Rad9 Is Required for B Cell Proliferation and Immunoglobulin Class Switch Recombination

B cell maturation and B cell-mediated antibody response require programmed DNA modifications such as the V(D)J recombination, the immunoglobulin (Ig) class switch recombination, and the somatic hypermutation to generate functional Igs. Many protein factors involved in DNA damage repair have been shown to be critical for the maturation and activation of B cells. Rad9 plays an important role in both DNA repair and cell cycle checkpoint control. However, its role in Ig generation has not been reported. In this study, we generated a conditional knock-out mouse line in which Rad9 is deleted specifically in B cells and investigated the function of Rad9 in B cells. The Rad9^−/− B cells isolated from the conditional knock-out mice displayed impaired growth response and enhanced DNA lesions. Impaired Ig production in response to immunization in Rad9^−/− mice was also detected. In addition, the Ig class switch recombination is deficient in Rad9^−/− B cells. Taken together, Rad9 plays dual roles in generating functional antibodies and in maintaining the integrity of the whole genome in B cells.     

A DNA Damage-Regulated BRCT-Containing Protein, TopBP1, Is Required for Cell Survival

BRCA1 carboxyl-terminal (BRCT) motifs are present in a number of proteins involved in DNA repair and/or DNA damage-signaling pathways. Human DNA topoisomerase II binding protein 1 (TopBP1) contains eight BRCT motifs and shares sequence similarity with the fission yeast Rad4/Cut5 protein and the budding yeast DPB11 protein, both of which are required for DNA damage and/or replication checkpoint controls. We report here that TopBP1 is phosphorylated in response to DNA double-strand breaks and replication blocks. TopBP1 forms nuclear foci and localizes to the sites of DNA damage or the arrested replication forks. In response to DNA strand breaks, TopBP1 phosphorylation depends on the ataxia telangiectasia mutated protein (ATM) in vivo. However, ATM-dependent phosphorylation of TopBP1 does not appear to be required for focus formation following DNA damage. Instead, focus formation relies on one of the BRCT motifs, BRCT5, in TopBP1. Antisense Morpholino oligomers against TopBP1 greatly reduced TopBP1 expression in vivo. Similar to that of ataxia telangiectasia-related protein (ATR), Chk1, or Hus1, downregulation of TopBP1 leads to reduced cell survival, probably due to increased apoptosis. Taken together, the data presented here suggest that, like its putative counterparts in yeast species, TopBP1 may be involved in DNA damage and replication checkpoint controls.     

Amoebic PI3K and PKC Is Required for Jurkat T Cell Death Induced by Entamoeba histolytica

The enteric protozoan parasite Entamoeba histolytica is the causative agent of human amebiasis. During infection, adherence of E. histolytica through Gal/GalNAc lectin on the surface of the amoeba can induce caspase-3-dependent or -independent host cell death. Phosphorylinositol 3-kinase (PI3K) and protein kinase C (PKC) in E. histolytica play an important function in the adhesion, killing, or phagocytosis of target cells. In this study, we examined the role of amoebic PI3K and PKC in amoeba-induced apoptotic cell death in Jurkat T cells. When Jurkat T cells were incubated with E. histolytica trophozoites, phosphatidylserine (PS) externalization and DNA fragmentation in Jurkat cells were markedly increased compared to those of cells incubated with medium alone. However, when amoebae were pretreated with a PI3K inhibitor, wortmannin before being incubated with E. histolytica, E. histolytica-induced PS externalization and DNA fragmentation in Jurkat cells were significantly reduced compared to results for amoebae pretreated with DMSO. In addition, pretreatment of amoebae with a PKC inhibitor, staurosporine strongly inhibited Jurkat T cell death. However, E. histolytica-induced cleavage of caspase-3, -6, and -7 were not inhibited by pretreatment of amoebae with wortmannin or staurosporin. In addition, we found that amoebic PI3K and PKC have an important role on amoeba adhesion to host compartment. These results suggest that amebic PI3K and PKC activation may play an important role in caspase-independent cell death in Entamoeba-induced apoptosis.     

Protection from Cell Death by mcl-1 Is Mediated by Membrane Hyperpolarization Induced By K+ Channel Activation

Mcl-1, a member of the Bcl-2 family, has been identified as an inhibitor of apoptosis induced by anticancer agents and radiation in myeloblastic leukemia cells. The molecular mechanism underlying this phenomenon, however, is not yet understood. In the present study, we report that hyperpolarization of the membrane potential is required for prevention of mcl-1 mediated cell death in murine myeloblastic FDC-P1 cells. In cells transfected with mcl-1, the membrane potential, measured by the whole-cell patch clamp, was hyperpolarized more than −30 mV compared with control cells. The membrane potential was repolarized by increased extracellular K⁺ concentration (56 mV per 10-fold change in K⁺ concentration). Using the cell-attached patch-clamp technique, K⁺ channel activity was 1.7 times higher in mcl-1 transfected cells (NP _o = 22.7 ± 3.3%) than control cells (NP _o = 13.2 ± 1.9%). Viabilities of control and mcl-1 transfected cells after treatment with the cytotoxin etoposide (20 μg/ml), were 37.9 ± 3.9% and 78.2 ± 2.0%, respectively. Suppression of K⁺ channel activity by 4-aminopyridine (4-AP) before etoposide treatment significantly reduced the viability of mcl-1 transfected cells to 49.0 ± 4.6%. These results indicate that as part of the prevention of cell death, mcl-1 causes a hyperpolarization of membrane potential through activation of K⁺ channel activity. Received: 30 March 1999/Revised: 20 July 1999     

DAAM1 Is a Formin Required for Centrosome Re-Orientation during Cell Migration

Background

Disheveled-associated activator of morphogenesis 1 (DAAM1) is a formin acting downstream of Wnt signaling that is important for planar cell polarity. It has been shown to promote proper cell polarization during embryonic development in both Xenopus and Drosophila. Importantly, DAAM1 binds to Disheveled (Dvl) and thus functions downstream of the Frizzled receptors. Little is known of how DAAM1 is localized and functions in mammalian cells. We investigate here how DAAM1 affects migration and polarization of cultured cells and conclude that it plays a key role in centrosome polarity.

Methodology/Principal Findings

Using a specific antibody to DAAM1, we find that the protein localizes to the acto-myosin system and co-localizes with ventral myosin IIB-containing actin stress fibers. These fibers are particularly evident in the sub-nuclear region. An N-terminal region of DAAM1 is responsible for this targeting and the DAAM1(1-440) protein can interact with myosin IIB fibers independently of either F-actin or RhoA binding. We also demonstrate that DAAM1 depletion inhibits Golgi reorientation in wound healing assays. Wound-edge cells exhibit multiple protrusions characteristic of unpolarized cell migration. Finally, in U2OS cells lines stably expressing DAAM1, we observe an enhanced myosin IIB stress fiber network which opposes cell migration.

Conclusions/Significance

This work highlights the importance of DAAM1 in processes underlying cell polarity and suggests that it acts in part by affecting the function of acto-myosin IIB system. It also emphasizes the importance of the N-terminal half of DAAM1. DAAM1 depletion strongly blocks centrosomal re-polarization, supporting the concept that DAAM1 signaling cooperates with the established Cdc42 associated polarity complex. These findings are also consistent with the observation that ablation of myosin IIB but not myosin IIA results in polarity defects downstream of Wnt signaling. The structure-function analysis of DAAM1 in cultured cells parallels more complex morphological events in the developing embryo.     

CLCA2 Interactor EVA1 Is Required for Mammary Epithelial Cell Differentiation

CLCA2 is a p53-, p63-inducible transmembrane protein that is frequently downregulated in breast cancer. It is induced during differentiation of human mammary epithelial cells, and its knockdown causes epithelial-to-mesenchymal transition (EMT). To determine how CLCA2 promotes epithelial differentiation, we searched for interactors using membrane dihybrid screening. We discovered a strong interaction with the cell junctional protein EVA1 (Epithelial V-like Antigen 1) and confirmed it by co-immunoprecipitation. Like CLCA2, EVA1 is a type I transmembrane protein that is regulated by p53 and p63. It is thought to mediate homophilic cell-cell adhesion in diverse epithelial tissues. We found that EVA1 is frequently downregulated in breast tumors and breast cancer cell lines, especially those of mesenchymal phenotype. Moreover, knockdown of EVA1 in immortalized human mammary epithelial cells (HMEC) caused EMT, implying that EVA1 is essential for epithelial differentiation. Both EVA1 and CLCA2 co-localized with E-cadherin at cell-cell junctions. The interacting domains were delimited by deletion analysis, revealing the site of interaction to be the transmembrane segment (TMS). The primary sequence of the CLCA2 TMS was found to be conserved in CLCA2 orthologs throughout mammals, suggesting that its interaction with EVA1 co-evolved with the mammary gland. A screen for other junctional interactors revealed that CLCA2 was involved in two different complexes, one with EVA1 and ZO-1, the other with beta catenin. Overexpression of CLCA2 caused downregulation of beta catenin and beta catenin-activated genes. Thus, CLCA2 links a junctional adhesion molecule to cytosolic signaling proteins that modulate proliferation and differentiation. These results may explain how attenuation of CLCA2 causes EMT and why CLCA2 and EVA1 are frequently downregulated in metastatic breast cancer cell lines.     

Lymphoid EVA1 Expression Is Required for DN1-DN3 Thymocytes Transition

Background

Thymus organogenesis and T lymphocyte development are accomplished together during fetal life. Proper development and maintenance of thymus architecture depend on signals generated by a sustained crosstalk between developing thymocytes and stromal elements. Any maturation impairment occurring in either cellular component leads to an aberrant thymic development. Gene expression occurring during T lymphocyte differentiation must be coordinated in a spatio-temporal fashion; one way in which this is achieved is through the regulation by cell-cell adhesion and interactions.

Principal Findings

We examined the role played by Epithelial V-like Antigen 1 (EVA1), an Ig adhesion molecule expressed on thymus epithelial cells (TEC) and immature thymocytes, in T cell development by employing RNA interference in vitro and in vivo models. Fetal liver derived haematopoietic progenitors depleted of Eva1, displayed a delayed DN1-DN3 transition and failed to generate CD4CD8 double positive T cells in OP9-DL1 coculture system. In addition, we could observe a coordinated Eva1 up-regulation in stromal and haematopoietic cells in coculture control experiments, suggesting a possible EVA1 involvement in TEC-haematopoietic cells crosstalk mechanisms. Similarly, Rag2-γc double knock out mice, transplanted with Eva1 depleted haematopoietic progenitors displayed a 10-fold reduction in thymus reconstitution and a time delayed thymocytes maturation compared to controls.

Conclusions

Our findings show that modulation of Eva1 expression in thymocytes is crucial for lymphocyte physiological developmental progression and stromal differentiation.     

Protection of Telomeres 1 Is Required for Telomere Integrity in the Moss Physcomitrella patens

In vertebrates, the single-stranded telomeric DNA binding protein Protection of Telomeres 1 (POT1) shields chromosome ends and prevents them from eliciting a DNA damage response. By contrast, Arabidopsis thaliana encodes two divergent full-length POT1 paralogs that do not exhibit telomeric DNA binding in vitro and have evolved to mediate telomerase regulation instead of chromosome end protection. To further investigate the role of POT1 in plants, we established the moss Physcomitrella patens as a new model for telomere biology and a counterpoint to Arabidopsis. The sequence and architecture of the telomere tract is similar in P. patens and Arabidopsis, but P. patens harbors only a single-copy POT1 gene. Unlike At POT1 proteins, Pp POT1 efficiently bound single-stranded telomeric DNA in vitro. Deletion of the P. patens POT1 gene resulted in the rapid onset of severe developmental defects and sterility. Although telomerase activity levels were unperturbed, telomeres were substantially shortened, harbored extended G-overhangs, and engaged in end-to-end fusions. We conclude that the telomere capping function of POT1 is conserved in early diverging land plants but is subsequently lost in Arabidopsis.     

Nubp1 Is Required for Lung Branching Morphogenesis and Distal Progenitor Cell Survival in Mice

The lung is a complex system in biology and medicine alike. Whereas there is a good understanding of the anatomy and histology of the embryonic and adult lung, less is known about the molecular details and the cellular pathways that ultimately orchestrate lung formation and affect its health. From a forward genetic approach to identify novel genes involved in lung formation, we identified a mutated Nubp1 gene, which leads to syndactyly, eye cataract and lung hypoplasia. In the lung, Nubp1 is expressed in progenitor cells of the distal epithelium. Nubp1(m1Nisw) mutants show increased apoptosis accompanied by a loss of the distal progenitor markers Sftpc, Sox9 and Foxp2. In addition, Nubp1 mutation disrupts localization of the polarity protein Par3 and the mitosis relevant protein Numb. Using knock-down studies in lung epithelial cells, we also demonstrate a function of Nubp1 in regulating centrosome dynamics and microtubule organization. Together, Nubp1 represents an essential protein for lung progenitor survival by coordinating vital cellular processes including cell polarity and centrosomal dynamics.     

PAF-Myc-Controlled Cell Stemness Is Required for Intestinal Regeneration and Tumorigenesis

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MyD88 Is Required for Protection from Lethal Infection with a Mouse-Adapted SARS-CoV

A novel human coronavirus, SARS-CoV, emerged suddenly in 2003, causing approximately 8000 human cases and more than 700 deaths worldwide. Since most animal models fail to faithfully recapitulate the clinical course of SARS-CoV in humans, the virus and host factors that mediate disease pathogenesis remain unclear. Recently, our laboratory and others developed a recombinant mouse-adapted SARS-CoV (rMA15) that was lethal in BALB/c mice. In contrast, intranasal infection of young 10-week-old C57BL/6 mice with rMA15 results in a nonlethal infection characterized by high titer replication within the lungs, lung inflammation, destruction of lung tissue, and loss of body weight, thus providing a useful model to identify host mediators of protection. Here, we report that mice deficient in MyD88 (MyD88^−/−), an adapter protein that mediates Toll-like receptor (TLR), IL-1R, and IL-18R signaling, are far more susceptible to rMA15 infection. The genetic absence of MyD88 resulted in enhanced pulmonary pathology and greater than 90% mortality by day 6 post-infection. MyD88^−/− mice had significantly higher viral loads in lung tissue throughout the course of infection. Despite increased viral loads, the expression of multiple proinflammatory cytokines and chemokines within lung tissue and recruitment of inflammatory monocytes/macrophages to the lung was severely impaired in MyD88^−/− mice compared to wild-type mice. Furthermore, mice deficient in chemokine receptors that contribute to monocyte recruitment to the lung were more susceptible to rMA15-induced disease and exhibited severe lung pathology similar to that seen in MyD88^−/−mice. These data suggest that MyD88-mediated innate immune signaling and inflammatory cell recruitment to the lung are required for protection from lethal rMA15 infection.