Signal integration in chemoreceptor complexes.

Motile bacteria use large receptor arrays to detect chemical and physical stimuli in their environment, process this complex information, and accordingly bias their swimming in a direction they deem favorable. The chemoreceptor molecules form tripod-like trimers of receptor dimers through direct contacts between their cytoplasmic tips. A pair of trimers, together with a dedicated kinase enzyme, form a core signaling complex. Hundreds of core complexes network to form extended arrays. While considerable progress has been made in revealing the hierarchical structure of the array, the molecular properties underlying signal processing in these structures remain largely unclear. Here we analyzed the signaling properties of nonnetworked core complexes in live cells by following both conformational and kinase control responses to attractant stimuli and to output-biasing lesions at various locations in the receptor molecule. Contrary to the prevailing view that individual receptors are binary two-state devices, we demonstrate that conformational coupling between the ligand binding and the kinase-control receptor domains is, in fact, only moderate. In addition, we demonstrate communication between neighboring receptors through their trimer-contact domains that biases them to adopt similar signaling states. Taken together, these data suggest a view of signaling in receptor trimers that allows significant signal integration to occur within individual core complexes.

Long-term positioning and polar preference of chemoreceptor clusters in E. coli.

The bacterial chemosensory arrays are a notable model for studying the basic principles of receptor clustering and cellular organization. Here, we provide a new perspective regarding the long-term dynamics of these clusters in growing E. coli cells. We demonstrate that pre-existing lateral clusters tend to avoid translocation to pole regions and, therefore, continually shuttle between the cell poles for many generations while being static relative to the local cell-wall matrix. We also show that the polar preference of clusters results fundamentally from reduced clustering efficiency in the lateral region, rather than a developmental-like progression of clusters. Furthermore, polar preference is surprisingly robust to structural alterations designed to probe preference due to curvature sorting, perturbing the cell envelope physiology affects the cluster-size distribution, and the size-dependent mobility of receptor complexes differs between polar and lateral regions. Thus, distinct envelope physiology in the polar and lateral cell regions may contribute to polar preference.

Dynamic Clustering of the Bacterial Sensory Kinase BaeS.

Several bacterial sensory-kinase receptors form clusters on the cell membrane. However, the dynamics of sensory-kinase clustering are largely unclear. Using measurements of fluorescence anisotropy and time-lapse imaging of Escherichia coli cells, we demonstrate that copper ions trigger self-association of BaeS receptors and lead to rapid formation of clusters, which can be reversibly dispersed by a metal chelator. Copper ions did not trigger self-association of other fluorescently tagged sensory kinases, and other divalent metal ions could not elicit self-association of BaeS. The histidine residues in the BaeS periplasmic domain are essential for copper binding in vitro and are important for the copper-induced BaeS responses in vivo. BaeS clustering was triggered also under conditions that directly triggered BaeS-dependent transcriptional responses. Thus, clustering of sensory kinase receptors can be dynamic and context dependent and can be triggered by specific environmental cues.

The role of motility and chemotaxis in the bacterial colonization of protected surfaces.

Internal epithelial surfaces in humans are both oxygenated and physically protected by a few hundred microns thick hydrogel mucosal layer, conditions that might support bacterial aerotaxis. However, the potential role of aerotaxis in crossing such a thin hydrogel layer is not clear. Here, we used a new setup to study the potential role of motility and chemotaxis in the bacterial colonization of surfaces covered by a thin hydrogel layer and subjected to a vertical oxygen gradient. Using the bacterium Escherichia coli, we show that both non-motile and motile-but-non-chemotactic bacteria could barely reach the surface. However, an acquired mutation in the non-chemotactic bacteria that altered their inherent swimming behavior led to a critical enhancement of surface colonization. Most chemotactic strains accumulated within the bulk of the hydrogel layer, except for the MG1655 strain, which showed a unique tendency to accumulate directly at the oxygenated surface and thus exhibited distinctly enhanced colonization. Even after a long period of bacterial growth, non-motile bacteria could not colonize the hydrogel. Thus, switching motility, which can be spontaneously acquired or altered in vivo, is critical for the colonization of such protected surfaces, whereas aerotaxis capacity clearly expedites surface colonization, and can lead to diverse colonization patterns.

The sensory histidine kinases TorS and EvgS tend to form clusters in Escherichia coli cells.

Microorganisms use multiple two-component sensory systems to detect changes in their environment and elicit physiological responses. Despite their wide spread and importance, the intracellular organization of two-component sensory proteins in bacteria remains little investigated. A notable exception is the well-studied clustering of the chemoreceptor-kinase complexes that mediate chemotaxis behaviour. However, these chemosensory complexes differ fundamentally from other systems, both structurally and functionally. Therefore, studying the organization of typical sensory kinases in bacteria is essential for understanding the general role of receptor clustering in bacterial sensory signalling. Here, by studying mYFP-tagged sensory kinases in Escherichia coli, we show that the tagged TorS and EvgS sensors have a clear tendency for self-association and clustering. These sensors clustered even when expressed at a level of a few hundred copies per cell. Moreover, the mYFP-tagged response regulator TorR showed clear TorS-dependent clustering, indicating that untagged TorS sensors also tend to form clusters. We also provide evidence for the functionality of these tagged sensors. Experiments with truncated TorS or EvgS proteins suggested that clustering of EvgS sensors depends on the cytoplasmic part of the protein, whereas clustering of TorS sensors can be potentially mediated by the periplasmic/transmembrane domain. Overall, these findings support the notion that sensor clustering plays a role in bacterial sensory signalling beyond chemotaxis.

The physical and functional thermal sensitivity of bacterial chemoreceptors.

The bacterium Escherichia coli exhibits chemotactic behavior at temperatures ranging from approximately 20 °C to at least 42 °C. This behavior is controlled by clusters of transmembrane chemoreceptors made from trimers of dimers that are linked together by cross-binding to cytoplasmic components. By detecting fluorescence energy transfer between various components of this system, we studied the underlying molecular behavior of these receptors in vivo and throughout their operating temperature range. We reveal a sharp modulation in the conformation of unclustered and clustered receptor trimers and, consequently, in kinase activity output. These modulations occurred at a characteristic temperature that depended on clustering and were lower for receptors at lower adaptational states. However, in the presence of dynamic adaptation, the response of kinase activity to a stimulus was sustained up to 45 °C, but sensitivity notably decreased. Thus, this molecular system exhibits a clear thermal sensitivity that emerges at the level of receptor trimers, but both receptor clustering and adaptation support the overall robust operation of the system at elevated temperatures.

Disrupted gene pattern in patients with repeated in vitro fertilization (IVF) failure.

BACKGROUND: Repeated implantation failure (RIF) is a severe obstacle in human assisted reproduction treatment. METHODS: Aiming to identify global gene profile in RIF patients, gene-array analyses were performed on endometrial samples collected on day 21 of the cycle from fertile women (n = 12) and from RIF patients (n = 20). Validation of cyclin E2, Slug, dickkopf homolog 1 (DKK1), lymphoid enhancer-binding factor 1 (LEF1) and secreted frizzled-related protein 1 (SFRP1) was carried out by real-time PCR. RESULTS: Gene-array analysis revealed 313 genes exhibiting modified expression levels in RIF patients. Of these, 288 genes (92%) were down-regulated and only 25 genes (8%) were up-regulated. Classification of the down-regulated genes to biological pathways revealed cell cycle, Wnt signaling and cellular adhesion pathways. Real-time PCR validation of cyclin E2, SFRP1 and LEF1 showed significantly lower expression levels in RIF-IVF patients as compared with fertile women. In addition, two up-regulated genes, Slug and DKK1, were also validated. Interestingly, about 8% of the down-regulated genes were estrogen-dependent. Western blot of estrogen receptor alpha revealed low expression of this protein in the RIF group. CONCLUSIONS: The evaluation of the endometrium of RIF patients by gene array analysis demonstrates that the expression of various genes is altered, including those belonging to the cell cycle, Wnt signaling and cellular adhesion pathways.

Transcription of steroidogenic acute regulatory protein in the rodent ovary and placenta: alternative modes of cyclic adenosine 3', 5'-monophosphate dependent and independent regulation.

Steroid hormone synthesis is a vital function of the adrenal cortex, serves a critical role in gonadal function, and maintains pregnancy if normally executed in the placenta. The substrate for the synthesis of all steroid hormones is cholesterol, and its conversion to the first steroid, pregnenolone, by the cholesterol side-chain cleavage cytochrome P450 (CYP11A1) enzyme complex takes place in the inner mitochondrial membranes. Steroidogenic acute regulatory protein (STAR) facilitates the rate-limiting transfer of cholesterol from the outer mitochondrial membrane to CYP11A1 located in the inner organelle membranes. The current study explored the mechanisms controlling transcription of the Star gene in primary cell cultures of mouse placental trophoblast giant cells and rat ovarian granulosa cells examined throughout the course of their functional differentiation. Our findings show that the cis-elements required for Star transcription in the rodent placenta and the ovary are centered in a relatively small proximal region of the promoter. In placental trophoblast giant cells, cAMP is required for activation of the Star promoter, and the cis-elements mediating a maximal response were defined as cAMP response element 2 and GATA. EMSA studies show that placental cAMP-responsive element binding protein (CREB)-1 and activating transcription factor-2 (ATF2) bind to a -81/-78 sequence, whereas GATA-2 binds to a -66/-61 sequence. In comparison, patterns of Star regulation in the ovary suggested tissue-specific and developmental controlled modes of Star transcription. During the follicular phase, FSH/cAMP induced CREB-1 dependent activity, whereas upon luteinization STAR expression becomes cAMP and CREB independent, a functional shift conferred by FOS-related antigen-2 displacement of CREB-1 binding, and the appearance of a new requirement for CCAAT enhancer-binding protein beta and steroidogenic factor 1 that bind to upstream elements (-117/-95). These findings suggest that during evolution, the promoters of the Star gene acquired nonconsensus sequence elements enabling expression of a single gene in different organs, or allowing dynamic temporal changes corresponding to progressing phases of differentiation in a given cell type.

Changing etiology of tubal pregnancy following IVF.

BACKGROUND: Tubal pregnancy (TP) is twice as common following IVF when compared with natural conception. This is surprising, since embryo transfer is aimed for an accurate area in the uterine cavity. We thus hypothesized that either the embryo or the Fallopian tube actively participates in a pathological process leading to implantation outside the uterine cavity. Since we recently found that E-cadherin expression is a useful marker of endometrial receptivity, we considered that it may have a role in TP following IVF. Therefore, the aim of this study was to compare E-cadherin expression and localization in tubal implantation sites from spontaneous TP and TP post-IVF. METHODS: We compared E-cadherin immunohistochemistry levels on cross-sections of Fallopian tubes in 11 spontaneous (antegrade) versus 13 post-IVF (retrograde) TP. The intensity of immunoreactivity was scored in a semi-qualitative blinded manner. RESULTS: The semi-quantitative intensity score in IVF tubal samples was more than double that observed in spontaneous TP (16.9 versus 7.3, respectively, P < 0.0005). E-cadherin showed the most intense immunostaining in cytotrophoblast cells of chorionic villi in ectopic TP post-IVF compared with negative or weak staining in spontaneous ectopic TP. CONCLUSIONS: E-cadherin can serve as a marker of implantation. Differential expression of this adhesion molecule in TP post-IVF, when compared with natural conception, may reflect a different mechanism of embryo implantation. Moreover, the observation that E-cadherin is mostly expressed in trophoblasts, and not in the tubal wall, suggests that the preimplantation embryo may actively participate in locating a suitable implantation site.

Heparanase improves mouse embryo implantation.

OBJECTIVE: To improve mouse embryonic implantation by recombinant heparanase supplementation. Heparanase, an endoglycosidase-degrading heparan sulfate proteoglycan, may have a role in embryonic implantation because of its enzymatic, angiogenic, and adhesive properties. Increasing endometrial receptivity could improve one of the most difficult pathologies in human fertility. DESIGN: Comparison between mouse blastocysts obtained after 24-hour incubation of morulae with or without heparanase. SETTING: Experimental laboratory in a medical center. ANIMAL(S): Mice. INTERVENTION(S): Morulae were flushed from CB6F1 female mice and incubated for 24 hours at 37 degrees C in M16 medium supplemented with 0.1 mg/mL heparanase (n = 203), with albumin (n = 60), or with medium alone (n = 258). MAIN OUTCOME MEASURE(S): Blastocysts were evaluated by heparanase immunostaining (n = 10), activity assay (n = 283), and transfer to foster mice uterine horns (n = 228). The number of implantation sites was compared. RESULT(S): Immunostaining demonstrated that heparanase is constitutively expressed in mouse morulae and blastocyts. Heparanase supplementation resulted in increased staining and enzymatic activity in blastocyts. Implantation rates for the heparanase, M16 medium, and albumin groups, were 36.9%, 17.8%, and 20%, respectively (P<.01). CONCLUSION(S): Heparanase was found to be constitutively expressed by blastocyst-stage embryos. Moreover, the amount of heparanase was markedly increased by incubation of morulae with recombinant heparanase, evaluated by immunostaining and enzymatic activity. Heparanase supplementation resulted in approximately a twofold increase in embryo implantation rate in vivo. Taken together, these data suggest that heparanase is actively involved in embryo implantation.

Mouse embryos generated from frozen-thawed oocytes can successfully survive a second cryopreservation.

BACKGROUND: To determine whether mouse embryos generated from frozen-thawed oocytes can successfully survive a second cryopreservation. METHODS: Immature C57BL6*BALB/c female mice underwent superovulation and the collected oocytes were divided into three groups. Group A oocytes (n = 107) underwent IVF. Group B oocytes (n = 167) underwent IVF and embryos generated were then cryopreserved. Group C oocytes (n = 94) were cryopreserved, thawed and underwent IVF. Two-four-cell stage embryos were re-cryopreserved and thawed. Embryos from all groups were then cultured to the blastocyst stage. RESULTS: Cleavage rates to the 2-4-cell stage were 78, 71 and 46% for groups A, B and C respectively. Blastulation rates from 2-4 cell-stage embryos were 37/83 (45%), 27/118 (23%) and 8/35 (23%) for groups A, B and C respectively. Development to blastocysts was observed in 37/107 oocytes (35%), 27/167 oocytes (16%) and only 8/94 oocytes (9%) for groups A, B and C respectively. CONCLUSION: Oocyte cryopreservation results in reduced fertilization rates. Embryo cryopreservation reduces blastulation rates by half regardless of whether the oocytes were fertilized fresh or frozen-thawed. Nevertheless, embryos generated from cryopreserved oocytes can survive cryopreservation and develop to the blastocyst stage at rates comparable with embryos obtained from fresh oocytes.

Transcription of cholesterol side-chain cleavage cytochrome P450 in the placenta: activating protein-2 assumes the role of steroidogenic factor-1 by binding to an overlapping promoter element.

Progesterone is essential to the sustenance of pregnancy in humans and other mammals. From the second trimester on, the human placenta is the sole origin of de novo synthesized steroid hormones. In mice, placentation at midgestation is accompanied by a temporal rise of steroid hormone synthesis commencing in the giant cells of the mouse trophoblast. In doing so, the giant trophoblasts, as any other steroidogenic cell, express high levels of the key steroidogenic enzyme, cholesterol side-chain cleavage cytochrome P450 (P450scc). Because steroidogenic factor 1 (SF-1), the transcription factor required for expression of P450scc in the adrenals and the gonads, is not expressed in the placenta, we hypothesized that placenta-specific nuclear factor(s) (PNF) assumes the role of SF-1 by binding to the same promoter region that harbors the SF-1 recognition site in the P450scc gene. To address this possibility, we used SCC1, a well conserved proximal region in the P450scc genes (-60/-32 in the rat gene) to purify PNF from human term placenta. Sequencing of the purified PNF revealed that it is the alpha isoform of the human activating protein-2 (AP-2alpha). Specific antibodies tested in EMSA confirmed that AP-2alpha is the predominant isoform that binds SCC1 in the human placenta, whereas AP-2gamma is the only mouse placental protein that binds this oligonucleotide. Functional studies showed that coexpression of the rat P450scc promoter (-378/+8 CAT) and AP-2 isoforms (alpha or gamma) in human embryonic kidney 293 cells results in a marked activation of chloramphenicol acetyltransferase (CAT) transcription that is dependent on an intact AP-2 motif, GCCTTGAGC. This motif conforms with consensus sequences previously determined for binding of the AP-2 alpha and gamma isoforms. Mutations of the AP-2 element ablated binding of AP-2 to SCC1, as well as severely diminished the promoter activity in primary mouse giant trophoblasts and human choriocarcinoma JAR cells. Collectively, these studies suggest that expression of placental P450scc is governed by AP-2 factors that bind to a cis-element that largely overlaps the sequence required for recognition of SF-1 in other steroidogenic tissues.

Uterine and placental expression of steroidogenic genes during rodent pregnancy.

The ontogeny and functional role of steroidogenesis during mammalian gestation is poorly understood. This review provides a summary of our recent findings on the spatio-temporal expression of key steroidogenic genes controlling progesterone synthesis in the uterus during mouse pregnancy. We have shown that onset of cholesterol side chain cleavage cytochrome P450 (P450scc) and a newly identified isoform of murine 3beta-hydroxysteroid dehydrogenase/isomerase type VI (3betaHSD VI) expression occurs upon decidualization of the uterine wall induced by implantation. This unexpected early expression of the enzymes in the maternal decidua is terminated at mid-pregnancy when the steroidogenic ability reappears in the extraembryonic giant cells at the time of placentation. The giant cells express another protein indispensable for steroid hormone synthesis in the adrenal and gonads, Steroidogenic Acute Regulatory (StAR) protein. Unlike the human placenta, the steroidogenic genes are not expressed in the cells of the mature mouse placenta during the second half of gestation. Finally, our studies suggest that transcriptional regulation of P450scc is mediated by a non-SF-1 protein that substitutes SF-1 functions in the extraembryonic cells. Collectively, the results of the present study suggest that, during early phases of pregnancy, local progesterone synthesis in the maternal decidua and the trophoblast layers surrounding the embryonal cavity is important for successful implantation and/or maintenance of pregnancy. We propose that the local production of progesterone acts as an immunosuppressant at the maternofetal interface preventing the rejection of the fetal allograft.