表面展示技术使外源蛋白固定化于细胞表面,从而生产微生物细胞表面蛋白。微生物细胞表面工程可用于细胞催化剂、细胞吸附剂、活疫苗、生物传感器的开发等。微生物细胞表面工程具有广阔的应用前景,但是国内对这一领域的研究刚起步。在介绍细胞表面工程的基础上,对微生物细胞表面工程技术进展进行了综述,展望了对该技术的发展。     

Single-cell unroofing: probing topology and nanomechanics of native membranes

Cell membranes separate the cell interior from the external environment. They are constituted by a variety of lipids; their composition determines the dynamics of membrane proteins and affects the ability of the cells to adapt. Even though the study of model membranes allows to understand the interactions among lipids and the overall mechanics, little is known about these properties in native membranes. To combine topology and nanomechanics analysis of native membranes, I designed a method to investigate the plasma membranes isolated from a variety of single cells. Five cell types were chosen and tested, revealing 20% variation in membrane thickness. I probed the resistance of the isolated membranes to indent, finding their line tension and spreading pressure. These results show that membranes isolated from neurons are stiffer and less diffusive than brain cancer cell membranes. This method gives direct quantitative insights on the mechanics of native cell membranes.     

Heat-shock protein 72 cell-surface expression on human lung carcinoma cells is associated with an increased sensitivity to lysis mediated by adherent natural killer cells

 The cell-surface expression patterns of major histocompatibility complex (MHC) class I, class II and heat-shock protein 72 (HSP72) molecules were measured on human lung (LX-1) and mammary (MX-1) carcinoma cells. No major differences were found in the MHC cell-surface expression pattern of both cell lines. However, they differ significantly in their capacity to express HSP72 on their cell surface. Under physiological conditions LX-1 cells express HSP72 molecules on more than 90% of the cells, whereas MX-1 cells exhibit no significant HSP72 cell-surface expression (less than 5%). These expression patterns remained stable in all further cell passages tested. The sensitivity to lysis mediated by an interleukin-2 (IL-2)-stimulated, adherent natural killer (NK) cell population could be correlated with the amount of cell-surface-expressed HSP72 molecules. By antibody-blocking studies, using HSP72-specific monoclonal antibody (mAb), a strong inhibition of lysis was only found with LX-1 cells but not with MX-1 cells. In contrast to the cell-surface expression, the cytoplasmic amount of HSP72 in MX-1 cells was twice as high compared to LX-1 cells under physiological conditions. After nonlethal heat-shock the rate of induction and the total cytoplasmic amounts of HSP72 were comparable in both cell lines. The clonogenic cell viability of LX-1 cells after incubation at temperatures ranging from 41°C to 44°C was significantly elevated compared to that of MX-1 cells. In conclusion we state the following: (i) HSP72 cell-surface expression on human carcinoma cells is independent of the cytoplasmic amount of HSP72; (ii) the cell-surface expression of HSP72 is associated with an increased sensitivity of tumour cells to lysis mediated by an IL-2-stimulated, adherent NK cell population; (iii) thermoresistance is not related to the cytoplasmic HSP72 level but might be related to the amount of HSP72 expressed on the cell surface. Received: 20 June 1996 / Accepted: 25 September 1996     

Cell monolayer deformation microscopy reveals mechanical fragility of cell monolayers following EMT

Tissue and cell mechanics are crucial factors in maintaining homeostasis and in development, with aberrant mechanics contributing to many diseases. During the epithelial-to-mesenchymal transition (EMT), a highly conserved cellular program in organismal development and cancer metastasis, cells gain the ability to detach from their original location and autonomously migrate. While a great deal of biochemical and biophysical changes at the single-cell level have been revealed, how the physical properties of multicellular assemblies change during EMT, and how this may affect disease progression, is unknown. Here we introduce cell monolayer deformation microscopy (CMDM), a new methodology to measure the planar mechanical properties of cell monolayers by locally applying strain and measuring their resistance to deformation. We employ this new method to characterize epithelial multicellular mechanics at early and late stages of EMT, finding the epithelial monolayers to be relatively compliant, ductile, and mechanically homogeneous. By comparison, the transformed mesenchymal monolayers, while much stiffer, were also more brittle, mechanically heterogeneous, displayed more viscoelastic creep, and showed sharp yield points at significantly lower strains. Here, CMDM measurements identify specific biophysical functional states of EMT and offer insight into how cell aggregates fragment under mechanical stress. This mechanical fingerprinting of multicellular assemblies using new quantitative metrics may also offer new diagnostic applications in healthcare to characterize multicellular mechanical changes in disease.     

Transcriptional regulation by the numbers: models

The expression of genes is regularly characterized with respect to how much, how fast, when and where. Such quantitative data demands quantitative models. Thermodynamic models are based on the assumption that the level of gene expression is proportional to the equilibrium probability that RNA polymerase (RNAP) is bound to the promoter of interest. Statistical mechanics provides a framework for computing these probabilities. Within this framework, interactions of activators, repressors, helper molecules and RNAP are described by a single function, the "regulation factor". This analysis culminates in an expression for the probability of RNA polymerase binding at the promoter of interest as a function of the number of regulatory proteins in the cell.     

Biomechanics of cell rolling: shear flow, cell-surface adhesion, and cell deformability

The mechanics of leukocyte (white blood cell; WBC) deformation and adhesion to endothelial cells (EC) has been investigated using a novel in vitro side-view flow assay. HL-60 cell rolling adhesion to surface-immobilized P-selectin was used to model the WBC-EC adhesion process. Changes in flow shear stress, cell deformability, or substrate ligand strength resulted in significant changes in the characteristic adhesion binding time, cell-surface contact and cell rolling velocity. A 2-D model indicated that cell-substrate contact area under a high wall shear stress (20 dyn/cm2) could be nearly twice of that under a low stress (0.5 dyn/cm2) due to shear flow-induced cell deformation. An increase in contact area resulted in more energy dissipation to both adhesion bonds and viscous cytoplasm, whereas the fluid energy that inputs to a cell decreased due to a flattened cell shape. The model also predicted a plateau of WBC rolling velocity as flow shear stresses further increased. Both experimental and computational studies have described how WBC deformation influences the WBC-EC adhesion process in shear flow.     

A soluble interleukin 2 receptor produced by a normal alloreactive human T cell clone binds interleukin 2 with low affinity

Several alloreactive human T cell clones derived from a rejected kidney graft were found to produce in their culture supernatants soluble interleukin 2 receptors (IL-2R) upon specific antigenic challenge (irradiated B cell line from the graft's donor). Among them, the 2B11, a high producer clone, was used to purify a soluble IL-2R preparation which was analyzed, in comparison with the high and low affinity cell-surface IL-2R expressed by 2B11 cells, for its parameters of interaction with a set of anti-IL-2R monoclonal antibodies (mAb) and IL-2. This soluble receptor purified by affinity chromatography (anti-IL-2R mAb column) and sodium dodecyl sulfate gel electrophoresis is composed of a single chain of 35,000 to 45,000 Da. Immunoradiometric assays (IRMA) at equilibrium were set up, using pairs of mAb directed against two separate epitopes on the Tac antigen of the human IL-2R, to measure the respective dissociation constant of these mAb for the soluble IL-2R. They were found to be identical to those found on the cell-surface IL-2R. A 1:1 stoichiometry between the two epitopes were found both on the membrane and soluble species. Competition experiments between membrane and soluble IL-2R for binding the mAb allowed the quantitative analysis of the concentration of soluble IL-2R without the need of amino acid analysis on purified material and set up a quantitative IRMA for the human soluble IL-2R (detection limit 5 pM). The affinity of the soluble IL-2R for IL-2 was determined by various techniques including an IRMA using an anti-IL-2R mAb and radiolabeled IL-2. The results obtained led us to conclude that the soluble IL-2R binds IL-2 with a dissociation constant (KD = 30 nM) identical to that found for the binding of IL-2 to low affinity cell-surface IL-2R (Tac antigen). Whereas 2.5% of cell-surface IL-2R expressed 2 days after antigenic stimulation of 2B11 cells were of high affinity for IL-2 (KD = 25 pM), no (less than 0.07%) high affinity binding sites could be detected on the purified soluble IL-2R. This soluble IL-2R therefore likely corresponds to a truncated, extracellular part of the membrane Tac antigen. The amounts of soluble Tac antigen produced by the 2B11 alloreactive human T cell clone did not exceed 1 nM and, as expected from the binding studies, did not affect IL-2-induced T cell proliferation. The physiologic and pathologic implications of our results are discussed.     

Leukemia inhibitory factor (LIF) concentration modulates embryonic stem cell self-renewal and differentiation independently of proliferation

A major limitation of the widespread use of stem cells in a variety of biotechnological applications is the relatively low level of knowledge about how to maintain these cells in vitro without losing the long-term multilineage growth properties required for their clinical utility. An experimental and theoretical framework for predicting and controlling the outcome of stem cell stimulation by exogenous cytokines would thus be useful. An emerging theme from recent hematopoietic stem cell (HSC)-expansion studies is that a net gain in HSC numbers requires the maintenance of critical signaling ligand(s) above a threshold level. These ligand-receptor complex thresholds can be maintained, for example, by high concentrations of soluble cytokines or by cytokine presentation on cell surfaces. According to such a model, when the relevant ligand-receptor interaction falls below this threshold level, the probability of a differentiation response is increased; otherwise, self-renewal is favored. Taking advantage of the ability of the cytokine leukemia inhibitory factor (LIF) to maintain embryonic stem (ES) cell pluripotentiality at high concentrations, we are testing this model by investigating critical parameters in the control of ES cell responses. We have developed quantitative assays of ES cell differentiation by measuring cell-surface alkaline phosphatase activity, cell-surface stage specific embryonic antigen (SSEA)-1 expression, and the ability of ES cells to form embryoid bodies. Examination of ES cell responses over a range of LIF concentrations shows that LIF supplementation has little effect on ES cell-growth rate but significantly alters the probability of a cell undergoing a self-renewal vs. a differentiation division. In vitro culture parameters such as inoculum cell density, medium exchange, as well as cell-intrinsic processes such as autocrine secretion are shown to affect this decision. In addition to yielding new information on stem cell regulation by exogenous factors, these studies provide important clues about culture of these cells and should stimulate further investigations into the mechanistic basis of stem cell differentiation control.     

A 50-Hz magnetic field induces structural and biophysical changes in membranes

The effects of a 50-Hz extremely low frequency magnetic field on cultured K562 cells growing in suspension were studied by means of scanning electron microscopy and electron paramagnetic resonance spectroscopy. Exposure of K562 cells at 2.5 mT for periods to 96 hours induced significant changes in cell-surface structure and physiology without modification of proliferative capability as indicated by quantitative analysis. Thus extremely low frequency fields seem able to induce injurious, sublethal cell alterations, and the plasma membrane seems to play an important role in this effect. © 1993 Wiley-Liss, Inc.     

A peptide filtering relation quantifies MHC class I peptide optimization

Major Histocompatibility Complex (MHC) class I molecules enable cytotoxic T lymphocytes to destroy virus-infected or cancerous cells, thereby preventing disease progression. MHC class I molecules provide a snapshot of the contents of a cell by binding to protein fragments arising from intracellular protein turnover and presenting these fragments at the cell surface. Competing fragments (peptides) are selected for cell-surface presentation on the basis of their ability to form a stable complex with MHC class I, by a process known as peptide optimization. A better understanding of the optimization process is important for our understanding of immunodominance, the predominance of some T lymphocyte specificities over others, which can determine the efficacy of an immune response, the danger of immune evasion, and the success of vaccination strategies. In this paper we present a dynamical systems model of peptide optimization by MHC class I. We incorporate the chaperone molecule tapasin, which has been shown to enhance peptide optimization to different extents for different MHC class I alleles. Using a combination of published and novel experimental data to parameterize the model, we arrive at a relation of peptide filtering, which quantifies peptide optimization as a function of peptide supply and peptide unbinding rates. From this relation, we find that tapasin enhances peptide unbinding to improve peptide optimization without significantly delaying the transit of MHC to the cell surface, and differences in peptide optimization across MHC class I alleles can be explained by allele-specific differences in peptide binding. Importantly, our filtering relation may be used to dynamically predict the cell surface abundance of any number of competing peptides by MHC class I alleles, providing a quantitative basis to investigate viral infection or disease at the cellular level. We exemplify this by simulating optimization of the distribution of peptides derived from Human Immunodeficiency Virus Gag-Pol polyprotein.     

Differential effects of 20-hydroxyecdysone on cell interactions and surface proteins in Drosophila cell lines

20-Hydroxyecdysone induces different cellular and biochemical responses in the Drosophila cell lines L3 and S3. The hormonal response in S3 cells includes mitotic arrest and aggregation, whereas L3 cells undergo mitotic arrest without aggregation. The possible involvement of 20-OH-ecdysone-modulated cell-surface proteins in mediating aggregation prompted us to compare the effects of hormonal stimulation on cell-surface proteins in these two cell lines. Radiolabeling of the cell-surface proteins revealed seven polypeptides modulated by 20-OH-ecdysone in S3 cells and three polypeptides so modulated in L3 cells. Increased and decreased labeling, as well as changes in migration of specific polypeptides on two-dimensional gels, were caused by the hormone. Analysis of radiolabelled cell-surface proteins by SDS-polyacrylamide gel electrophoresis revealed nine bands which were affected by 20-OH-ecdysone in S3 cells, whereas only three bands were altered by 20-OH-ecdysone in L3 cells. These observations are compared to earlier reports on the 20-OH-ecdysone-dependent modulation of cell-surface proteins in imaginal discs and other cell lines of Drosophila. We suggest that at least some of the cell-surface proteins which are modulated by 20-OH-ecdysone specifically in S3 cells may be mediators of the increase in cell-cell adhesions which occurs during hormone exposure.     

The spatial and mechanical challenges of female meiosis

Recent work shows that cytokinesis and other cellular morphogenesis events are tuned by an interplay among biochemical signals, cell shape, and cellular mechanics. In cytokinesis, this includes cross-talk between the cortical cytoskeleton and the mitotic spindle in coordination with cell cycle control, resulting in characteristic changes in cellular morphology and mechanics through metaphase and cytokinesis. The changes in cellular mechanics affect not just overall cell shape, but also mitotic spindle morphology and function. This review will address how these principles apply to oocytes undergoing the asymmetric cell divisions of meiosis I and II. The biochemical signals that regulate cell cycle timing during meiotic maturation and egg activation are crucial for temporal control of meiosis. Spatial control of the meiotic divisions is also important, ensuring that the chromosomes are segregated evenly and that meiotic division is clearly asymmetric, yielding two daughter cells - oocyte and polar body - with enormous volume differences. In contrast to mitotic cells, the oocyte does not undergo overt changes in cell shape with its progression through meiosis, but instead maintains a relatively round morphology with the exception of very localized changes at the time of polar body emission. Placement of the metaphase-I and -II spindles at the oocyte periphery is clearly important for normal polar body emission, although this is likely not the only control element. Here, consideration is given to how cellular mechanics could contribute to successful mammalian female meiosis, ultimately affecting egg quality and competence to form a healthy embryo.     

Human retinoblastoma: in vitro differentiation and immunoglobulin superfamily antigen modulation by retinoic acid

 Suspension and attachment cultures of Y79 human retinoblastoma cells were treated with all-trans retinoic acid (RA) for up to 10 days to assess its effect on growth and cell-surface expression of immunoglobulin superfamily antigens MHC class I and class II, ICAM-1, NCAM and Thy1. RA up to 10 μM induced growth inhibition, and marked morphological differentiation with extension of prominent processes resembling neurites was seen in attachment cultures. However, above 10 μM RA produced extensive cell death. We also observed increased cell-surface expression of MHC class I, ICAM-1, NCAM and Thy1 on Y79 cells treated with 10 μM over 10 days; constitutive MHC class II expression was not apparent, nor did RA treatment appear to induce Y79 cells to express MHC class immunoreactivity. The up-modulation of cell-adhesion molecules (NCAM, ICAM-1 and Thy1) and immune recognition molecules (NCAM, ICAM-1 and MHC class I), associated with reduced growth and tumour cell differentiation, suggests that RA may have a potential role in regulating the growth and development of retinoblastoma tumours. Received: 29 August 1996 / Accepted: 16 January 1997     

Cell-surface Processing of the Metalloprotease Pro-ADAMTS9 Is Influenced by the Chaperone GRP94/gp96

A disintegrin-like and metalloprotease domain with thrombospondin type 1 motifs 9 (ADAMTS9) is a highly conserved metalloprotease that has been identified as a tumor suppressor gene and is required for normal mouse development. The secreted ADAMTS9 zymogen undergoes proteolytic excision of its N-terminal propeptide by the proprotein convertase furin. However, in contrast to other metalloproteases, propeptide excision occurs at the cell surface and leads to decreased activity of the zymogen. Here, we investigated the potential cellular mechanisms regulating ADAMTS9 biosynthesis and cell-surface processing by analysis of molecular complexes formed by a construct containing the propeptide and catalytic domain of pro-ADAMTS9 (Pro-Cat) in HEK293F cells. Cross-linking of cellular proteins bound to Pro-Cat followed by mass spectrometric analysis identified UDP-glucose:glycoprotein glucosyltransferase I, heat shock protein gp96 (GRP94), BiP (GRP78), and ERdj3 (Hsp40 homolog) as associated proteins. gp96 and BiP were present at the cell surface in an immunoprecipitable complex with pro-ADAMTS9 and furin. Treatment with geldanamycin, an inhibitor of the HSP90α family (including gp96), led to decreased furin processing of pro-ADAMTS9 and accumulation of the unprocessed pro-ADAMTS9 at the cell surface. gp96 siRNA down-regulated the levels of cell-surface pro-ADAMTS9 and furin, whereas the levels of cell-surface pro-ADAMTS9, but not of cell-surface furin, were decreased upon treatment with BiP siRNA. These data identify for the first time the cellular chaperones associated with secretion of an ADAMTS protease and suggest a role for gp96 in modulating pro-ADAMTS9 processing.     

Changes in surface topologies of chondrocytes subjected to mechanical forces: an AFM analysis

The cartilage is composed of chondrocytes embedded in a matrix of collagen fibrils interspersed within a network of proteoglycans and is constantly exposed to biomechanical forces during normal joint movement. Characterization of the surface morphology, cytoskeletal structure, adherance and elastic properties of these mechanosensitive cells are crucial in understanding the effects of mechanical forces around a cell and how a cell responds to changes in its physical environment. In this work, we employed the atomic force microscope (AFM) to image cultured chondrocytes before and after subjecting them to mechanical forces in the presence or absence of interleukin-1β to mimic inflammatory conditions. Nanoscale imaging and quantitative measurements from AFM data revealed that there are distinct changes in cell-surface topology and cytoskeleton arrangement in the cells following treatment with mechanical forces, IL-1β or both. Our findings for the first time demonstrate that cultured chondrocytes are amenable to high-resolution AFM imaging and dynamic tensile forces may help overcome the effect of inflammatory factors on chondrocyte response.     

An AFM-based pit-measuring method for indirect measurements of cell-surface membrane vesicles

Circulating membrane vesicles, which are shed from many cell types, have multiple functions and have been correlated with many diseases. Although circulating membrane vesicles have been extensively characterized, the status of cell-surface membrane vesicles prior to their release is less understood due to the lack of effective measurement methods. Recently, as a powerful, micro- or nano-scale imaging tool, atomic force microscopy (AFM) has been applied in measuring circulating membrane vesicles. However, it seems very difficult for AFM to directly image/identify and measure cell-bound membrane vesicles due to the similarity of surface morphology between membrane vesicles and cell surfaces. Therefore, until now no AFM studies on cell-surface membrane vesicles have been reported. In this study, we found that air drying can induce the transformation of most cell-surface membrane vesicles into pits that are more readily detectable by AFM. Based on this, we developed an AFM-based pit-measuring method and, for the first time, used AFM to indirectly measure cell-surface membrane vesicles on cultured endothelial cells. Using this approach, we observed and quantitatively measured at least two populations of cell-surface membrane vesicles, a nanoscale population (<500 nm in diameter peaking at ∼250 nm) and a microscale population (from 500 nm to ∼2 μm peaking at ∼0.8 μm), whereas confocal microscopy only detected the microscale population. The AFM-based pit-measuring method is potentially useful for studying cell-surface membrane vesicles and for investigating the mechanisms of membrane vesicle formation/release.