Generation of Transgenic Fluorescent Reporter Lines for Studying Hematopoietic Development in the Mouse.

Hematopoiesis in the mouse and other mammals occurs in several waves and arises from distinct anatomic sites. Transgenic mice expressing fluorescent reporter proteins at various points in the hematopoietic hierarchy, from hematopoietic stem cell to more restricted progenitors to each of the final differentiated cell types, have provided valuable tools for tagging, tracking, and isolating these cells. In this chapter, we discuss general considerations in designing a transgene, survey available fluorescent probes, and describe methods for confirming and analyzing transgene expression in the hematopoietic tissues of the embryo, fetus, and postnatal/adult animal.

Generation of transgenic mouse fluorescent reporter lines for studying hematopoietic development.

During the development of the hematopoietic system, at least eight distinct lineages are generated in the mouse embryo. Transgenic mice expressing fluorescent proteins at various points in the hematopoietic hierarchy, from hematopoietic stem cell to multipotent progenitors to each of the final differentiated cell types, have provided valuable tools for tagging, tracking, and isolating these cells. In this chapter, we discuss general considerations in designing a transgene and survey available fluorescent probes and methods for confirming and analyzing transgene expression in the hematopoietic systems of the embryo, fetus, and postnatal/adult animal.

Phosphoketolases from Lactococcus lactis, Leuconostoc mesenteroides and Pseudomonas aeruginosa: dissimilar sequences, similar substrates but distinct enzymatic characteristics.

Phosphoketolases (PKs) are large thiamine pyrophosphate (TPP)-dependent enzymes playing key roles in a number of essential pathways of carbohydrate metabolism. The putative PK genes of Lactococcus lactis (Ll) and Leuconostoc mesenteroides (Lm) were cloned in a prokaryotic vector, and the encoded proteins were expressed and purified yielding high purity proteins termed PK-Ll and PK-Lm, respectively. Similarly, the PK gene of Pseudomonas aeruginosa was expressed, and the corresponding protein (PK-Pa) was purified to homogeneity. The amino acid sequences predicted on the basis of genes' nucleotide sequences were confirmed by mass spectrometry and display low relative similarities. Circular dichroism (CD) spectra of these proteins predict higher α-helix than ß-strand contents. In addition, it is predicted that PK-Ll contains tightly packed domains. Enzymatic analysis showed that all three recombinant proteins, despite their dissimilar amino acid sequences, are active PKs and accept both xylulose 5-phosphate (X5P) and fructose 6-phosphate (F6P) as substrates. However, they display substantially higher preference for X5P than for F6P. Kinetic measurements indicated that PK-Pa has the lowest Km values for X5P and F6P suggesting the highest capacity for substrate binding. PK-Ll has the largest kcat values for both substrates. Nevertheless, in terms of substrate specificity constant, PK-Pa has been found to be the most active PK against X5P. Structural models for all three analysed PKs predict similar folds in spite of amino acid sequence dissimilarities and contribute to understanding the enzymatic peculiarities of PK-Pa compared to PK-Ll and PK-Lm.

Analysis of primitive erythroid cell proliferation and enucleation using a cyan fluorescent reporter in transgenic mice.

Primitive erythropoiesis is a vital process for mammalian embryonic development. Here we report the generation and characterization of a new transgenic mouse line that expresses a histone H2B-CFP fusion protein in the nuclei of primitive erythroid cells. We demonstrate the potential of this ε-globin-histone H2B-CFP line for multicolor imaging and flow cytometry analysis. The ε-globin-H2B-CFP line was used to analyze the cell cycle distribution and proliferation of CFP-expressing primitive erythroblasts from E8.5-E13.5. We also evaluated phagocytosis of extruded CFP-positive nuclei by macrophages in fetal liver and placenta. The ε-globin-H2B-CFP transgenic mouse line adds to the available tools for studying the development of the primitive erythroid lineage.

Serine dephosphorylation of receptor protein tyrosine phosphatase alpha in mitosis induces Src binding and activation.

Receptor protein tyrosine phosphatase alpha (RPTPalpha) is the mitotic activator of the protein tyrosine kinase Src. RPTPalpha serine hyperphosphorylation was proposed to mediate mitotic activation of Src. We raised phosphospecific antibodies to the two main serine phosphorylation sites, and we discovered that RPTPalpha Ser204 was almost completely dephosphorylated in mitotic NIH 3T3 and HeLa cells, whereas Ser180 and Tyr789 phosphorylation were only marginally reduced in mitosis. Concomitantly, Src pTyr527 and pTyr416 were dephosphorylated, resulting in 2.3-fold activation of Src in mitosis. Using inhibitors and knockdown experiments, we demonstrated that dephosphorylation of RPTPalpha pSer204 in mitosis was mediated by PP2A. Mutation of Ser204 to Ala did not activate RPTPalpha, and intrinsic catalytic activity of RPTPalpha was not affected in mitosis. Interestingly, binding of endogenous Src to RPTPalpha was induced in mitosis. GRB2 binding to RPTPalpha, which was proposed to compete with Src binding to RPTPalpha, was only modestly reduced in mitosis, which could not account for enhanced Src binding. Moreover, we demonstrate that Src bound to mutant RPTPalpha-Y789F, lacking the GRB2 binding site, and mutant Src with an impaired Src homology 2 (SH2) domain bound to RPTPalpha, illustrating that Src binding to RPTPalpha is not mediated by a pTyr-SH2 interaction. Mutation of RPTPalpha Ser204 to Asp, mimicking phosphorylation, reduced coimmunoprecipitation with Src, suggesting that phosphorylation of Ser204 prohibits binding to Src. Based on our results, we propose a new model for mitotic activation of Src in which PP2A-mediated dephosphorylation of RPTPalpha pSer204 facilitates Src binding, leading to RPTPalpha-mediated dephosphorylation of Src pTyr527 and pTyr416 and hence modest activation of Src.

Catalytically active membrane-distal phosphatase domain of receptor protein-tyrosine phosphatase alpha is required for Src activation.

Receptor protein-tyrosine phosphatase alpha (RPTPalpha) is a transmembrane protein with tandem cytoplasmic phosphatase domains. Most of the catalytic activity is contained by the membrane-proximal catalytic domain (D1). We found a spontaneous Arg554 to His mutation in the pTyr recognition loop of the membrane-distal phosphatase domain (D2) of a human patient. This mutation was not linked to the disease. Here, we report that the R554H mutation abolished RPTPalpha-D2 catalytic activity. The R554H mutation impaired Src binding to RPTPalpha. RPTPalpha, with a catalytic site cysteine to serine mutation in D2, also displayed diminished binding to Src. Concomitant with decreased Src binding of the R554H and C723S mutants compared with wild-type RPTPalpha, enhanced phosphorylation of the inhibitory Src Tyr527 site was observed, as well as reduced Src activation. To confirm that catalytic activity of RPTPalpha-D2 was required for these effects, we analyzed a third mutant, RPTPalpha-R729K, which had an inactive D2. Again, Src binding was reduced and Tyr527 phosphorylation was enhanced. Our results suggest that a catalytically active D2 is required for RPTPalpha to bind and dephosphorylate its well-characterized substrate, Src.

Analysis of molecular determinants of PRL-3.

In order to analyse whether a C-terminal polybasic sequence represents a nuclear localization signal (NLS) we obtained several truncated and mutant forms of protein of regerating liver (PRL)-3 and evaluated their subcellular localization as compared to the wild-type form. Our results invalidate the hypothesis that this is an NLS. We also analysed the influence of the C- and N-terminal residues on the phosphatase activity of PRL-3. Our results provide in vitro evidence that the C-terminal CAAX motif, besides directing the protein farnesylation, plays an additional regulatory role by inhibiting the catalytic efficiency of PRL-3. Taking into account the results we obtained, as well as reported data, we propose a hypothetical molecular mechanism for the nucleocytoplasmic localization and transfer of PRL-3.

Laminin-alpha4 and integrin-linked kinase mutations cause human cardiomyopathy via simultaneous defects in cardiomyocytes and endothelial cells.

BACKGROUND: Extracellular matrix proteins, such as laminins, and endothelial cells are known to influence cardiomyocyte performance; however, the underlying molecular mechanisms remain poorly understood. METHODS AND RESULTS: We used a forward genetic screen in zebrafish to identify novel genes required for myocardial function and were able to identify the lost-contact (loc) mutant, which encodes a nonsense mutation in the integrin-linked kinase (ilk) gene. This loc/ilk mutant is associated with a severe defect in cardiomyocytes and endothelial cells that leads to severe myocardial dysfunction. Additional experiments revealed the epistatic regulation between laminin-alpha4 (Lama4), integrin, and Ilk, which led us to screen for mutations in the human ILK and LAMA4 genes in patients with severe dilated cardiomyopathy. We identified 2 novel amino acid residue-altering mutations (2828C>T [Pro943Leu] and 3217C>T [Arg1073X]) in the integrin-interacting domain of the LAMA4 gene and 1 mutation (785C>T [Ala262Val]) in the ILK gene. Biacore quantitative protein/protein interaction data, which have been used to determine the equilibrium dissociation constants, point to the loss of integrin-binding capacity in case of the Pro943Leu (Kd=5+/-3 micromol/L) and Arg1073X LAMA4 (Kd=1+/-0.2 micromol/L) mutants compared with the wild-type LAMA4 protein (Kd=440+/-20 nmol/L). Additional functional data point to the loss of endothelial cells in affected patients as a direct consequence of the mutant genes, which ultimately leads to heart failure. CONCLUSIONS: This is the first report on mutations in the laminin, integrin, and ILK system in human cardiomyopathy, which has consequences for endothelial cells as well as for cardiomyocytes, thus providing a new genetic basis for dilated cardiomyopathy in humans.