Visualization of endothelial cell cycle dynamics in mouse using the Flt-1/eGFP-anillin system.

Endothelial cell proliferation is a key process during vascular growth but its kinetics could only be assessed in vitro or ex vivo so far. To enable the monitoring and quantification of cell cycle kinetics in vivo, we have generated transgenic mice expressing an eGFP-anillin construct under control of the endothelial-specific Flt-1 promoter. This construct labels the nuclei of endothelial cells in late G1, S and G2 phase and changes its localization during the different stages of M phase, thereby enabling the monitoring of EC proliferation and cytokinesis. In Flt-1/eGFP-anillin mice, we found eGFP+ signals specifically in Ki67+/PECAM+ endothelial cells during vascular development. Quantification using this cell cycle reporter in embryos revealed a decline in endothelial cell proliferation between E9.5 to E12.5. By time-lapse microscopy, we determined the length of different cell cycle phases in embryonic endothelial cells in vivo and found a M phase duration of about 80 min with 2/3 covering karyokinesis and 1/3 cytokinesis. Thus, we have generated a versatile transgenic system for the accurate assessment of endothelial cell cycle dynamics in vitro and in vivo.

Live monitoring of small vessels during development and disease using the flt-1 promoter element.

Vessel formation is of critical importance for organ function in the normal and diseased state. In particular, the labeling and quantitation of small vessels prove to be technically challenging using current approaches. We have, therefore, established a transgenic embryonic stem (ES) cell line and a transgenic mouse model where the vascular endothelial growth factor receptor VEGFR-1 (flt-1) promoter drives the expression of the live reporter eGFP. Fluorescence microscopy and immunostainings revealed endothelial-specific eGFP labeling of vascular networks. The expression pattern recapitulates that of the endogenous flt-1 gene, because small and large vessels are labeled by eGFP during embryonic development; after birth, the expression becomes more restricted to small vessels. We have explored this in the cardiovascular system more in detail and found that all small vessels and capillaries within the heart are strongly eGFP+. In addition, myocardial injuries have been induced in transgenic mice and prominent vascular remodeling, and an increase in endothelial cell area within the peri-infarct area could be observed underscoring the utility of this mouse model. Thus, the transgenic flt-1/eGFP models are powerful tools to investigate and quantify vascularization in vivo and to probe the effect of different compounds on vessel formation in vitro.

Transmembrane segment II of NhaA Na+/H+ antiporter lines the cation passage, and Asp65 is critical for pH activation of the antiporter.

The crystal structure of Escherichia coli NhaA determined at pH 4 has provided insights into the mechanism of activity of a pH-regulated Na+/H+ antiporter. However, because NhaA is activated at physiological pH (pH 5.5-8.5), many questions related to the active state of NhaA have remained elusive. Our experimental results at physiological pH and computational analyses reveal that amino acid residues in transmembrane segment II contribute to the cation pathway of NhaA and its pH regulation: 1) transmembrane segment II is a highly conserved helix and the conserved amino acid residues are located on one side of the helix facing either the cytoplasmic or periplasmic funnels of NhaA structure. 2) Cys replacements of the conserved residues and measuring their antiporter activity in everted membrane vesicles showed that D65C, L67C, E78C, and E82C increased the apparent K(m) to Na+ and Li+ and changed the pH response of the antiporter. 3) Introduced Cys replacements, L60C, N64C, F71C, F72C, and E78C, were significantly alkylated by [14C]N-ethylmaleimide implying the presence of water-filled cavities in NhaA. 4) Several Cys replacements were modified by MTSES and/or MTSET, membrane impermeant, negatively and positively charged reagents, respectively, that could reach Cys replacements from the periplasm only via water-filled funnel(s). Remarkably, the reactivity of D65C to MTSES increased with increasing pH and chemical modification by MTSES but not by MTSET, decreased the apparent K(m) of the antiporter at pH 7.5 (10-fold) but not at pH 8.5, implying the importance of Asp(65) negative charge for pH activation of the antiporter.

PECAM/eGFP transgenic mice for monitoring of angiogenesis in health and disease.

For the monitoring of vascular growth as well as adaptive or therapeutic (re)vascularization endothelial-specific reporter mouse models are valuable tools. However, currently available mouse models have limitations, because not all endothelial cells express the reporter in all developmental stages. We have generated PECAM/eGFP embryonic stem (ES) cell and mouse lines where the reporter gene labels PECAM+ endothelial cells and vessels with high specificity. Native eGFP expression and PECAM staining were highly co-localized in vessels of various organs at embryonic stages E9.5, E15.5 and in adult mice. Expression was found in large and small arteries, capillaries and in veins but not in lymphatic vessels. Also in the bone marrow arteries and sinusoidal vessel were labeled, moreover, we could detect eGFP in some CD45+ hematopoietic cells. We also demonstrate that this labeling is very useful to monitor sprouting in an aortic ring assay as well as vascular remodeling in a murine injury model of myocardial infarction. Thus, PECAM/eGFP transgenic ES cells and mice greatly facilitate the monitoring and quantification of endothelial cells ex vivo and in vivo during development and injury.

Beta-sheet-dependent dimerization is essential for the stability of NhaA Na+/H+ antiporter.

A structural model of the NhaA dimer showed that a beta-hairpin of each monomer combines to form a beta-sheet at the periplasmic side of the membrane. By Cys scanning the entire beta-hairpin and testing each Cys replacement for functionality and intermolecular cross-linking, we found that Gln47 and Arg49 are critical for the NhaA dimer and that K57C causes an acidic shift of 1 pH unit to the pH dependence of NhaA. Comparing the growth of the NhaA variants with the previously isolated beta-hairpin deleted mutant (Delta(P45-N58)) and the wild type validated that NhaA dimers have an advantage over monomers in growth under extreme stress conditions and unraveled that during this growth the apparent Km for Na+ of Delta(P45-N58) was increased 50-fold as compared with the wild type. Remarkably, the effect of the extreme stress on the NhaA variants is reversible. Testing the temperature stability (4-55 degrees C) of the NhaA variants in dodecyl maltoside micells showed that the mutants impaired in dimerization were much less temperature-stable than the wild type. We suggest that NhaA dimers are crucial for the stability of the antiporter under extreme stress conditions.

NhaA crystal structure: functional-structural insights.

Na(+)/H(+) antiporters are integral membrane proteins that exchange Na(+) for H(+) across the cytoplasmic membrane and many intracellular membranes. They are essential for Na(+), pH and volume homeostasis, which are crucial processes for cell viability. Accordingly, antiporters are important drug targets in humans and underlie salt-resistance in plants. Many Na(+)/H(+) antiporters are tightly regulated by pH. Escherichia coli NhaA Na(+)/H(+) antiporter, a prototype pH-regulated antiporter, exchanges 2 H(+) for 1 Na(+) (or Li(+)). The NhaA crystal structure has provided insights into the pH-regulated mechanism of antiporter action and opened up new in silico and in situ avenues of research. The monomer is the functional unit of NhaA yet the dimer is essential for the stability of the antiporter under extreme stress conditions. Ionizable residues of NhaA that strongly interact electrostatically are organized in a transmembrane fashion in accordance with the functional organization of the cation-binding site, ;pH sensor', the pH transduction pathway and the pH-induced conformational changes. Remarkably, NhaA contains an inverted topology motive of transmembrane segments, which are interrupted by extended mid-membrane chains that have since been found to vary in other ion-transport proteins. This novel structural fold creates a delicately balanced electrostatic environment in the middle of the membrane, which might be essential for ion binding and translocation. Based on the crystal structure of NhaA, a model structure of the human Na(+)/H(+) exchanger (NHE1) was constructed, paving the way to a rational drug design.

Functional and structural interactions of the transmembrane domain X of NhaA, Na+/H+ antiporter of Escherichia coli, at physiological pH.

The 3D structure of Escherichia coli NhaA, determined at pH 4, provided the first structural insights into the mechanism of antiport and pH regulation of a Na+/H+ antiporter. However, because NhaA is activated at physiological pH (pH 7.0-8.5), many questions pertaining to the active state of NhaA have remained open, including the physiological role of helix X. Using a structural-based evolutionary approach in silico, we identified a segment of most conserved residues in the middle of helix X. These residues were then used as targets for functional studies at physiological pH. Cysteine-scanning mutagenesis showed that Gly303, in the middle of the conserved segment, is an essential residue and Cys replacement of Lys300 retains only Li+/H+ antiporter activity, with a 20-fold increase in the apparent KM for Li+. Cys replacements of Leu296 and Gly299 increase the apparent KM of the Na+/H+ antiporter for both Na+ and Li+. Accessibility test to N-ethylmaleimide and 2-sulfonatoethyl methanethiosulfonate showed that G299C, K300C, and G303C are accessible to the cytoplasm. Suppressor mutations and site-directed chemical cross-linking identified a functional and/or structural interaction between helix X (G295C) and helix IVp (A130C). While these results were in accordance with the acid-locked crystal structure, surprisingly, conflicting data were also obtained; E78C of helix II cross-links very efficiently with several Cys replacements of helix X, and E78K/K300E is a suppressor mutation of K300E. These results reveal that, at alkaline pH, the distance between the conserved center of helix X and E78 of helix II is drastically decreased, implying a pH-induced conformational change of one or both helices.

Model structure of the Na+/H+ exchanger 1 (NHE1): functional and clinical implications.

Eukaryotic Na(+)/H(+) exchangers are transmembrane proteins that are vital for cellular homeostasis and play key roles in pathological conditions such as cancer and heart diseases. Using the crystal structure of the Na(+)/H(+) antiporter from Escherichia coli (EcNhaA) as a template, we predicted the three-dimensional structure of human Na(+)/H(+) exchanger 1 (NHE1). Modeling was particularly challenging because of the extremely low sequence identity between these proteins, but the model structure is supported by evolutionary conservation analysis and empirical data. It also revealed the location of the binding site of NHE inhibitors; which we validated by conducting mutagenesis studies with EcNhaA and its specific inhibitor 2-aminoperimidine. The model structure features a cluster of titratable residues that are evolutionarily conserved and are located in a conserved region in the center of the membrane; we suggest that they are involved in the cation binding and translocation. We also suggest a hypothetical alternating-access mechanism that involves conformational changes.

Roles of NhaA, NhaB, and NhaD Na+/H+ antiporters in survival of Vibrio cholerae in a saline environment.

Vibrio cholerae, the causative agent of cholera, is a normal inhabitant of aquatic environments, where it survives in a wide range of conditions of pH and salinity. In this work, we investigated the role of three Na+/H+ antiporters on the survival of V. cholerae in a saline environment. We have previously cloned the Vc-nhaA gene encoding the V. cholerae homolog of Escherichia coli. Here we identified two additional antiporter genes, designated Vc-nhaB and Vc-nhaD, encoding two putative proteins of 530 and 477 residues, respectively, highly homologous to the respective antiporters of Vibrio species and E. coli. We showed that both Vc-NhaA and Vc-NhaB confer Na+ resistance and that Vc-NhaA displays an antiport activity in E. coli, which is similar in magnitude, kinetic parameters, and pH regulation to that of E. coli NhaA. To determine the roles of the Na+/H+ antiporters in V. cholerae, we constructed nhaA, nhaB, and nhaD mutants (single, double, and triple mutants). In contrast to E. coli, the inactivation of the three putative antiporter genes (Vc-nhaABD) in V. cholerae did not alter the bacterial exponential growth in the presence of high Na+ concentrations and had only a slight effect in the stationary phase. In contrast, a pronounced and similar Li+-sensitive phenotype was found with all mutants lacking Vc-nhaA during the exponential phase of growth and also with the triple mutant in the stationary phase of growth. By using 2-n-nonyl-4-hydroxyquinoline N-oxide, a specific inhibitor of the electron-transport-linked Na+ pump NADH-quinone oxidoreductase (NQR), we determined that in the absence of NQR activity, the Vc-NhaA Na+/H+ antiporter activity becomes essential for the resistance of V. cholerae to Na+ at alkaline pH. Since the ion pump NQR is Na+ specific, we suggest that its activity masks the Na+/H+ but not the Li+/H+ antiporter activities. Our results indicate that the Na+ resistance of the human pathogen V. cholerae requires a complex molecular system involving multiple antiporters and the NQR pump.

Unraveling functional and structural interactions between transmembrane domains IV and XI of NhaA Na+/H+ antiporter of Escherichia coli.

A functionally important, interface domain between transmembrane segments (TMSs) IV and XI of the NhaA Na+/H+ antiporter of Escherichia coli has been unraveled. Scanning by single Cys replacements identified new mutations (F136C, G125C, and A137C) that cluster in one face of TMS IV and increase dramatically the Km of the antiporter. Whereas G125C, in addition, causes a drastic alkaline shift to the pH dependence of the antiporter, G338C alleviates the pH control of NhaA. Scanning by double Cys replacements (21 pairs of one replacement per TMS) identified genetically eight pairs of residues that showed very strong negative complementation. Cross-linking of the double mutants identified six double mutants (T132C/G338C, D133C/G338C, F136C/S342C, T132C/S342C, A137C/S342C, and A137C/G338C) of which pronounced intramolecular cross-linking defined an interface domain between the two TMSs. Remarkably, cross-linking by a short and rigid reagent (N,N'-o-phenylenedimaleimide) revived the Li+/H+ antiport activity, whereas a shorter reagent (1,2-ethanediyl bismethanethiosulfonate) revived both Na+/H+ and Li+/H+ antiporter activities and even the pH response of the dead mutant T132C/G338C. Hence, cross-linking at this position restores an active conformation of NhaA.