Lats2/Kpm is required for embryonic development, proliferation control and genomic integrity

The Drosophila melanogaster warts/lats tumour suppressor has two mammalian counterparts LATS1/Warts-1 and LATS2/Kpm. Here, we show that mammalian Lats orthologues exhibit distinct expression profiles according to germ cell layer origin. Lats2(-/-) embryos show overgrowth in restricted tissues of mesodermal lineage; however, lethality ultimately ensues on or before embryonic day 12.5 preceded by defective proliferation. Lats2(-/-) mouse embryonic fibroblasts (MEFs) acquire growth advantages and display a profound defect in contact inhibition of growth, yet exhibit defective cytokinesis. Lats2(-/-) embryos and MEFs display centrosome amplification and genomic instability. Lats2 localizes to centrosomes and overexpression of Lats2 suppresses centrosome overduplication induced in wild-type MEFs and reverses centrosome amplification inherent in Lats2(-/-) MEFs. These findings indicate an essential role of Lats2 in the integrity of processes that govern centrosome duplication, maintenance of mitotic fidelity and genomic stability.     

The p11 subunit of annexin II heterotetramer is regulated by basic carboxypeptidase

The Ca(2+)-dependent phospholipid-binding protein annexin II heterotetramer (AIIt) is composed of two copies of annexin II and a p11 dimer. The interaction of the carboxyl-terminal lysine residues of the p11 subunit of AIIt with the lysine-binding kringle domains of plasminogen is believed to play a key role in plasminogen binding and stimulation of the tPA-catalyzed cleavage of plasminogen to plasmin. In the current report, we show that AIIt-stimulated plasminogen activation is regulated by basic carboxypeptidases, in vitro. The incubation of AIIt with a 1/400 molar ratio of carboxypeptidase B for periods as short as 2 min resulted in a significant loss in AIIt-stimulated plasminogen activation. Carboxypeptidase B (CpB) as well as thrombin-activated fibrinolysis inhibitor (TAFIa) and carboxypeptidase N (CpN) rapidly reduced AIIt-stimulated plasminogen activation by 80%. The molar ratio of carboxypeptidase/AIIt for half-maximal inhibition of AIIt was 1/4700, 1/700, and 1/500 for CpB, TAFIa, and CpN, respectively. Treatment of AIIt with carboxypeptidase resulted in loss of both carboxyl-terminal lysine residues from the p11 subunit, which correlated with a decrease in the k(cat) and an increase in the K(m) for plasminogen activation. The data reveal a novel mechanism for the regulation of AIIt-stimulated plasminogen activation.     

Morphometric analysis on ecomorphologically equivalent cichlid species from Lakes Malawi and Tanganyika

Landmark-based geometric morphometric techniques were used to test the hypothesis that Petrochromis spp. from Lake Tanganyika are ecomorphologically equivalent to Petrotilapia spp. from Lake Malawi. Both genera are epilithic algal feeders and inhabit the rocky shores of their respective lakes. We investigated the morphological component of the ecomorphology hypothesis by investigating body shape, using landmark-based morphometric techniques. A MANOVA revealed significant differences among species and an ordination of all species along the first two CV axes showed clear separation of the two genera in the morphospace with Petrochromis fasciolatus as an intermediate. A thin-plate spline analysis revealed that Petrochromis spp. had a deeper, broader anterior body, larger gape, shorter anal fin base and narrower caudal peduncle than Petrotilapia spp. Basically, differences between lakes were found, but there were no similarities or clusters of presumptive ecomorphs. Based on such results, we reject the hypothesis of morphological equivalence between these two genera. However, considering the non-significant difference in body shape revealed between P. fasciolatus , Petrotilapia genalutea and Petrotilapia 'mumbo blue', we conclude that these three species represent morphological equivalence and hence display a best example of convergent evolution.     

Adaptive Autoimmunity and Foxp3-Based Immunoregulation in Zebrafish

Background

Jawed vertebrates generate their immune-receptor repertoire by a recombinatorial mechanism that has the potential to produce harmful autoreactive lymphocytes. In mammals, peripheral tolerance to self-antigens is enforced by Foxp3⁺ regulatory T cells. Recombinatorial mechanisms also operate in teleosts, but active immunoregulation is thought to be a late incorporation to the vertebrate lineage.

Methods/Principal Findings

Here we report the characterization of adaptive autoimmunity and Foxp3-based immunoregulation in the zebrafish. We found that zebrafish immunization with an homogenate of zebrafish central nervous system (zCNS) triggered CNS inflammation and specific antibodies. We cloned the zebrafish ortholog for mammalian Foxp3 (zFoxp3) which induced a regulatory phenotype on mouse T cells and controlled IL-17 production in zebrafish embryos.

Conclusions/Significance

Our findings demonstrate the acquisition of active mechanisms of self-tolerance early in vertebrate evolution, suggesting that active regulatory mechanisms accompany the development of the molecular potential for adaptive autoimmunity. Moreover, they identify the zebrafish as a tool to study the molecular pathways controlling adaptive immunity.     

TIM-4 expressed on APCs induces T cell expansion and survival

TIM (T cell, Ig, mucin) proteins can regulate T cell immune responses. Tim-4 mRNA is not expressed in T cells, but exclusively in APCs. Tim-4 is a ligand for Tim-1 and Tim-4.Ig fusion protein was shown to either inhibit or expand T cells. However, the molecular basis for such opposite effects was not defined. By generating mAbs, we show that expression of Tim-4 protein is restricted to CD11c(+) and CD11b(+) cells and is up-regulated upon activation. We show that Tim-4 specifically phosphorylates Tim-1 and induces T cell expansion by enhancing cell division and reducing apoptosis. Tim-4 also induces the phosphorylation of signaling molecules LAT, Akt, and ERK1/2 in T cells. Tim-4, expressed on APCs, is a costimulatory molecule that promotes T cell expansion and survival by cross-linking Tim-1 on T cells.     

p22 is a novel plasminogen fragment with antiangiogenic activity.

Tumor or tumor-associated cells cleave circulating plasminogen into three or four kringle-containing antiangiogenic fragments, collectively referred to as angiostatin. Angiostatin blocks tumor growth and metastasis by preventing the growth of endothelial cells that are critical for tumor vascularization. Here, we show that cancer and normal cells convert plasminogen into a novel 22 kDa fragment (p22). Production of this plasminogen fragment in a cell-free system has allowed characterization of the structure and activity of the protein. p22 consists of amino acid residues 78-180 of plasminogen and therefore embodies the first plasminogen kringle (residues 84-162) as well as additional N- and C-terminal residues. Circular dichroism and intrinsic fluorescence spectrum analysis have defined structural differences between p22 and recombinant plasminogen kringle 1 (rK1), therefore suggesting a unique conformation for kringle 1 within p22. Proliferation of capillary endothelial cells but not cells of other lineages was selectively inhibited by p22 in vitro. In addition, p22 prevented vascular growth of chick chorioallantoic membranes (CAMs) in vivo. Furthermore, administration of p22 at low dose suppressed the growth of murine Lewis lung carcinoma (LLC) metastatic foci in vivo. This is the first identification of a single kringle-containing antiangiogenic plasminogen fragment produced under physiological conditions.     

Regulation of plasmin-dependent fibrin clot lysis by annexin II heterotetramer

In a previous report we showed that plasmin-dependent lysis of a fibrin polymer, produced from purified components, was totally blocked if annexin II heterotetramer (AIIt) was present during fibrin polymer formation. Here, we show that AIIt inhibits fibrin clot lysis by stimulation of plasmin autodegradation, which results in a loss of plasmin activity. Furthermore, the C-terminal lysine residues of its p11 subunit play an essential role in the inhibition of fibrin clot lysis by AIIt. We also found that AIIt binds to fibrin with a K(d) of 436 nm and a stoichiometry of about 0.28 mol of AIIt/mol of fibrin monomer. The binding of AIIt to fibrin was not dependent on the C-terminal lysines of the p11 subunit. Furthermore, in the presence of plasminogen, the binding of AIIt to fibrin was increased to about 1.3 mol of AIIt/mol of fibrin monomer, suggesting that AIIt and plasminogen do not compete for identical sites on fibrin. Immunohistochemical identification of p36 and p11 subunits of AIIt in a pathological clot provides important evidence for its role as a physiological fibrinolytic regulator. These results suggest that AIIt may play a key role in the regulation of plasmin activity on the fibrin clot surface.