Lack of clpC does not affect sporulation efficiency in Bacillus subtilis cells.

Fine-tuning in regulation of Clp protein content in Bacillus subtilis.

Clp-controlled proteolysis in Bacillus subtilis seems to play a substantial role, particularly under stress conditions. Calibrated Western blot analyses were used to estimate the approximate numbers of heat-inducible Clp molecules within a single cell. According to these numbers, the different Clp ATPases do not seem to compete for the proteolytic subunit ClpP. Coimmunoprecipitation experiments revealed the predicted specific ClpX-ClpP, ClpC-ClpP, and ClpE-ClpP interactions. ClpE and ClpX are rapidly degraded in wild-type cells during permanent heat stress but remained almost stable in a clpP mutant, suggesting ClpP-dependent degradation. In particular, ClpCP appeared to be involved in the degradation of the short-lived ClpE ATPase, indicating a negative "autoregulatory" circuit for this particular Clp ATPase at the posttranslational level. Analysis of the half-life of stress-inducible clp mRNAs during exponential growth and heat shock revealed precise regulation of the synthesis of each Clp protein at the posttranscriptional level as well to meet the needs of B. subtilis.

The first gene of the Bacillus subtilis clpC operon, ctsR, encodes a negative regulator of its own operon and other class III heat shock genes.

The Bacillus subtilis clpC operon is regulated by two stress induction pathways relying on either sigmaB or a class III stress induction mechanism acting at a sigmaA-like promoter. When the clpC operon was placed under the control of the isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible Pspac promoter, dramatic repression of the natural clpC promoters fused to a lacZ reporter gene was noticed after IPTG induction. This result strongly indicated negative regulation of the clpC operon by one of its gene products. Indeed, the negative regulator could be identified which is encoded by the first gene of the clpC operon, ctsR, containing a predicted helix-turn-helix DNA-binding motif. Deletion of ctsR abolished the negative regulation and resulted in high expression of both the clpC operon and the clpP gene under nonstressed conditions. Nevertheless, a further increase in clpC and clpP mRNA levels was observed after heat shock, even in the absence of sigmaB, suggesting a second induction mechanism at the vegetative promoter. Two-dimensional gel analysis and mRNA studies showed that the expression of other class III stress genes was at least partially influenced by the ctsR deletion. Studies with different clpC promoter fragments either fused to the reporter gene bgaB or used in gel mobility shift experiments with the purified CtsR protein revealed a possible target region where the repressor seemed to bind in vivo and in vitro. Our data demonstrate that the CtsR protein acts as a global repressor of the clpC operon, as well as other class III heat shock genes, by preventing unstressed transcription from either the sigmaB- or sigmaA-dependent promoter and might be inactivated or dissociate under inducing stress conditions.

High-Throughput Genetic Screens Identify a Large and Diverse Collection of New Sporulation Genes in Bacillus subtilis

The differentiation of the bacterium Bacillus subtilis into a dormant spore is among the most well-characterized developmental pathways in biology. Classical genetic screens performed over the past half century identified scores of factors involved in every step of this morphological process. More recently, transcriptional profiling uncovered additional sporulation-induced genes required for successful spore development. Here, we used transposon-sequencing (Tn-seq) to assess whether there were any sporulation genes left to be discovered. Our screen identified 133 out of the 148 genes with known sporulation defects. Surprisingly, we discovered 24 additional genes that had not been previously implicated in spore formation. To investigate their functions, we used fluorescence microscopy to survey early, middle, and late stages of differentiation of null mutants from the B. subtilis ordered knockout collection. This analysis identified mutants that are delayed in the initiation of sporulation, defective in membrane remodeling, and impaired in spore maturation. Several mutants had novel sporulation phenotypes. We performed in-depth characterization of two new factors that participate in cell-cell signaling pathways during sporulation. One (SpoIIT) functions in the activation of σE in the mother cell; the other (SpoIIIL) is required for σG activity in the forespore. Our analysis also revealed that as many as 36 sporulation-induced genes with no previously reported mutant phenotypes are required for timely spore maturation. Finally, we discovered a large set of transposon insertions that trigger premature initiation of sporulation. Our results highlight the power of Tn-seq for the discovery of new genes and novel pathways in sporulation and, combined with the recently completed null mutant collection, open the door for similar screens in other, less well-characterized processes.

From Cell Differentiation to Cell Collectives: Bacillus subtilis Uses Division of Labor to Migrate

The organization of cells, emerging from cell-cell interactions, can give rise to collective properties. These properties are adaptive when together cells can face environmental challenges that they separately cannot. One particular challenge that is important for microorganisms is migration. In this study, we show how flagellum-independent migration is driven by the division of labor of two cell types that appear during Bacillus subtilis sliding motility. Cell collectives organize themselves into bundles (called "van Gogh bundles") of tightly aligned cell chains that form filamentous loops at the colony edge. We show, by time-course microscopy, that these loops migrate by pushing themselves away from the colony. The formation of van Gogh bundles depends critically on the synergistic interaction of surfactin-producing and matrix-producing cells. We propose that surfactin-producing cells reduce the friction between cells and their substrate, thereby facilitating matrix-producing cells to form bundles. The folding properties of these bundles determine the rate of colony expansion. Our study illustrates how the simple organization of cells within a community can yield a strong ecological advantage. This is a key factor underlying the diverse origins of multicellularity.

Porcine circovirus type 2 DNA influences cytoskeleton rearrangements in plasmacytoid and monocyte-derived dendritic cells.

Functional disruption of dendritic cells (DC) is an important strategy for viral pathogens to evade host defences. In this context, porcine circovirus type 2 (PCV2), a single-stranded DNA virus, impairs plasmacytoid DC (pDC) and conventional DC activation by certain viruses or Toll-like receptor (TLR) ligands. This inhibitory capacity is associated with the viral DNA, but the impairment does not affect all signalling cascades; TLR7 ligation by small chemical molecules will still induce interleukin-6 (IL-6) and tumour necrosis factor-α secretion, but not interferon-α or IL-12. In this study, the molecular mechanisms by which silencing occurs were investigated. PP2, a potent inhibitor of the Lyn and Hck kinases, produced a similar profile to the PCV2 DNA interference with cytokine secretion by pDC, efficiently inhibiting cell activation induced through TLR9, but not TLR7, ligation. Confocal microscopy and cytometry analysis strongly suggested that PCV2 DNA impairs actin polymerization and endocytosis in pDC and monocyte-derived DC, respectively. Altogether, this study delineates for the first time particular molecular mechanisms involved in PCV2 interference with DC danger recognition, which may be responsible for the virus-induced immunosuppression observed in infected pigs.

Fission Yeast Scm3: A CENP-A Receptor Required for Integrity of Subkinetochore Chromatin

The mechanisms ensuring specific incorporation of CENP-A at centromeres are poorly understood. Mis16 and Mis18 are required for CENP-A localization at centromeres and form a complex that is conserved from fission yeast to human. Fission yeast sim1 mutants that alleviate kinetochore domain silencing are defective in Scm3(Sp), the ortholog of budding yeast Scm3(Sc). Scm3(Sp) depends on Mis16/18 for its centromere localization and like them is recruited to centromeres in late anaphase. Importantly, Scm3(Sp) coaffinity purifies with CENP-A(Cnp1) and associates with CENP-A(Cnp1) in vitro, yet localizes independently of intact CENP-A(Cnp1) chromatin and is differentially released from chromatin. While Scm3(Sc) has been proposed to form a unique hexameric nucleosome with CENP-A(Cse4) and histone H4 at budding yeast point centromeres, we favor a model in which Scm3(Sp) acts as a CENP-A(Cnp1) receptor/assembly factor, cooperating with Mis16 and Mis18 to receive CENP-A(Cnp1) from the Sim3 escort and mediate assembly of CENP-A(Cnp1) into subkinetochore chromatin.

Mechanism and Regulation of DNA-Protein Crosslink Repair by the DNA-Dependent Metalloprotease SPRTN

Covalent DNA-protein crosslinks (DPCs) are toxic DNA lesions that interfere with essential chromatin transactions, such as replication and transcription. Little was known about DPC-specific repair mechanisms until the recent identification of a DPC-processing protease in yeast. The existence of a DPC protease in higher eukaryotes is inferred from data in Xenopus laevis egg extracts, but its identity remains elusive. Here we identify the metalloprotease SPRTN as the DPC protease acting in metazoans. Loss of SPRTN results in failure to repair DPCs and hypersensitivity to DPC-inducing agents. SPRTN accomplishes DPC processing through a unique DNA-induced protease activity, which is controlled by several sophisticated regulatory mechanisms. Cellular, biochemical, and structural studies define a DNA switch triggering its protease activity, a ubiquitin switch controlling SPRTN chromatin accessibility, and regulatory autocatalytic cleavage. Our data also provide a molecular explanation on how SPRTN deficiency causes the premature aging and cancer predisposition disorder Ruijs-Aalfs syndrome.

Disturbance in cerebral spinal fluid sphingolipid content is associated with memory impairment in subjects infected with the human immunodeficiency virus

Despite widespread use of antiretroviral therapies to control replication of the human immunodeficiency virus (HIV), dysfunctions of cognition that are collectively termed HIV-associated neurocognitive disorders (HAND) still occur in approximately 50% of those infected by the virus. Currently there is not a biomarker that can identify HIV-infected people who are at risk for the development of HAND. Previous studies have identified particular sphingolipid species that are dysregulated in HAND, but the neurocognitive correlates of these biochemical findings are not currently understood. To address this question, we compared cerebrospinal fluid (CSF) levels of sphingomyelin, ceramide, and sterol species with performance on standard neurological tests designed to assess the function of multiple cognitive and motor domains in HIV-infected subjects. We found that sphingomyelin:ceramide ratios for acyl chain lengths of C16∶0, C18∶0, C22∶0, and C24∶0 were associated with worse performance on several indices of memory. The most striking finding was for the acyl chain of C18∶0 that consistently associatedwith performance onmultiple tests of memory. These findings suggest that the sphingomyelin:ceramide ratio for C18∶0 may be a reasonable surrogate marker for memory dysfunction in HIV-infected subjects.

Adult c-Kit(+) progenitor cells are necessary for maintenance and regeneration of olfactory neurons.

The olfactory epithelium houses chemosensory neurons, which transmit odor information from the nose to the brain. In adult mammals, the olfactory epithelium is a uniquely robust neuroproliferative zone, with the ability to replenish its neuronal and non-neuronal populations due to the presence of germinal basal cells. The stem and progenitor cells of these germinal layers, and their regulatory mechanisms, remain incompletely defined. Here we show that progenitor cells expressing c-Kit, a receptor tyrosine kinase marking stem cells in a variety of embryonic tissues, are required for maintenance of the adult neuroepithelium. Mouse genetic fate-mapping analyses show that embryonically, a c-Kit(+) population contributes to olfactory neurogenesis. In adults under conditions of normal turnover, there is relatively sparse c-Kit(+) progenitor cell (ckPC) activity. However, after experimentally induced neuroepithelial injury, ckPCs are activated such that they reconstitute the neuronal population. There are also occasional non-neuronal cells found to arise from ckPCs. Moreover, the selective depletion of the ckPC population, utilizing temporally controlled targeted diphtheria toxin A expression, results in failure of neurogenesis after experimental injury. Analysis of this model indicates that most ckPCs reside among the globose basal cell populations and act downstream of horizontal basal cells, which can serve as stem cells. Identification of the requirement for olfactory c-Kit-expressing progenitors in olfactory maintenance provides new insight into the mechanisms involved in adult olfactory neurogenesis. Additionally, we define an important and previously unrecognized site of adult c-Kit activity.

Evolution of novel cooperative swarming in the bacterium Myxococcus xanthus

Cooperation among individuals is necessary for evolutionary transitions to higher levels of biological organization. In such transitions, groups of individuals at one level (such as single cells) cooperate to form selective units at a higher level (such as multicellular organisms). Though the evolution of cooperation is difficult to observe directly in higher eukaryotes, microorganisms do offer such an opportunity. Here we report the evolution of novel cooperative behaviour in experimental lineages of the bacterium Myxococcus xanthus. Wild-type strains of M. xanthus exhibit socially dependent swarming across soft surfaces by a mechanism known as ‘S-motility’ that requires the presence of extracellular type IV pili. In lineages of M. xanthus unable to make pili, a new mechanistic basis for cooperative swarming evolved. Evolved swarming is mediated, at least in part, by enhanced production of an extracellular fibril matrix that binds cells—and their evolutionary interests—together. Though costly to individuals, fibril production greatly enhanced population expansion in groups of interconnected cells. These results show that fundamental transitions to primitive cooperation can readily occur in bacteria.

Rho, Rac and Cdc42 regulate actin organization and cell adhesion in macrophages.

Rho family proteins are known to regulate actin organization in fibroblasts, but their functions in cells of haematopoietic origin have not been studied in detail. Bac1.2F5 cells are a colony-stimulating factor-1 (CSF-1)-dependent murine macrophage cell line; CSF-1 stimulates their proliferation and motility, and acts as a chemoattractant. CSF-1 rapidly induced actin reorganization in Bac1 cells: it stimulated the formation of filopodia, lamellipodia and membrane ruffles at the plasma membrane, as well as the appearance of fine actin cables within the cell interior. Microinjection of constitutively activated (V12)Rac1 stimulated lamellipodium formation and membrane ruffling. The dominant inhibitory Rac mutant, N17Rac1, inhibited CSF-1-induced lamellipodium formation, and also induced cell rounding. V12Cdc42 induced the formation of long filopodia, while the dominant inhibitory mutant N17Cdc42 prevented CSF-1-induced formation of filopodia but not lamellipodia. V14RhoA stimulated actin cable assembly and cell contraction, while the Rho inhibitor, C3 transferase, induced the loss of actin cables. Bac1 cells had cell-to-substratum adhesion sites containing beta1 integrin, pp125FAK, paxillin, vinculin, and tyrosine phosphorylated proteins. These 'focal complexes' were present in growing and CSF-1-starved cells, but were disassembled in cells injected with N17Cdc42 or N17Rac1. Interestingly, beta1 integrin did not disperse until long after focal phosphotyrosine and vinculin staining had disappeared. We conclude that in Bac1 macrophages Cdc42, Rac and Rho regulate the formation of distinct actin filament-based structures, and that Cdc42 and Rac are also required for the assembly of adhesion sites to the extracellular matrix.

Genetic disruption of aurora B uncovers an essential role for aurora C during early mammalian development.

Mitosis is controlled by multiple kinases that drive cell cycle progression and prevent chromosome mis-segregation. Aurora kinase B interacts with survivin, borealin and incenp to form the chromosomal passenger complex (CPC), which is involved in the regulation of microtubule-kinetochore attachments and cytokinesis. Whereas genetic ablation of survivin, borealin or incenp results in early lethality at the morula stage, we show here that aurora B is dispensable for CPC function during early cell divisions and aurora B-null embryos are normally implanted. This is due to a crucial function of aurora C during these early embryonic cycles. Expression of aurora C decreases during late blastocyst stages resulting in post-implantation defects in aurora B-null embryos. These defects correlate with abundant prometaphase figures and apoptotic cell death of the aurora B-deficient inner cell mass. Conditional deletion of aurora B in somatic cells that do not express aurora C results in chromosomal misalignment and lack of chromosome segregation. Re-expression of wild-type, but not kinase-dead, aurora C rescues this defect, suggesting functional overlap between these two kinases. Finally, aurora B-null cells partially arrest in the presence of nocodazole, suggesting that this kinase is not essential for the spindle assembly checkpoint.

Clathrin is required for the function of the mitotic spindle

Clathrin has an established function in the generation of vesicles that transfer membrane and proteins around the cell. The formation of clathrin-coated vesicles occurs continuously in non-dividing cells, but is shut down during mitosis, when clathrin concentrates at the spindle apparatus. Here, we show that clathrin stabilizes fibres of the mitotic spindle to aid congression of chromosomes. Clathrin bound to the spindle directly by the amino-terminal domain of clathrin heavy chain. Depletion of clathrin heavy chain using RNA interference prolonged mitosis; kinetochore fibres were destabilized, leading to defective congression of chromosomes to the metaphase plate and persistent activation of the spindle checkpoint. Normal mitosis was rescued by clathrin triskelia but not the N-terminal domain of clathrin heavy chain, indicating that stabilization of kinetochore fibres was dependent on the unique structure of clathrin. The importance of clathrin for normal mitosis may be relevant to understanding human cancers that involve gene fusions of clathrin heavy chain.

Vertebrate rod photoreceptors express both BK and IK calcium-activated potassium channels, but only BK channels are involved in receptor potential regulation.

In salamander rods, Ca(2+)-activated K(+) current (I(KCa)) provides an effective "clamp" of the dark membrane potential to its normal resting level. By a combination of electrophysiological, pharmacological, and immunohistochemical approaches, we show that salamander rods functionally express large-conductance Ca(2+)- and voltage-dependent potassium (BK) channel and intermediate-conductance Ca(2+)-dependent potassium (IK) channel, but not small-conductance Ca(2+)-dependent potassium channel (SK) subtypes. Application of 100 nM iberiotoxin and 100 nM clotrimazole reduced net I(KCa) to 36% and 63%, respectively, whereas the current was unaffected by application of 1 microM apamin. Consistently, anti- SK1, -SK2, and -SK3 antibodies were unable to stain rod photoreceptors, whereas both anti-BK and -SK4/ IK1 antibodies heavily stained the ellipsoid region of the inner segments of the rods. Moreover, by using current-clamp experiments, it was clearly seen that the strong clamping effect of the total I(KCa) was lost when IbTx, but not CLTZ, was applied to the bath. This behavior strongly suggests that of BK and IK channels, only the former are responsible for the clamping effect on the photoreceptor membrane potential.

Live-cell delamination counterbalances epithelial growth to limit tissue overcrowding

The development and maintenance of an epithelium requires finely balanced rates of growth and cell death. However, the mechanical and biochemical mechanisms that ensure proper feedback control of tissue growth, which when deregulated contribute to tumorigenesis, are poorly understood. Here we use the fly notum as a model system to identify a novel process of crowding-induced cell delamination that balances growth to ensure the development of well-ordered cell packing. In crowded regions of the tissue, a proportion of cells undergo a serial loss of cell–cell junctions and a progressive loss of apical area, before being squeezed out by their neighbours. This path of delamination is recapitulated by a simple computational model of epithelial mechanics, in which stochastic cell loss relieves overcrowding as the system tends towards equilibrium. We show that this process of delamination is mechanistically distinct from apoptosis-mediated cell extrusion and precedes the first signs of cell death. Overall, this analysis reveals a simple mechanism that buffers epithelia against variations in growth. Because live-cell delamination constitutes a mechanistic link between epithelial hyperplasia and cell invasion, this is likely to have important implications for our understanding of the early stages of cancer development.

Reprogramming factor stoichiometry influences the epigenetic state and biological properties of induced pluripotent stem cells.

We compared two genetically highly defined transgenic systems to identify parameters affecting reprogramming of somatic cells to a pluripotent state. Our results demonstrate that the level and stoichiometry of reprogramming factors during the reprogramming process strongly influence the resulting pluripotency of iPS cells. High expression of Oct4 and Klf4 combined with lower expression of c-Myc and Sox2 produced iPS cells that efficiently generated "all-iPSC mice" by tetraploid (4n) complementation, maintained normal imprinting at the Dlk1-Dio3 locus, and did not create mice with tumors. Loss of imprinting (LOI) at the Dlk1-Dio3 locus did not strictly correlate with reduced pluripotency though the efficiency of generating "all-iPSC mice" was diminished. Our data indicate that stoichiometry of reprogramming factors can influence epigenetic and biological properties of iPS cells. This concept complicates efforts to define a "generic" epigenetic state of iPSCs and ESCs and should be considered when comparing different iPS and ES cell lines.

GEMC1 is a TopBP1 interacting protein required for chromosomal DNA replication

Many of the factors required for chromosomal DNA replication have been identified in unicellular eukaryotes. However, DNA replication is poorly understood in multicellular organisms. Here, we report the identification of GEMC1 (geminin coiled-coil containing protein 1), a novel vertebrate protein required for chromosomal DNA replication. GEMC1 is highly conserved in vertebrates and is preferentially expressed in proliferating cells. Using Xenopus laevis egg extract we show that Xenopus GEMC1 (xGEMC1) binds to the checkpoint and replication factor TopBP1, which promotes binding of xGEMC1 to chromatin during pre-replication complex (pre-RC) formation. We demonstrate that xGEMC1 interacts directly with replication factors such as Cdc45 and the kinase Cdk2-CyclinE, through which it is heavily phosphorylated. Phosphorylated xGEMC1 stimulates initiation of DNA replication, whereas depletion of xGEMC1 prevents the onset of DNA replication owing to the impairment of Cdc45 loading onto chromatin. Similarly, inhibition of GEMC1 expression with morpholino and siRNA oligos prevents DNA replication in embryonic and somatic vertebrate cells. These data suggest that GEMC1 promotes initiation of chromosomal DNA replication in multicellular organisms by mediating TopBP1- and Cdk2-dependent recruitment of Cdc45 onto replication origins.

Genome-wide identification of genes directly regulated by the pleiotropic transcription factor Spx in Bacillus subtilis

The transcriptional regulator Spx plays a key role in maintaining the redox homeostasis of Bacillus subtilis cells exposed to disulfide stress. Defects in Spx were previously shown to lead to differential expression of numerous genes but direct and indirect regulatory effects could not be distinguished. Here we identified 283 discrete chromosomal sites potentially bound by the Spx-RNA polymerase (Spx-RNAP) complex using chromatin immunoprecipitation of Spx. Three quarters of these sites were located near Sigma(A)-dependent promoters, and upon diamide treatment, the fraction of the Spx-RNAP complex increased in parallel with the number and occupancy of DNA sites. Correlation of Spx-RNAP-binding sites with gene differential expression in wild-type and Δspx strains exposed or not to diamide revealed that 144 transcription units comprising 275 genes were potentially under direct Spx regulation. Spx-controlled promoters exhibited an extended -35 box in which nucleotide composition at the -43/-44 positions strongly correlated with observed activation. In vitro transcription confirmed activation by oxidized Spx of seven newly identified promoters, of which one was also activated by reduced Spx. Our study globally characterized the Spx regulatory network, revealing its role in the basal expression of some genes and its complex interplay with other stress responses.

A key role for Ctf4 in coupling the MCM2-7 helicase to DNA polymerase alpha within the eukaryotic replisome.

The eukaryotic replisome is a crucial determinant of genome stability, but its structure is still poorly understood. We found previously that many regulatory proteins assemble around the MCM2-7 helicase at yeast replication forks to form the replisome progression complex (RPC), which might link MCM2-7 to other replisome components. Here, we show that the RPC associates with DNA polymerase alpha that primes each Okazaki fragment during lagging strand synthesis. Our data indicate that a complex of the GINS and Ctf4 components of the RPC is crucial to couple MCM2-7 to DNA polymerase alpha. Others have found recently that the Mrc1 subunit of RPCs binds DNA polymerase epsilon, which synthesises the leading strand at DNA replication forks. We show that cells lacking both Ctf4 and Mrc1 experience chronic activation of the DNA damage checkpoint during chromosome replication and do not complete the cell cycle. These findings indicate that coupling MCM2-7 to replicative polymerases is an important feature of the regulation of chromosome replication in eukaryotes, and highlight a key role for Ctf4 in this process.

Transformation of intact yeast cells treated with alkali cations.

Intact yeast cells treated with alkali cations took up plasmid DNA. Li+, Cs+, Rb+, K+, and Na+ were effective in inducing competence. Conditions for the transformation of Saccharomyces cerevisiae D13-1A with plasmid YRp7 were studied in detail with CsCl. The optimum incubation time was 1 h, and the optimum cell concentration was 5 x 10(7) cells per ml. The optimum concentration of Cs+ was 1.0 M. Transformation efficiency increased with increasing concentrations of plasmid DNA. Polyethylene glycol was absolutely required. Heat pulse and various polyamines or basic proteins stimulated the uptake of plasmid DNA. Besides circular DNA, linear plasmid DNA was also taken up by Cs+-treated yeast cells, although the uptake efficiency was considerably reduced. The transformation efficiency with Cs+ or Li+ was comparable with that of conventional protoplast methods for a plasmid containing ars1, although not for plasmids containing a 2 microns origin replication.

GATA-4 and GATA-6 modulate tissue-specific transcription of the human gene for P450c17 by direct interaction with Sp1.

Cytochrome P450c17 catalyzes steroidogenic 17alpha-hydroxylase and 17,20 lyase activities. Expression of the gene for P450c17 is cAMP dependent, tissue specific, developmentally programmed, and varies among species. Binding of Sp1, Sp3, and NF1-C (nuclear factor 1-C) to the first 227 bp of 5'flanking DNA (-227/LUC) is crucial for basal transcription in human NCI-H295A adrenal cells. Human placental JEG-3 cells contain Sp1, Sp3, and NF1, but do not express -227/LUC, even when transfected with a vector expressing steroidogenic factor 1 (SF-1). Therefore, other factors are essential for basal expression of P450c17. Deoxyribonuclease I footprinting and EMSAs identified a GATA consensus site at -64/-58 and an SF-1 site at -58/-50. RT-PCR identified GATA-4, GATA-6, and SF-1 in NCI-H295A cells and GATA-2 and GATA-3, but not GATA-4, GATA-6, or SF-1 in JEG-3 cells. Cotransfection of either GATA-4 or GATA-6 without SF-1 activated -227/LUC in JEG-3 cells, but cotransfection of GATA-2 or GATA-3 with or without SF-1 did not. Surprisingly, mutation of the GATA binding site in -227/LUC increased GATA-4 or GATA-6 induced activity, whereas mutation of the Sp1/Sp3 site decreased it. Furthermore, promoter constructs including the GATA site, but excluding the Sp1/Sp3 site at -196/-188, were not activated by GATA-4 or GATA-6, suggesting an interaction between Sp1/Sp3 and GATA-4 or GATA-6. Glutathione-S-transferase pull-down experiments and coimmunoprecipitation demonstrated interaction between GATA-4 or GATA-6 and Sp1, but not Sp3. Chromatin immunoprecipitation assays confirmed that this GATA-4/6 interaction with Sp1 occurred at the Sp site in the P450c17 promoter in NCI-H295A cells. Demethylation with 5-aza-2-deoxycytidine permitted JEG-3 cells to express endogenous P450c17, SF-1, GATA-4, GATA-6, and transfected -227/LUC. Thus, GATA-4 or GATA-6 and Sp1 together regulate expression of P450c17 in adrenal NCI-H295A cells and methylation of P450c17, GATA-4 and GATA-6 silence the expression of P450c17 in placental JEG-3 cells.

Exploitative and Hierarchical Antagonism in a Cooperative Bacterium

Social organisms that cooperate with some members of their own species, such as close relatives, may fail to cooperate with other genotypes of the same species. Such noncooperation may take the form of outright antagonism or social exploitation. Myxococcus xanthus is a highly social prokaryote that cooperatively develops into spore-bearing, multicellular fruiting bodies in response to starvation. Here we have characterized the nature of social interactions among nine developmentally proficient strains of M. xanthus isolated from spatially distant locations. Strains were competed against one another in all possible pairwise combinations during starvation-induced development. In most pairings, at least one competitor exhibited strong antagonism toward its partner and a majority of mixes showed bidirectional antagonism that decreased total spore production, even to the point of driving whole populations to extinction. Differential response to mixing was the primary determinant of competitive superiority rather than the sporulation efficiencies of unmixed populations. In some competitive pairings, the dominant partner sporulated more efficiently in mixed populations than in clonal isolation. This finding represents a novel form of exploitation in bacteria carried out by socially competent genotypes and is the first documentation of social exploitation among natural bacterial isolates. Patterns of antagonistic superiority among these strains form a highly linear dominance hierarchy. At least some competition pairs construct chimeric, rather than segregated, fruiting bodies. The cooperative prokaryote M. xanthus has diverged into a large number of distinct social types that cooperate with clone-mates but exhibit intense antagonism toward distinct social types of the same species. Most lengthy migration events in nature may thus result in strong antagonism between migratory and resident populations, and this antagonism may have large effects on local population sizes and dynamics. Intense mutual antagonism appears to be more prevalent in this prokaryotic social species than has been observed in the eukaryotic social slime mold Dictyostelium discoideum, which also exhibits multicellular development. The finding of several cases of facultative social exploitation among these natural isolates suggests that such exploitation may occur frequently in nature in many prokaryotes with cooperative traits.

Krüppel-like factor 2 (KLF2) regulates B-cell reactivity, subset differentiation, and trafficking molecule expression.

The transcription factor Krüppel-like factor 2 (KLF2) is critical for normal trafficking of T lymphocytes, but its role in B cells is unclear. We report that B cell-specific KLF2 deficiency leads to decreased expression of the trafficking molecules CD62L and β7-integrin, yet expression of sphingosine-1 phosphate receptor 1 (which is a critical target of KLF2 in T cells) was, unexpectedly, minimally altered. Unexpectedly, Klf2 deletion led to a drastic reduction in the B1 B-cell pool and a substantial increase in transitional and marginal zone B-cell numbers. In addition, we observed that KLF2-deficient B cells showed increased apoptosis and impaired proliferation after B-cell receptor cross-linking. Gene expression analysis indicated that KLF2-deficient follicular B cells display numerous characteristics shared by normal marginal zone B cells, including reduced expression of several signaling molecules that may contribute to defective activation of these cells. Hence, our data indicate that KLF2 plays a critical role in dictating normal subset differentiation and functional reactivity of mature B cells.

Extensive diversity of Ig-superfamily proteins in the immune system of insects.

The extensive somatic diversification of immune receptors is a hallmark of higher vertebrates. However, whether molecular diversity contributes to immune protection in invertebrates is unknown. We present evidence that Drosophila immune-competent cells have the potential to express more than 18,000 isoforms of the immunoglobulin (Ig)-superfamily receptor Down syndrome cell adhesion molecule (Dscam). Secreted protein isoforms of Dscam were detected in the hemolymph, and hemocyte-specific loss of Dscam impaired the efficiency of phagocytic uptake of bacteria, possibly due to reduced bacterial binding. Importantly, the molecular diversity of Dscam transcripts generated through a mechanism of alternative splicing is highly conserved across major insect orders, suggesting an unsuspected molecular complexity of the innate immune system of insects.

Evidence that the Ipl1-Sli15 (Aurora Kinase-INCENP) Complex Promotes Chromosome Bi-orientation by Altering Kinetochore-Spindle Pole Connections

How sister kinetochores attach to microtubules from opposite spindle poles during mitosis (bi-orientation) remains poorly understood. In yeast, the ortholog of the Aurora B-INCENP protein kinase complex (Ipl1-Sli15) may have a role in this crucial process, because it is necessary to prevent attachment of sister kinetochores to microtubules from the same spindle pole. We investigated IPL1 function in cells that cannot replicate their chromosomes but nevertheless duplicate their spindle pole bodies (SPBs). Kinetochores detach from old SPBs and reattach to old and new SPBs with equal frequency in IPL1+ cells, but remain attached to old SPBs in ipl1 mutants. This raises the possibility that Ipl1-Sli15 facilitates bi-orientation by promoting turnover of kinetochore-SPB connections until traction of sister kinetochores toward opposite spindle poles creates tension in the surrounding chromatin.

The Drosophila hindgut lacks constitutively active adult stem cells but proliferates in response to tissue damage.

The adult Drosophila hindgut was recently reported to contain active, tissue-replenishing stem cells, like those of the midgut, but located within an anterior ring so as to comprise a single giant crypt. In contrast to this view, we observed no active stem cells and little cell turnover in adult hindgut tissue based on clonal marking and BrdU incorporation studies. Again contradicting the previous proposal, we showed that the adult hindgut is not generated by anterior stem cells during larval/pupal development. However, severe tissue damage within the hindgut elicits cell proliferation within a ring of putative quiescent stem cells at the anterior of the pylorus. Thus, the hindgut does not provide a model of tissue maintenance by constitutively active stem cells, but has great potential to illuminate mechanisms of stress-induced tissue repair.

The adaptor protein Tks5/Fish is required for podosome formation and function, and for the protease-driven invasion of cancer cells.

Tks5/Fish is a scaffolding protein with five SH3 domains and one PX domain. In Src-transformed cells, Tks5/Fish localizes to podosomes, discrete protrusions of the ventral membrane. We generated Src-transformed cells with reduced Tks5/Fish levels. They no longer formed podosomes, did not degrade gelatin, and were poorly invasive. We detected Tks5/Fish expression in podosomes in invasive cancer cells, as well as in human breast cancer and melanoma samples. Tks5/Fish expression was also required for protease-driven matrigel invasion in human cancer cells. Finally, coexpression of Tks5/Fish and Src in epithelial cells resulted in the appearance of podosomes. Thus, Tks5/Fish appears to be required for podosome formation, for degradation of the extracellular matrix, and for invasion of some cancer cells.

Alpha-Synuclein Induces Lysosomal Rupture and Cathepsin Dependent Reactive Oxygen Species Following Endocytosis

α-synuclein dysregulation is a critical aspect of Parkinson's disease pathology. Recent studies have observed that α-synuclein aggregates are cytotoxic to cells in culture and that this toxicity can be spread between cells. However, the molecular mechanisms governing this cytotoxicity and spread are poorly characterized. Recent studies of viruses and bacteria, which achieve their cytoplasmic entry by rupturing intracellular vesicles, have utilized the redistribution of galectin proteins as a tool to measure vesicle rupture by these organisms. Using this approach, we demonstrate that α-synuclein aggregates can induce the rupture of lysosomes following their endocytosis in neuronal cell lines. This rupture can be induced by the addition of α-synuclein aggregates directly into cells as well as by cell-to-cell transfer of α-synuclein. We also observe that lysosomal rupture by α-synuclein induces a cathepsin B dependent increase in reactive oxygen species (ROS) in target cells. Finally, we observe that α-synuclein aggregates can induce inflammasome activation in THP-1 cells. Lysosomal rupture is known to induce mitochondrial dysfunction and inflammation, both of which are well established aspects of Parkinson's disease, thus connecting these aspects of Parkinson's disease to the propagation of α-synuclein pathology in cells.

Kinesin-2 controls development and patterning of the vertebrate skeleton by Hedgehog- and Gli3-dependent mechanisms.

Hedgehog signaling plays an essential role in patterning of the vertebrate skeleton. Here we demonstrate that conditional inactivation of the Kif3a subunit of the kinesin-2 intraflagellar transport motor in mesenchymal skeletal progenitor cells results in severe patterning defects in the craniofacial area, the formation of split sternum and the development of polydactyly. These deformities are reminiscent of those previously described in mice with deregulated hedgehog signaling. We show that in Kif3a-deficient mesenchymal tissues both the repressor function of Gli3 transcription factor and the activation of the Shh transcriptional targets Ptch and Gli1 are compromised. Quantitative analysis of gene expression demonstrates that the Gli1 transcript level is dramatically reduced, whereas Gli3 expression is not significantly affected by kinesin-2 depletion. However, the motor appears to be required for the efficient cleavage of the full-length Gli3 transcription factor into a repressor form.

Single-Cell Expression Analyses during Cellular Reprogramming Reveal an Early Stochastic and a Late Hierarchic Phase

During cellular reprogramming, only a small fraction of cells become induced pluripotent stem cells (iPSCs). Previous analyses of gene expression during reprogramming were based on populations of cells, impeding single-cell level identification of reprogramming events. We utilized two gene expression technologies to profile 48 genes in single cells at various stages during the reprogramming process. Analysis of early stages revealed considerable variation in gene expression between cells in contrast to late stages. Expression of Esrrb, Utf1, Lin28, and Dppa2 is a better predictor for cells to progress into iPSCs than expression of the previously suggested reprogramming markers Fbxo15, Fgf4, and Oct4. Stochastic gene expression early in reprogramming is followed by a late hierarchical phase with Sox2 being the upstream factor in a gene expression hierarchy. Finally, downstream factors derived from the late phase, which do not include Oct4, Sox2, Klf4, c-Myc, and Nanog, can activate the pluripotency circuitry.

Localization and requirement for Myosin II at the dorsal-ventral compartment boundary of the Drosophila wing.

As organisms develop, their tissues can become separated into distinct cell populations through the establishment of compartment boundaries. Compartment boundaries have been discovered in a wide variety of tissues, but in many cases the molecular mechanisms that separate cells remain poorly understood. In the Drosophila wing, a stripe of Notch activation maintains the dorsal-ventral compartment boundary, through a process that depends on the actin cytoskeleton. Here, we show that the dorsal-ventral boundary exhibits a distinct accumulation of Myosin II, and that this accumulation is regulated downstream of Notch signaling. Conversely, the dorsal-ventral boundary is depleted for the Par-3 homologue Bazooka. We further show that mutations in the Myosin heavy chain subunit encoded by zipper can impair dorsal-ventral compartmentalization without affecting anterior-posterior compartmentalization. These observations identify a distinct accumulation and requirement for Myosin activity in dorsal-ventral compartmentalization, and suggest a novel mechanism in which contractile tension along an F-actin cable at the compartment boundary contributes to compartmentalization.

MLL1 Inhibition Reprograms Epiblast Stem Cells to Naive Pluripotency.

The interconversion between naive and primed pluripotent states is accompanied by drastic epigenetic rearrangements. However, it is unclear whether intrinsic epigenetic events can drive reprogramming to naive pluripotency or if distinct chromatin states are instead simply a reflection of discrete pluripotent states. Here, we show that blocking histone H3K4 methyltransferase MLL1 activity with the small-molecule inhibitor MM-401 reprograms mouse epiblast stem cells (EpiSCs) to naive pluripotency. This reversion is highly efficient and synchronized, with more than 50% of treated EpiSCs exhibiting features of naive embryonic stem cells (ESCs) within 3 days. Reverted ESCs reactivate the silenced X chromosome and contribute to embryos following blastocyst injection, generating germline-competent chimeras. Importantly, blocking MLL1 leads to global redistribution of H3K4me1 at enhancers and represses lineage determinant factors and EpiSC markers, which indirectly regulate ESC transcription circuitry. These findings show that discrete perturbation of H3K4 methylation is sufficient to drive reprogramming to naive pluripotency.

Lipid Desaturation Is a Metabolic Marker and Therapeutic Target of Ovarian Cancer Stem Cells.

Lack of sensitive single-cell analysis tools has limited the characterization of metabolic activity in cancer stem cells. By hyperspectral-stimulated Raman scattering imaging of single living cells and mass spectrometry analysis of extracted lipids, we report here significantly increased levels of unsaturated lipids in ovarian cancer stem cells (CSCs) as compared to non-CSCs. Higher lipid unsaturation levels were also detected in CSC-enriched spheroids compared to monolayer cultures of ovarian cancer cell lines or primary cells. Inhibition of lipid desaturases effectively eliminated CSCs, suppressed sphere formation in vitro, and blocked tumor initiation capacity in vivo. Mechanistically, we demonstrate that nuclear factor κB (NF-κB) directly regulates the expression levels of lipid desaturases, and inhibition of desaturases blocks NF-κB signaling. Collectively, our findings reveal that increased lipid unsaturation is a metabolic marker for ovarian CSCs and a target for CSC-specific therapy.

Embryonic stem cells require Wnt proteins to prevent differentiation to epiblast stem cells

Pluripotent stem cells exist in naive and primed states, epitomized by mouse embryonic stem cells (ESCs) and the developmentally more advanced epiblast stem cells (EpiSCs; ref. ). In the naive state of ESCs, the genome has an unusual open conformation and possesses a minimum of repressive epigenetic marks. In contrast, EpiSCs have activated the epigenetic machinery that supports differentiation towards the embryonic cell types. The transition from naive to primed pluripotency therefore represents a pivotal event in cellular differentiation. But the signals that control this fundamental differentiation step remain unclear. We show here that paracrine and autocrine Wnt signals are essential self-renewal factors for ESCs, and are required to inhibit their differentiation into EpiSCs. Moreover, we find that Wnt proteins in combination with the cytokine LIF are sufficient to support ESC self-renewal in the absence of any undefined factors, and support the derivation of new ESC lines, including ones from non-permissive mouse strains. Our results not only demonstrate that Wnt signals regulate the naive-to-primed pluripotency transition, but also identify Wnt as an essential and limiting ESC self-renewal factor.

Centrifugal elutriation as a method for isolation of large numbers of functionally intact human peripheral blood monocytes.

Centrifugal elutriation was used further to isolate human peripheral blood monocytes (HPBM) from mononuclear-enriched cells harvested as a secondary component following platelet concentration collection samples. HPBM were recovered in either one or two populations consisting of either total HPBM or small (SM) and large monocytes (LM). The elutriation was carried out at 3,500 +/- 5 rpm for the separation of lymphocytes and HPBM in Ca++- and Mg++-free PBS without EDTA. An average of 5.05 +/- 1.50 X 10(8) HPBM were recovered in the total HPBM with a purity of 95% +/- 3%. The SM and LM were obtained by splitting the total HPBM into two equal populations with an HPBM purity of 92% +/- 3% and 93% +/- 3, respectively, by nonspecific esterase staining. The elutriation media were shown to have no effect on viability by trypan blue exclusion. All three HPBM populations were shown to be histochemically (lack of reactivity to leu-1 and leu-7) and functionally (depletion of NK cell activity) purified from the lymphocyte population. The HPBM populations were enriched in HLA-Dr, OKM-1, OKM-5, MY-8, and leu M-3 monoclonal antibody marker staining. There were no differences in percent positive cells between SM and LM populations for any of the monocyte-specific monoclonal antibodies. All three monocyte populations mediated antibody-dependent cell-mediated cytotoxicity to human red blood cells, with LM mediating more lysis (27.0% +/- 5%) than SM (7% +/- 3%).(ABSTRACT TRUNCATED AT 250 WORDS)

Role of the Single-Stranded DNA–Binding Protein SsbB in Pneumococcal Transformation: Maintenance of a Reservoir for Genetic Plasticity

Bacteria encode a single-stranded DNA (ssDNA) binding protein (SSB) crucial for genome maintenance. In Bacillus subtilis and Streptococcus pneumoniae, an alternative SSB, SsbB, is expressed uniquely during competence for genetic transformation, but its precise role has been disappointingly obscure. Here, we report our investigations involving comparison of a null mutant (ssbB(-)) and a C-ter truncation (ssbBΔ7) of SsbB of S. pneumoniae, the latter constructed because SSBs' acidic tail has emerged as a key site for interactions with partner proteins. We provide evidence that SsbB directly protects internalized ssDNA. We show that SsbB is highly abundant, potentially allowing the binding of ~1.15 Mb ssDNA (half a genome equivalent); that it participates in the processing of ssDNA into recombinants; and that, at high DNA concentration, it is of crucial importance for chromosomal transformation whilst antagonizing plasmid transformation. While the latter observation explains a long-standing observation that plasmid transformation is very inefficient in S. pneumoniae (compared to chromosomal transformation), the former supports our previous suggestion that SsbB creates a reservoir of ssDNA, allowing successive recombination cycles. SsbBΔ7 fulfils the reservoir function, suggesting that SsbB C-ter is not necessary for processing protein(s) to access stored ssDNA. We propose that the evolutionary raison d'être of SsbB and its abundance is maintenance of this reservoir, which contributes to the genetic plasticity of S. pneumoniae by increasing the likelihood of multiple transformation events in the same cell.

CCL2/monocyte chemoattractant protein-1 mediates enhanced transmigration of human immunodeficiency virus (HIV)-infected leukocytes across the blood-brain barrier: a potential mechanism of HIV-CNS invasion and NeuroAIDS.

Encephalitis and dementia associated with acquired immunodeficiency syndrome (AIDS) are characterized by leukocyte infiltration into the CNS, microglia activation, aberrant chemokine expression, blood-brain barrier (BBB) disruption, and eventual loss of neurons. Little is known about whether human immunodeficiency virus 1 (HIV-1) infection of leukocytes affects their ability to transmigrate in response to chemokines and to alter BBB integrity. We now demonstrate that HIV infection of human leukocytes results in their increased transmigration across our tissue culture model of the human BBB in response to the chemokine CCL2, as well as in disruption of the BBB, as evidenced by enhanced permeability, reduction of tight junction proteins, and expression of matrix metalloproteinases (MMP)-2 and MMP-9. HIV-infected cells added to our model did not transmigrate in the absence of CCL2, nor did this condition alter BBB integrity. The chemokines CXCL10/interferon-gamma-inducible protein of 10 kDa, CCL3/macrophage inflammatory protein-1alpha, or CCL5/RANTES (regulated on activation normal T-cell expressed and secreted) did not enhance HIV-infected leukocyte transmigration or BBB permeability. The increased capacity of HIV-infected leukocytes to transmigrate in response to CCL2 correlated with their increased expression of CCR2, the chemokine receptor for CCL2. These data suggest that CCL2, but not other chemokines, plays a key role in infiltration of HIV-infected leukocytes into the CNS and the subsequent pathology characteristic of NeuroAIDS.

Memory and Modularity in Cell-Fate Decision Making

Genetically identical cells sharing an environment can display markedly different phenotypes. It is often unclear how much of this variation derives from chance, external signals, or attempts by individual cells to exert autonomous phenotypic programs. By observing thousands of cells for hundreds of consecutive generations under constant conditions, we dissect the stochastic decision between a solitary, motile state and a chained, sessile state in Bacillus subtilis. We show that the motile state is 'memoryless', exhibiting no autonomous control over the time spent in the state. In contrast, the time spent as connected chains of cells is tightly controlled, enforcing coordination among related cells in the multicellular state. We show that the three-protein regulatory circuit governing the decision is modular, as initiation and maintenance of chaining are genetically separable functions. As stimulation of the same initiating pathway triggers biofilm formation, we argue that autonomous timing allows a trial commitment to multicellularity that external signals could extend.

Staphylococcus aureus infection dynamics

Staphylococcus aureus is a human commensal that can also cause systemic infections. This transition requires evasion of the immune response and the ability to exploit different niches within the host. However, the disease mechanisms and the dominant immune mediators against infection are poorly understood. Previously it has been shown that the infecting S. aureus population goes through a population bottleneck, from which very few bacteria escape to establish the abscesses that are characteristic of many infections. Here we examine the host factors underlying the population bottleneck and subsequent clonal expansion in S. aureus infection models, to identify underpinning principles of infection. The bottleneck is a common feature between models and is independent of S. aureus strain. Interestingly, the high doses of S. aureus required for the widely used "survival" model results in a reduced population bottleneck, suggesting that host defences have been simply overloaded. This brings into question the applicability of the survival model. Depletion of immune mediators revealed key breakpoints and the dynamics of systemic infection. Loss of macrophages, including the liver Kupffer cells, led to increased sensitivity to infection as expected but also loss of the population bottleneck and the spread to other organs still occurred. Conversely, neutrophil depletion led to greater susceptibility to disease but with a concomitant maintenance of the bottleneck and lack of systemic spread. We also used a novel microscopy approach to examine abscess architecture and distribution within organs. From these observations we developed a conceptual model for S. aureus disease from initial infection to mature abscess. This work highlights the need to understand the complexities of the infectious process to be able to assign functions for host and bacterial components, and why S. aureus disease requires a seemingly high infectious dose and how interventions such as a vaccine may be more rationally developed.

Cyclin A Regulates Kinetochore-Microtubules to Promote Faithful Chromosome Segregation

The most conspicuous event in the cell cycle is the alignment of chromosomes in metaphase. Chromosome alignment fosters faithful segregation through the formation of bi-oriented attachments of kinetochores to spindle microtubules. Notably, numerous kinetochore-microtubule (k-MT) attachment errors are present in early mitosis (prometaphase), and the persistence of those errors is the leading cause of chromosome mis-segregation in aneuploid human tumour cells that continually mis-segregate whole chromosomes and display chromosomal instability. How robust error correction is achieved in prometaphase to ensure error-free mitosis remains unknown. Here we show that k-MT attachments in prometaphase cells are considerably less stable than in metaphase cells. The switch to more stable k-MT attachments in metaphase requires the proteasome-dependent destruction of cyclin A in prometaphase. Persistent cyclin A expression prevents k-MT stabilization even in cells with aligned chromosomes. By contrast, k-MTs are prematurely stabilized in cyclin-A-deficient cells. Consequently, cells lacking cyclin A display higher rates of chromosome mis-segregation. Thus, the stability of k-MT attachments increases decisively in a coordinated fashion among all chromosomes as cells transit from prometaphase to metaphase. Cyclin A creates a cellular environment that promotes microtubule detachment from kinetochores in prometaphase to ensure efficient error correction and faithful chromosome segregation.

Phosphorylation of p35 and p39 by Cdk5 determines the subcellular location of the holokinase in a phosphorylation-site-specific manner.

Cdk5 is a member of the cyclin-dependent kinase (Cdk) family, which is activated by neuronal activators p35 or p39. Cdk5 regulates a variety of neuronal activities including migration, synaptic activity and neuronal death. p35 and p39 impart cytoplasmic membrane association of p35-Cdk5 and p39-Cdk5, respectively, through their myristoylation, but it is not clearly understood how the cellular localization is related to different functions. We investigated the role of Cdk5 activity in the subcellular localization of p35-Cdk5 and p39-Cdk5. Cdk5 activity affected the localization of p35-Cdk5 and p39-Cdk5 through phosphorylation of p35 or p39. Using unphosphorylated or phosphomimetic mutants of p35 and p39, we found that phosphorylation at Ser8, common to p35 and p39, by Cdk5 regulated the cytoplasmic localization and perinuclear accumulation of unphosphorylated S8A mutants, and whole cytoplasmic distribution of phosphomimetic S8E mutants. Cdk5 activity was necessary to retain Cdk5-activator complexes in the cytoplasm. Nevertheless, small but distinct amounts of p35 and p39 were detected in the nucleus. In particular, nuclear p35 and p39 were increased when the Cdk5 activity was inhibited. p39 had a greater propensity to accumulate in the nucleus than p35, and phosphorylation at Thr84, specific to p39, regulated the potential nuclear localization activity of the Lys cluster in p39. These results suggest that the subcellular localization of the Cdk5-activator complexes is determined by its kinase activity, and also implicate a role for p39-Cdk5 in the nucleus.

An actomyosin-based barrier inhibits cell mixing at compartmental boundaries in Drosophila embryos

Partitioning tissues into compartments that do not intermix is essential for the correct morphogenesis of animal embryos and organs. Several hypotheses have been proposed to explain compartmental cell sorting, mainly differential adhesion, but also regulation of the cytoskeleton or of cell proliferation. Nevertheless, the molecular and cellular mechanisms that keep cells apart at boundaries remain unclear. Here we demonstrate, in early Drosophila melanogaster embryos, that actomyosin-based barriers stop cells from invading neighbouring compartments. Our analysis shows that cells can transiently invade neighbouring compartments, especially when they divide, but are then pushed back into their compartment of origin. Actomyosin cytoskeletal components are enriched at compartmental boundaries, forming cable-like structures when the epidermis is mitotically active. When MyoII (non-muscle myosin II) function is inhibited, including locally at the cable by chromophore-assisted laser inactivation (CALI), in live embryos, dividing cells are no longer pushed back, leading to compartmental cell mixing. We propose that local regulation of actomyosin contractibility, rather than differential adhesion, is the primary mechanism sorting cells at compartmental boundaries.

Stem cell characteristics in prostate cancer cell lines.

BACKGROUND Recent studies indicate the presence of a small, stem-like cell population in several human cancers that is crucial for the tumour (re)population. OBJECTIVE Six established prostate cancer (PCa) cell lines-DU145, DuCaP, LAPC-4, 22Rv1, LNCaP, and PC-3-were examined for their stem cell properties in vitro. DESIGN, SETTINGS, AND PARTICIPANTS The colony-forming efficiency and self-renewal ability of morphologically distinguishable holoclones and paraclones were tested with low-density plating and serial passaging. Expression of the putative stem cell marker CD133 and breast cancer resistance protein (BCRP) was examined with flow cytometry, and immunohistochemical stainings were made for CD133, alpha2-integrin, nestin, BCRP, cytokeratin 5 (CK5), and cytokeratin 18 (CK18). RESULTS AND LIMITATIONS Five out of six cell lines formed clear holo-, mero-, and paraclones. Unlike paraclones, we can maintain DU145 holoclones in culture for several passages, which is indicative of self-renewal ability. Using fluorescence-activated cell sorting (FACS) analysis only in DU145 cells, a small fraction (0.01%) of CD133(+) cells was detected. CD133(+) cells; however, like DU145 BCRP(+) (0.15%) cells, they were not more clonogenic, and they did not show more holoclone formation than the marker-negative cells or unselected cells. Immunohistochemistry revealed alpha2-integrin and BCRP as potential stem cell markers and CK5 with the combination of CK18 to distinguish transient amplifying cells. CONCLUSIONS These results indicate the possible presence of stem-like cells in several established PCa cell lines. CD133 selection does not enrich for stem-like cells in PCa cell lines.

Distinct RNA-dependent RNA polymerases are required for RNAi triggered by double-stranded RNA versus truncated transgenes in Paramecium tetraurelia

In many eukaryotes, RNA-dependent RNA polymerases (RdRPs) play key roles in the RNAi pathway. They have been implicated in the recognition and processing of aberrant transcripts triggering the process, and in amplification of the silencing response. We have tested the functions of RdRP genes from the ciliate Paramecium tetraurelia in experimentally induced and endogenous mechanisms of gene silencing. In this organism, RNAi can be triggered either by high-copy, truncated transgenes or by directly feeding cells with double-stranded RNA (dsRNA). Surprisingly, dsRNA-induced silencing depends on the putatively functional RDR1 and RDR2 genes, which are required for the accumulation of both primary siRNAs and a distinct class of small RNAs suggestive of secondary siRNAs. In contrast, a third gene with a highly divergent catalytic domain, RDR3, is required for siRNA accumulation when RNAi is triggered by truncated transgenes. Our data further implicate RDR3 in the accumulation of previously described endogenous siRNAs and in the regulation of the surface antigen gene family. While only one of these genes is normally expressed in any clonal cell line, the knockdown of RDR3 leads to co-expression of multiple antigens. These results provide evidence for a functional specialization of Paramecium RdRP genes in distinct RNAi pathways operating during vegetative growth.

Role of fascin in filopodial protrusion

In this study, the mechanisms of actin-bundling in filopodia were examined. Analysis of cellular localization of known actin cross-linking proteins in mouse melanoma B16F1 cells revealed that fascin was specifically localized along the entire length of all filopodia, whereas other actin cross-linkers were not. RNA interference of fascin reduced the number of filopodia, and remaining filopodia had abnormal morphology with wavy and loosely bundled actin organization. Dephosphorylation of serine 39 likely determined cellular filopodia frequency. The constitutively active fascin mutant S39A increased the number and length of filopodia, whereas the inactive fascin mutant S39E reduced filopodia frequency. Fluorescence recovery after photobleaching of GFP-tagged wild-type and S39A fascin showed that dephosphorylated fascin underwent rapid cycles of association to and dissociation from actin filaments in filopodia, with t1/2 < 10 s. We propose that fascin is a key specific actin cross-linker, providing stiffness for filopodial bundles, and that its dynamic behavior allows for efficient coordination between elongation and bundling of filopodial actin filaments.

Identification and transmission of Piper yellow mottle virus and Cucumber mosaic virus infecting black pepper (Piper nigrum) in Sri Lanka

Sri Lankan black pepper with symptoms of yellow mottle disease contained a mixture of viruses: Piper yellow mottle virus (PYMV) particles (30 × 130 nm), Cucumber mosaic virus (CMV, 30 nm diameter isometric particles), and unidentified, isometric virus-like particles (30 nm diameter). An effective purification procedure is described for PYMV. Immunosorbent and conventional electron microscopy successfully detected badnavirus particles only when at least partially purified extracts were used. PYMV was confirmed as the cause of the disease, with the other two viruses apparently playing no part in producing symptoms. PYMV was transmitted by grafting, by the insect vectors citrus mealy bug (Planococcus citri) and black pepper lace bug (Diconocoris distanti), but not by mechanical inoculation or through seeds. The CMV isolate was transmitted to indicator plants by mechanical inoculation and by the vector Aphis gossypii, but not by Myzus persicae; but neither mechanical nor insect transmission of CMV to black pepper was successful. A sensitive polymerase chain reaction assay was developed to detect PYMV in black pepper.

Cnn1 inhibits the interactions between the KMN complexes of the yeast kinetochore

Kinetochores attach the replicated chromosomes to the mitotic spindle and orchestrate their transmission to the daughter cells. Kinetochore–spindle binding and chromosome segregation are mediated by the multi-copy KNL1Spc105, MIS12Mtw1 and NDC80Ndc80 complexes that form the so-called KMN network. KMN–spindle attachment is regulated by the Aurora BIpl1 and MPS1Mps1 kinases. It is unclear whether other mechanisms exist that support KMN activity during the cell cycle. Using budding yeast, we show that kinetochore protein Cnn1 localizes to the base of the Ndc80 complex and promotes a functionally competent configuration of the KMN network. Cnn1 regulates KMN activity in a spatiotemporal manner by inhibiting the interaction between its complexes. Cnn1 activity peaks in anaphase and is driven by the Cdc28, Mps1 and Ipl1 kinases.

Deficiency in l-serine deaminase results in abnormal growth and cell division of Escherichia coli K-12.

The loss of the ability to deaminate l-serine severely impairs growth and cell division in Escherichia coli K-12. A strain from which the three genes (sdaA, sdaB, tdcG) coding for this organism's three l-serine deaminases had been deleted grows well in glucose minimal medium but, on subculture into minimal medium with glucose and casamino acids, it makes very large, abnormally shaped cells, many of which lyse. When inoculated into Luria-Bertani (LB) broth with or without glucose, it makes very long filaments. Provision of S-adenosylmethionine restores cell division in LB broth with glucose, and repairs much of the difficulty in growth in medium with casamino acids. We suggest that replication of E. coli is regulated by methylation, that an unusually high intracellular l-serine concentration, in the presence of other amino acids, starves the cell for S-adenosylmethionine and that it is the absence of S-adenosylmethionine and/or of C1-tetrahydrofolate derivatives that prevents normal cell division.

Genome-wide analysis reveals SR protein cooperation and competition in regulated splicing.

SR proteins are well-characterized RNA binding proteins that promote exon inclusion by binding to exonic splicing enhancers (ESEs). However, it has been unclear whether regulatory rules deduced on model genes apply generally to activities of SR proteins in the cell. Here, we report global analyses of two prototypical SR proteins, SRSF1 (SF2/ASF) and SRSF2 (SC35), using splicing-sensitive arrays and CLIP-seq on mouse embryo fibroblasts (MEFs). Unexpectedly, we find that these SR proteins promote both inclusion and skipping of exons in vivo, but their binding patterns do not explain such opposite responses. Further analyses reveal that loss of one SR protein is accompanied by coordinated loss or compensatory gain in the interaction of other SR proteins at the affected exons. Therefore, specific effects on regulated splicing by one SR protein actually depend on a complex set of relationships with multiple other SR proteins in mammalian genomes.

Spatiotemporal control of mitosis by the conserved spindle matrix protein Megator

A putative spindle matrix has been hypothesized to mediate chromosome motion, but its existence and functionality remain controversial. In this report, we show that Megator (Mtor), the Drosophila melanogaster counterpart of the human nuclear pore complex protein translocated promoter region (Tpr), and the spindle assembly checkpoint (SAC) protein Mad2 form a conserved complex that localizes to a nuclear derived spindle matrix in living cells. Fluorescence recovery after photobleaching experiments supports that Mtor is retained around spindle microtubules, where it shows distinct dynamic properties. Mtor/Tpr promotes the recruitment of Mad2 and Mps1 but not Mad1 to unattached kinetochores (KTs), mediating normal mitotic duration and SAC response. At anaphase, Mtor plays a role in spindle elongation, thereby affecting normal chromosome movement. We propose that Mtor/Tpr functions as a spatial regulator of the SAC, which ensures the efficient recruitment of Mad2 to unattached KTs at the onset of mitosis and proper spindle maturation, whereas enrichment of Mad2 in a spindle matrix helps confine the action of a diffusible "wait anaphase" signal to the vicinity of the spindle.

Translational control of intron splicing in eukaryotes

Most eukaryotic genes are interrupted by non-coding introns that must be accurately removed from pre-messenger RNAs to produce translatable mRNAs. Splicing is guided locally by short conserved sequences, but genes typically contain many potential splice sites, and the mechanisms specifying the correct sites remain poorly understood. In most organisms, short introns recognized by the intron definition mechanism cannot be efficiently predicted solely on the basis of sequence motifs. In multicellular eukaryotes, long introns are recognized through exon definition and most genes produce multiple mRNA variants through alternative splicing. The nonsense-mediated mRNA decay (NMD) pathway may further shape the observed sets of variants by selectively degrading those containing premature termination codons, which are frequently produced in mammals. Here we show that the tiny introns of the ciliate Paramecium tetraurelia are under strong selective pressure to cause premature termination of mRNA translation in the event of intron retention, and that the same bias is observed among the short introns of plants, fungi and animals. By knocking down the two P. tetraurelia genes encoding UPF1, a protein that is crucial in NMD, we show that the intrinsic efficiency of splicing varies widely among introns and that NMD activity can significantly reduce the fraction of unspliced mRNAs. The results suggest that, independently of alternative splicing, species with large intron numbers universally rely on NMD to compensate for suboptimal splicing efficiency and accuracy.

Nonproductive human immunodeficiency virus type 1 infection in nucleoside-treated G0 lymphocytes.

Productive infection by human immunodeficiency virus type 1 (HIV-1) requires the activation of target cells. Infection of quiescent peripheral CD4 lymphocytes by HIV-1 results in incomplete, labile, reverse transcripts. We have previously identified G1b as the cell cycle stage required for the optimal completion of the reverse transcription process in T lymphocytes. However, the mechanism(s) involved in the blockage of reverse transcription remains undefined. In this study we investigated whether nucleotide levels influence viral reverse transcription in G0 cells. For this purpose the role of the enzyme ribonucleotide reductase was bypassed, by adding exogenous deoxyribonucleosides to highly purified T cells in the G0 or the G1a phase of the cell cycle. Our data showed a significant increase in the efficiency of the reverse transcription process following the addition of the deoxyribonucleosides. To define the stability and functionality of these full reverse transcripts, we used an HIV-1 reporter virus that expresses the murine heat-stable antigen on the surfaces of infected cells. Following activation of infected quiescent cells treated with exogenous nucleosides, no increased rescue of productive infection was seen. Thus, in addition to failure to complete reverse transcription, there was an additional nonreversible blockage of productive infection in quiescent T cells. These experiments have important relevance in the gene therapy arena, in terms of improving the ability of lentivirus vectors to enter metabolically inactive cells, such as hematopoietic stem cells.

PrimPol Bypasses UV Photoproducts during Eukaryotic Chromosomal DNA Replication

DNA damage can stall the DNA replication machinery, leading to genomic instability. Thus, numerous mechanisms exist to complete genome duplication in the absence of a pristine DNA template, but identification of the enzymes involved remains incomplete. Here, we establish that Primase-Polymerase (PrimPol; CCDC111), an archaeal-eukaryotic primase (AEP) in eukaryotic cells, is involved in chromosomal DNA replication. PrimPol is required for replication fork progression on ultraviolet (UV) light-damaged DNA templates, possibly mediated by its ability to catalyze translesion synthesis (TLS) of these lesions. This PrimPol UV lesion bypass pathway is not epistatic with the Pol η-dependent pathway and, as a consequence, protects xeroderma pigmentosum variant (XP-V) patient cells from UV-induced cytotoxicity. In addition, we establish that PrimPol is also required for efficient replication fork progression during an unperturbed S phase. These and other findings indicate that PrimPol is an important player in replication fork progression in eukaryotic cells.

Eic1 links Mis18 with the CCAN/Mis6/Ctf19 complex to promote CENP-A assembly

CENP-A chromatin forms the foundation for kinetochore assembly. Replication-independent incorporation of CENP-A at centromeres depends on its chaperone HJURP(Scm3), and Mis18 in vertebrates and fission yeast. The recruitment of Mis18 and HJURP(Scm3) to centromeres is cell cycle regulated. Vertebrate Mis18 associates with Mis18BP1(KNL2), which is critical for the recruitment of Mis18 and HJURP(Scm3). We identify two novel fission yeast Mis18-interacting proteins (Eic1 and Eic2), components of the Mis18 complex. Eic1 is essential to maintain Cnp1(CENP-A) at centromeres and is crucial for kinetochore integrity; Eic2 is dispensable. Eic1 also associates with Fta7(CENP-Q/Okp1), Cnl2(Nkp2) and Mal2(CENP-O/Mcm21), components of the constitutive CCAN/Mis6/Ctf19 complex. No Mis18BP1(KNL2) orthologue has been identified in fission yeast, consequently it remains unknown how the key Cnp1(CENP-A) loading factor Mis18 is recruited. Our findings suggest that Eic1 serves a function analogous to that of Mis18BP1(KNL2), thus representing the functional counterpart of Mis18BP1(KNL2) in fission yeast that connects with a module within the CCAN/Mis6/Ctf19 complex to allow the temporally regulated recruitment of the Mis18/Scm3(HJURP) Cnp1(CENP-A) loading factors. The novel interactions identified between CENP-A loading factors and the CCAN/Mis6/Ctf19 complex are likely to also contribute to CENP-A maintenance in other organisms.

Podoplanin-Rich Stromal Networks Induce Dendritic Cell Motility via Activation of the C-type Lectin Receptor CLEC-2

To initiate adaptive immunity, dendritic cells (DCs) move from parenchymal tissues to lymphoid organs by migrating along stromal scaffolds that display the glycoprotein podoplanin (PDPN). PDPN is expressed by lymphatic endothelial and fibroblastic reticular cells and promotes blood-lymph separation during development by activating the C-type lectin receptor, CLEC-2, on platelets. Here, we describe a role for CLEC-2 in the morphodynamic behavior and motility of DCs. CLEC-2 deficiency in DCs impaired their entry into lymphatics and trafficking to and within lymph nodes, thereby reducing T cell priming. CLEC-2 engagement of PDPN was necessary for DCs to spread and migrate along stromal surfaces and sufficient to induce membrane protrusions. CLEC-2 activation triggered cell spreading via downregulation of RhoA activity and myosin light-chain phosphorylation and triggered F-actin-rich protrusions via Vav signaling and Rac1 activation. Thus, activation of CLEC-2 by PDPN rearranges the actin cytoskeleton in DCs to promote efficient motility along stromal surfaces.

An efficient nonviral method to generate integration-free human-induced pluripotent stem cells from cord blood and peripheral blood cells.

The generation of induced pluripotent stem cells (iPSCs) provides the opportunity to use patient-specific somatic cells, which are a valuable source for disease modeling and drug discovery. To promote research involving these cells, it is important to make iPSCs from easily accessible and less invasive tissues, like blood. We have recently reported the efficient generation of human iPSCs from adult fibroblasts using a combination of plasmids encoding OCT3/4, SOX2, KLF4, L-MYC, LIN28, and shRNA for TP53. We herein report a modified protocol enabling efficient iPSC induction from CD34+ cord blood cells and from peripheral blood isolated from healthy donors using these plasmid vectors. The original plasmid mixture could induce iPSCs; however, the efficiency was low. The addition of EBNA1, an essential factor for episomal amplification of the vectors, by an extra plasmid greatly increased the efficiency of iPSC induction, especially when the induction was performed from αβT cells. This improvement enabled the establishment of blood-derived iPSCs from seven healthy donors ranging in age from their 20s to their 60s. This induction method will be useful for the derivation of patient-specific integration-free iPSCs and would also be applicable to the generation of clinical-grade iPSCs in the future.

A core subunit of Polycomb repressive complex 1 is broadly conserved in function but not primary sequence.

Polycomb Group (PcG) proteins mediate heritable gene silencing by modifying chromatin structure. An essential PcG complex, PRC1, compacts chromatin and inhibits chromatin remodeling. In Drosophila melanogaster, the intrinsically disordered C-terminal region of PSC (PSC-CTR) mediates these noncovalent effects on chromatin, and is essential for viability. Because the PSC-CTR sequence is poorly conserved, the significance of its effects on chromatin outside of Drosophila was unclear. The absence of folded domains also made it difficult to understand how the sequence of PSC-CTR encodes its function. To determine the mechanistic basis and extent of conservation of PSC-CTR activity, we identified 17 metazoan PSC-CTRs spanning chordates to arthropods, and examined their sequence features and biochemical properties. PSC-CTR sequences are poorly conserved, but are all highly charged and structurally disordered. We show that active PSC-CTRs--which bind DNA tightly and inhibit chromatin remodeling efficiently--are distinguished from less active ones by the absence of extended negatively charged stretches. PSC-CTR activity can be increased by dispersing its contiguous negative charge, confirming the importance of this property. Using the sequence properties defined as important for PSC-CTR activity, we predicted the presence of active PSC-CTRs in additional diverse genomes. Our analysis reveals broad conservation of PSC-CTR activity across metazoans. This conclusion could not have been determined from sequence alignments. We further find that plants that lack active PSC-CTRs instead possess a functionally analogous PcG protein, EMF1. Thus, our study suggests that a disordered domain with dispersed negative charges underlies PRC1 activity, and is conserved across metazoans and plants.

Divalent cations differentially regulate integrin alphaIIb cytoplasmic tail binding to beta3 and to calcium- and integrin-binding protein.

We have used recombinant or synthetic alphaIIb and beta3 integrin cytoplasmic peptides to study their in vitro complexation and ligand binding capacity by surface plasmon resonance. alpha.beta heterodimerization occurred in a 1:1 stoichiometry with a weak KD in the micromolar range. Divalent cations were not required for this association but stabilized the alpha.beta complex by decreasing the dissociation rate. alpha.beta complexation was impaired by the R995A substitution or the KVGFFKR deletion in alphaIIb but not by the beta3 S752P mutation. Recombinant calcium- and integrin-binding protein (CIB), an alphaIIb-specific ligand, bound to the alphaIIb cytoplasmic peptide in a Ca2+- or Mn2+-independent, one-to-one reaction with a KD value of 12 microM. In contrast, in vitro liquid phase binding of CIB to intact alphaIIbbeta3 occurred preferentially with Mn2+-activated alphaIIbbeta3 conformers, as demonstrated by enhanced coimmunoprecipitation of CIB with PAC-1-captured Mn2+-activated alphaIIbbeta3, suggesting that Mn2+ activation of intact alphaIIbbeta3 induces the exposure of a CIB-binding site, spontaneously exposed by the free alphaIIb peptide. Since CIB did not stimulate PAC-1 binding to inactive alphaIIbbeta3 nor prevented activated alphaIIbbeta3 occupancy by PAC-1, we conclude that CIB does not regulate alphaIIbbeta3 inside-out signaling, but rather is involved in an alphaIIbbeta3 post-receptor occupancy event.

Phosphorylation of centromeric histone H3 variant regulates chromosome segregation in Saccharomyces cerevisiae

The centromeric histone H3 variant (CenH3) is essential for chromosome segregation in eukaryotes. We identify posttranslational modifications of Saccharomyces cerevisiae CenH3, Cse4. Functional characterization of cse4 phosphorylation mutants shows growth and chromosome segregation defects when combined with kinetochore mutants okp1 and ame1. Using a phosphoserine-specific antibody, we show that the association of phosphorylated Cse4 with centromeres increases in response to defective microtubule attachment or reduced cohesion. We determine that evolutionarily conserved Ipl1/Aurora B contributes to phosphorylation of Cse4, as levels of phosphorylated Cse4 are reduced at centromeres in ipl1 strains in vivo, and in vitro assays show phosphorylation of Cse4 by Ipl1. Consistent with these results, we observe that a phosphomimetic cse4-4SD mutant suppresses the temperature-sensitive growth of ipl1-2 and Ipl1 substrate mutants dam1 spc34 and ndc80, which are defective for chromosome biorientation. Furthermore, cell biology approaches using a green fluorescent protein-labeled chromosome show that cse4-4SD suppresses chromosome segregation defects in dam1 spc34 strains. On the basis of these results, we propose that phosphorylation of Cse4 destabilizes defective kinetochores to promote biorientation and ensure faithful chromosome segregation. Taken together, our results provide a detailed analysis, in vivo and in vitro, of Cse4 phosphorylation and its role in promoting faithful chromosome segregation.

Derivation conditions impact X-inactivation status in female human induced pluripotent stem cells.

Female human induced pluripotent stem cell (hiPSC) lines exhibit variability in X-inactivation status. The majority of hiPSC lines maintain one transcriptionally active X (Xa) and one inactive X (Xi) chromosome from donor cells. However, at low frequency, hiPSC lines with two Xas are produced, suggesting that epigenetic alterations of the Xi occur sporadically during reprogramming. We show here that X-inactivation status in female hiPSC lines depends on derivation conditions. hiPSC lines generated by the Kyoto method (retroviral or episomal reprogramming), which uses leukemia inhibitory factor (LIF)-expressing SNL feeders, frequently had two Xas. Early passage Xa/Xi hiPSC lines generated on non-SNL feeders were converted into Xa/Xa hiPSC lines after several passages on SNL feeders, and supplementation with recombinant LIF caused reactivation of some of X-linked genes. Thus, feeders are a significant factor affecting X-inactivation status. The efficient production of Xa/Xa hiPSC lines provides unprecedented opportunities to understand human X-reactivation and -inactivation.

Sustained survivin expression from OX40 costimulatory signals drives T cell clonal expansion.

Sustained signaling from the T cell receptor (TCR) and costimulatory molecules is thought necessary for generating high numbers of effector T cells. Here, we show that Survivin is controlled in peripheral T cells by OX40 cosignaling via sustained PI3k and PKB activation. Survivin is induced by OX40 independent of mitotic progression in late G1, and blocking Survivin suppresses S-phase transition and division of T cells and leads to apoptosis. Moreover, Survivin expression alone is sufficient to restore proliferation and to antagonize apoptosis in costimulation-deficient T cells and can rescue T cell expansion in vivo. Survivin allows effector T cells to accumulate in large numbers, but Bcl-2 family proteins are required for T cell survival after the phase of active division. Thus, sustained Survivin expression from costimulatory signaling maintains T cell division over time and regulates the extent of clonal expansion.

Stress and host immunity amplify Mycobacterium tuberculosis phenotypic heterogeneity and induce nongrowing metabolically active forms.

Nonreplicating and metabolically quiescent bacteria are implicated in latent tuberculosis infections and relapses following "sterilizing" chemotherapy. However, evidence linking bacterial dormancy and persistence in vivo is largely inconclusive. Here we measure the single-cell dynamics of Mycobacterium tuberculosis replication and ribosomal activity using quantitative time-lapse microscopy and a reporter of ribosomal RNA gene expression. Single-cell dynamics exhibit heterogeneity under standard growth conditions, which is amplified by stressful conditions such as nutrient limitation, stationary phase, intracellular replication, and growth in mouse lungs. Additionally, the lungs of chronically infected mice harbor a subpopulation of nongrowing but metabolically active bacteria, which are absent in mice lacking interferon-γ, a cytokine essential for antituberculosis immunity. These cryptic bacterial forms are prominent in mice treated with the antituberculosis drug isoniazid, suggesting a role in postchemotherapeutic relapses. Thus, amplification of bacterial phenotypic heterogeneity in response to host immunity and drug pressure may contribute to tuberculosis persistence.

Analysis of Epstein-Barr virus-regulated host gene expression changes through primary B-cell outgrowth reveals delayed kinetics of latent membrane protein 1-mediated NF-κB activation.

Epstein-Barr virus (EBV) is an oncogenic human herpesvirus that dramatically reorganizes host gene expression to immortalize primary B cells. In this study, we analyzed EBV-regulated host gene expression changes following primary B-cell infection, both during initial proliferation and through transformation into lymphoblastoid cell lines (LCLs). While most EBV-regulated mRNAs were changed during the transition from resting, uninfected B cells through initial B-cell proliferation, a substantial number of mRNAs changed uniquely from early proliferation through LCL outgrowth. We identified constitutively and dynamically EBV-regulated biological processes, protein classes, and targets of specific transcription factors. Early after infection, genes associated with proliferation, stress responses, and the p53 pathway were highly enriched. However, the transition from early to long-term outgrowth was characterized by genes involved in the inhibition of apoptosis, the actin cytoskeleton, and NF-κB activity. It was previously thought that the major viral protein responsible for NF-κB activation, latent membrane protein 1 (LMP1), is expressed within 2 days after infection. Our data indicate that while this is true, LCL-level LMP1 expression and NF-κB activity are not evident until 3 weeks after primary B-cell infection. Furthermore, heterologous NF-κB activation during the first week after infection increased the transformation efficiency, while early NF-κB inhibition had no effect on transformation. Rather, inhibition of NF-κB was not toxic to EBV-infected cells until LMP1 levels and NF-κB activity were high. These data collectively highlight the dynamic nature of EBV-regulated host gene expression and support the notion that early EBV-infected proliferating B cells have a fundamentally distinct growth and survival phenotype from that of LCLs.

Identification of a human HLA-E-restricted CD8+ T cell subset in volunteers immunized with Salmonella enterica serovar Typhi strain Ty21a typhoid vaccine.

Our previous studies in volunteers immunized with Salmonella enterica serovar Typhi (S. Typhi) have suggested an important role for CD8+ T cells in host defense. In this study we describe a novel subset of nonclassical human HLA-E-restricted S. Typhi-specific CD8+ T cells derived from PBMC of Ty21a typhoid vaccinees. CD3+CD8+CD4-CD56- T cells effectively killed S. Typhi-infected targets regardless of whether they share classical HLA class I molecules with them, by a FAS-independent, granule-dependent mechanism, as evidenced by induction of granzyme B release and the blocking effects of concanamycin and strontium ions. The expression of HLA-E Ags, but not CD1-a, -b, or -c, on the membrane of S. Typhi-infected targets rendered them susceptible to lysis. Moreover, anti-HLA-E Abs partially blocked these responses. We also demonstrated that presentation of S. Typhi Ags via HLA-E could stimulate IFN-gamma production. Increases in the net frequency of IFN-gamma spot-forming cells were observed in the presence of targets coated with peptides that contain S. Typhi GroEL HLA-E binding motifs. These results demonstrate that HLA-E binds nonamer peptides derived from bacterial proteins and trigger CD8+-mediated lysis and IFN-gamma production when exposed to infected targets, raising the possibility that this novel effector mechanism might contribute to host defense against intracellular bacterial infections.

Katanin p60-like1 promotes microtubule growth and terminal dendrite stability in the larval class IV sensory neurons of Drosophila.

Dendrite shape is considered a defining component of neuronal function. Yet, the mechanisms specifying diverse dendritic morphologies, and the extent to which their function depends on these morphologies, remain unclear. Here, we demonstrate a requirement for the microtubule-severing protein katanin p60-like 1 (Kat-60L1) in regulating the elaborate dendrite morphology and nocifensive functions of Drosophila larval class IV dendritic arborization neurons. Kat-60L1 mutants exhibit diminished responsiveness to noxious mechanical and thermal stimuli. Class IV dendrite branch number and length are also reduced, supporting a correspondence between neuronal function and the full extent of the dendritic arbor. These arborization defects occur particularly in late larval development, and live imaging reveals that Kat-60L1 is required for dynamic, filopodia-like nascent branches to stabilize during this stage. Mutant dendrites exhibit fewer EB1-GFP-labeled microtubules, suggesting that Kat-60L1 increases polymerizing microtubules to establish terminal branch stability and full arbor complexity. Although loss of the related microtubule-severing protein Spastin also reduces the class IV dendrite arbor, microtubule polymerization within dendrites is unaffected. Conversely, Spastin overexpression destroys stable microtubules within these neurons, while Kat-60L1 has no effect. Kat-60L1 thus sculpts the class IV dendritic arbor through microtubule regulatory mechanisms distinct from Spastin. Our data support differential roles of microtubule-severing proteins in regulating neuronal morphology and function, and provide evidence that dendritic arbor development is the product of multiple pathways functioning at distinct developmental stages.

Induction of pluripotent stem cells from mouse embryonic fibroblasts by Oct4 and Klf4 with small-molecule compounds.

Somatic cells can be induced into pluripotent stem cells (iPSCs) with a combination of four transcription factors, Oct4/Sox2/Klf4/c-Myc or Oct4/Sox2/Nanog/LIN28. This provides an enabling platform to obtain patient-specific cells for various therapeutic and research applications. However, several problems remain for this approach to be therapeutically relevant due to drawbacks associated with efficiency and viral genome integration. Recently, it was shown that neural progenitor cells (NPCs) transduced with Oct4/Klf4 can be reprogrammed into iPSCs. However, NPCs express Sox2 endogenously, possibly facilitating reprogramming in the absence of exogenous Sox2. In this study, we identified a small-molecule combination, BIX-01294 and BayK8644, that enables reprogramming of Oct4/Klf4-transduced mouse embryonic fibroblasts, which do not endogenously express the factors essential for reprogramming. This study demonstrates that small molecules identified through a phenotypic screen can compensate for viral transduction of critical factors, such as Sox2, and improve reprogramming efficiency.

The Sp1-like protein BTEB3 inhibits transcription via the basic transcription element box by interacting with mSin3A and HDAC-1 co-repressors and competing with Sp1.

Sp1-like proteins are characterized by three conserved C-terminal zinc finger motifs that bind GC-rich sequences found in promoters of numerous genes essential for mammalian cell homeostasis. These proteins behave as transcriptional activators or repressors. Although significant information has been reported on the molecular mechanisms by which Sp1-like activators function, relatively little is known about mechanisms for repressor proteins. Here we report the functional characterization of BTEB3, a ubiquitously expressed Sp1-like transcriptional repressor. GAL4 assays show that the N terminus of BTEB3 contains regions that can act as direct repressor domains. Immunoprecipitation assays reveal that BTEB3 interacts with the co-repressor mSin3A and the histone deacetylase protein HDAC-1. Gel shift assays demonstrate that BTEB3 specifically binds the BTE site, a well characterized GC-rich DNA element, with an affinity similar to that of Sp1. Reporter and gel shift assays in Chinese hamster ovary cells show that BTEB3 can also mediate repression by competing with Sp1 for BTE binding. Thus, the characterization of this protein expands the repertoire of BTEB-like members of the Sp1 family involved in transcriptional repression. Furthermore, our results suggest a mechanism of repression for BTEB3 involving direct repression by the N terminus via interaction with mSin3A and HDAC-1 and competition with Sp1 via the DNA-binding domain.

Klebsiella aerogenes urease gene cluster: sequence of ureD and demonstration that four accessory genes (ureD, ureE, ureF, and ureG) are involved in nickel metallocenter biosynthesis.

The region located immediately upstream from the Klebsiella aerogenes urease structural genes was sequenced and shown to possess an open reading frame capable of encoding a 29.8-kDa peptide. Deletions were generated in this gene, denoted ureD, and in each of the genes (ureE, ureF, and ureG) located immediately downstream of the three structural genes. Transformation of the mutated plasmids into Escherichia coli resulted in high levels of urease expression, but the enzyme was inactive (deletions in ureD, ureF, or ureG) or only partially active (deletions in ureE). Ureases were purified from the recombinant cells and shown to be identical to control enzyme when analyzed by gel filtration chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis; however, in every case the activity levels correlated to nickel contents as analyzed by atomic absorption analysis. UreD, UreE, UreF, and UreG peptides were tentatively identified by gel electrophoretic comparison of mutant and control cell extracts, by in vivo expression of separately cloned genes, or by in vitro transcription-translation analyses; the assignments were confirmed for UreE and UreG by amino-terminal sequencing. The latter peptides (apparent M(r)s, 23,900 and 28,500) were present at high levels comparable to those of the urease subunits, whereas the amounts of UreF (apparent M(r), 27,000) and UreD (apparent M(r), 29,300) were greatly reduced, perhaps because of the lack of good ribosome binding sites in the regions upstream of these open reading frames. These results demonstrate that all four accessory genes are necessary for the functional incorporation of the urease metallocenter.

Biochemical analysis of TssK, a core component of the bacterial Type VI secretion system, reveals distinct oligomeric states of TssK and identifies a TssK–TssFG subcomplex

Gram-negative bacteria use the Type VI secretion system (T6SS) to inject toxic proteins into rival bacteria or eukaryotic cells. However, the mechanism of the T6SS is incompletely understood. In the present study, we investigated a conserved component of the T6SS, TssK, using the antibacterial T6SS of Serratia marcescens as a model system. TssK was confirmed to be essential for effector secretion by the T6SS. The native protein, although not an integral membrane protein, appeared to localize to the inner membrane, consistent with its presence within a membrane-anchored assembly. Recombinant TssK purified from S. marcescens was found to exist in several stable oligomeric forms, namely trimer, hexamer and higher-order species. Native-level purification of TssK identified TssF and TssG as interacting proteins. TssF and TssG, conserved T6SS components of unknown function, were required for T6SS activity, but not for correct localization of TssK. A complex containing TssK, TssF and TssG was subsequently purified in vitro, confirming that these three proteins form a new subcomplex within the T6SS. Our findings provide new insight into the T6SS assembly, allowing us to propose a model whereby TssK recruits TssFG into the membrane-associated T6SS complex and different oligomeric states of TssK may contribute to the dynamic mechanism of the system.

Human Macrophages Infected with a High Burden of ESAT-6-Expressing M. tuberculosis Undergo Caspase-1- and Cathepsin B-Independent Necrosis

Mycobacterium tuberculosis (Mtb) infects lung macrophages, which instead of killing the pathogen can be manipulated by the bacilli, creating an environment suitable for intracellular replication and spread to adjacent cells. The role of host cell death during Mtb infection is debated because the bacilli have been shown to be both anti-apoptotic, keeping the host cell alive to avoid the antimicrobial effects of apoptosis, and pro-necrotic, killing the host macrophage to allow infection of neighboring cells. Since mycobacteria activate the NLRP3 inflammasome in macrophages, we investigated whether Mtb could induce one of the recently described inflammasome-linked cell death modes pyroptosis and pyronecrosis. These are mediated through caspase-1 and cathepsin-B, respectively. Human monocyte-derived macrophages were infected with virulent (H37Rv) Mtb at a multiplicity of infection (MOI) of 1 or 10. The higher MOI resulted in strongly enhanced release of IL-1β, while a low MOI gave no IL-1β response. The infected macrophages were collected and cell viability in terms of the integrity of DNA, mitochondria and the plasma membrane was determined. We found that infection with H37Rv at MOI 10, but not MOI 1, over two days led to extensive DNA fragmentation, loss of mitochondrial membrane potential, loss of plasma membrane integrity, and HMGB1 release. Although we observed plasma membrane permeabilization and IL-1β release from infected cells, the cell death induced by Mtb was not dependent on caspase-1 or cathepsin B. It was, however, dependent on mycobacterial expression of ESAT-6. We conclude that as virulent Mtb reaches a threshold number of bacilli inside the human macrophage, ESAT-6-dependent necrosis occurs, activating caspase-1 in the process.

Wnt7a signaling promotes dendritic spine growth and synaptic strength through Ca²⁺/Calmodulin-dependent protein kinase II.

The balance between excitatory and inhibitory synapses is crucial for normal brain function. Wnt proteins stimulate synapse formation by increasing synaptic assembly. However, it is unclear whether Wnt signaling differentially regulates the formation of excitatory and inhibitory synapses. Here, we demonstrate that Wnt7a preferentially stimulates excitatory synapse formation and function. In hippocampal neurons, Wnt7a increases the number of excitatory synapses, whereas inhibitory synapses are unaffected. Wnt7a or postsynaptic expression of Dishevelled-1 (Dvl1), a core Wnt signaling component, increases the frequency and amplitude of miniature excitatory postsynaptic currents (mEPSCs), but not miniature inhibitory postsynaptic currents (mIPSCs). Wnt7a increases the density and maturity of dendritic spines, whereas Wnt7a-Dvl1-deficient mice exhibit defects in spine morphogenesis and mossy fiber-CA3 synaptic transmission in the hippocampus. Using a postsynaptic reporter for Ca(2+)/Calmodulin-dependent protein kinase II (CaMKII) activity, we demonstrate that Wnt7a rapidly activates CaMKII in spines. Importantly, CaMKII inhibition abolishes the effects of Wnt7a on spine growth and excitatory synaptic strength. These data indicate that Wnt7a signaling is critical to regulate spine growth and synaptic strength through the local activation of CaMKII at dendritic spines. Therefore, aberrant Wnt7a signaling may contribute to neurological disorders in which excitatory signaling is disrupted.

Inter-viral conflicts that exploit host CRISPR immune systems of Sulfolobus.

Infection of Sulfolobus islandicus REY15A with mixtures of different Sulfolobus viruses, including STSV2, did not induce spacer acquisition by the host CRISPR immune system. However, coinfection with the tailed fusiform viruses SMV1 and STSV2 generated hyperactive spacer acquisition in both CRISPR loci, exclusively from STSV2, with the resultant loss of STSV2 but not SMV1. SMV1 was shown to activate adaptation while itself being resistant to CRISPR-mediated adaptation and DNA interference. Exceptionally, a single clone S-1 isolated from an SMV1 + STSV2-infected culture, that carried STSV2-specific spacers and had lost STSV2 but not SMV1, acquired spacers from SMV1. This effect was also reproducible on reinfecting wild-type host cells with a variant SMV1 isolated from the S-1 culture. The SMV1 variant lacked a virion protein ORF114 that was shown to bind DNA. This study also provided evidence for: (i) limits on the maximum sizes of CRISPR loci; (ii) spacer uptake strongly retarding growth of infected cultures; (iii) protospacer selection being essentially random and non-directional, and (iv) the reversible uptake of spacers from STSV2 and SMV1. A hypothesis is presented to explain the interactive conflicts between SMV1 and the host CRISPR immune system.

Sphingosine Kinase 1 Regulates the Akt/FOXO3a/Bim Pathway and Contributes to Apoptosis Resistance in Glioma Cells

The aim of this study was to investigate the mechanism through which Sphingosine kinase-1 (SPHK1) exerts its anti-apoptosis activity in glioma cancer cells. We here report that dysregulation of SPHK1 alters the sensitivity of glioma to apoptosis both in vitro and in vivo. Further mechanistic study examined the expression of Bcl-2 family members, including Bcl-2, Mcl-1, Bax and Bim, in SPHK1-overexpressing glioma cells and revealed that only pro-apoptotic Bim was downregulated by SPHK1. Moreover, the transcriptional level of Bim was also altered by SPHK1 in glioma cells. We next confirmed the correlation between SPHK1 and Bim expression in primary glioma specimens. Importantly, increasing SPHK1 expression in glioma cells markedly elevated Akt activity and phosphorylated inactivation of FOXO3a, which led to downregulation of Bim. A pharmacological approach showed that these effects of SPHK1 were dependent on phosphatidylinositol 3-kinase (PI3K). Furthermore, effects of SPHK1 on Akt/FOXO3a/Bim pathway could be reversed by SPHK1 specific RNA interference or SPHK1 inhibitor. Collectively, our results indicate that regulation of the Akt/FOXO3a/Bim pathway may be a novel mechanism by which SPHK1 protects glioma cells from apoptosis, thereby involved in glioma tumorigenesis.

Rictor, a Novel Binding Partner of mTOR, Defines a Rapamycin-Insensitive and Raptor-Independent Pathway that Regulates the Cytoskeleton

The mammalian TOR (mTOR) pathway integrates nutrient- and growth factor-derived signals to regulate growth, the process whereby cells accumulate mass and increase in size. mTOR is a large protein kinase and the target of rapamycin, an immunosuppressant that also blocks vessel restenosis and has potential anticancer applications. mTOR interacts with the raptor and GbetaL proteins to form a complex that is the target of rapamycin. Here, we demonstrate that mTOR is also part of a distinct complex defined by the novel protein rictor (rapamycin-insensitive companion of mTOR). Rictor shares homology with the previously described pianissimo from D. discoidieum, STE20p from S. pombe, and AVO3p from S. cerevisiae. Interestingly, AVO3p is part of a rapamycin-insensitive TOR complex that does not contain the yeast homolog of raptor and signals to the actin cytoskeleton through PKC1. Consistent with this finding, the rictor-containing mTOR complex contains GbetaL but not raptor and it neither regulates the mTOR effector S6K1 nor is it bound by FKBP12-rapamycin. We find that the rictor-mTOR complex modulates the phosphorylation of Protein Kinase C alpha (PKCalpha) and the actin cytoskeleton, suggesting that this aspect of TOR signaling is conserved between yeast and mammals.

Th2-mediated host protective immunity to intestinal nematode infections.

Despite many years of study, relatively little is known about the effector mechanisms that operate against intestine-dwelling nematodes. Most of the current understanding comes from studies of laboratory model systems in rodents. It is clear that when an intestinal helminth infection takes place the immune system generates a strong Th2-mediated response, which regulates a variety of responses characteristic of helminth infections such as eosinophilia, intestinal mastocytosis and elevated IgE production. The ability to modulate the host's immune response in vivo with cytokine-specific monoclonal antibodies and recombinant cytokines, together with the use of animals with disruption of key genes involved in the immune response, have provided powerful tools with which to dissect the potential effector mechanisms operating. In the absence of a T-cell compartment the host is unable to expel the parasite. If a Th1-dominated response is generated, protective immunity is almost universally compromised. Thus, it it would appear that some aspect of a Th2-mediated response controls effector mechanisms. Although it is clear that for some infections the mast cell appears to be involved in protection, probably through the generation of a non-specific inflammatory response, how these cells become activated remains unclear. Data from infections in transgenic animals suggest that activation is not through the high-affinity receptor for IgE. Such studies also call into doubt the importance of conventional interactions between effector leucocytes and antibody. There is little evidence to support a protective role for eosinophilia in any system. New data also imply that, although interleukin 4 (IL-4) is generally important (and can exert effects independent of an adaptive immune response), it is not always sufficient to mediate protection; other Th2 cytokines (e.g. IL-13) may warrant closer investigation. It is apparent that a number of potential Th2-controlled effector mechanisms (some of which may be particularly important at mucosal surfaces) remain to be explored. Overall, it is likely that worm expulsion is the result of a combination of multiple mechanisms, some of which are more critical to some species of parasite than to others.

Replisome stalling and stabilization at CGG repeats, which are responsible for chromosomal fragility

Expanded CGG repeats cause chromosomal fragility and hereditary neurological disorders in humans. Replication forks stall at CGG repeats in a length-dependent manner in primate cells and in yeast. Saccharomyces cerevisiae proteins Tof1 and Mrc1 facilitate replication fork progression through CGG repeats. Remarkably, the fork-stabilizing role of Mrc1 does not involve its checkpoint function. Thus, chromosomal fragility might occur when forks stalled at expanded CGG repeats escape the S-phase checkpoint.

Nanoparticle conjugation of CpG enhances adjuvancy for cellular immunity and memory recall at low dose.

In subunit vaccines, strong CD8(+) T-cell responses are desired, yet they are elusive at reasonable adjuvant doses. We show that targeting adjuvant to the lymph node (LN) via ultrasmall polymeric nanoparticles (NPs), which rapidly drain to the LN after intradermal injection, greatly enhances adjuvant efficacy at low doses. Coupling CpG-B or CpG-C oligonucleotides to NPs led to better dual-targeting of adjuvant and antigen (codelivered on separate NPs) in cross-presenting dendritic cells compared with free adjuvant. This led to enhanced dendritic cell maturation and T helper 1 (Th1)-cytokine secretion, in turn driving stronger effector CD8(+) T-cell activation with enhanced cytolytic profiles and, importantly, more powerful memory recall. With only 4 μg CpG, NP-CpG-B could substantially protect mice from syngeneic tumor challenge, even after 4 mo of vaccination, compared with free CpG-B. Together, these results show that nanocarriers can enhance vaccine efficacy at a low adjuvant dose for inducing potent and long-lived cellular immunity.

Fasting Activation of AgRP Neurons Requires NMDA Receptors and Involves Spinogenesis and Increased Excitatory Tone

AgRP neuron activity drives feeding and weight gain whereas that of nearby POMC neurons does the opposite. However, the role of excitatory glutamatergic input in controlling these neurons is unknown. To address this question, we generated mice lacking NMDA receptors (NMDARs) on either AgRP or POMC neurons. Deletion of NMDARs from AgRP neurons markedly reduced weight, body fat and food intake whereas deletion from POMC neurons had no effect. Activation of AgRP neurons by fasting, as assessed by c-Fos, Agrp and Npy mRNA expression, AMPA receptor-mediated EPSCs, depolarization and firing rates, required NMDARs. Furthermore, AgRP but not POMC neurons have dendritic spines and increased glutamatergic input onto AgRP neurons caused by fasting was paralleled by an increase in spines, suggesting fasting induced synaptogenesis and spinogenesis. Thus glutamatergic synaptic transmission and its modulation by NMDARs play key roles in controlling AgRP neurons and determining the cellular and behavioral response to fasting.

Ligand binding determines whether CD46 is internalized by clathrin-coated pits or macropinocytosis.

CD46 is a ubiquitous human cell surface receptor for the complement components C3b and C4b and for various pathogens, including the measles virus and human herpes virus 6. Ligand binding to CD46 affects (i) protection of autologous cells from complement attack by breakdown of complement components, (ii) intracellular signals that affect the regulation of immune cell function, (iii) antigen presentation, and (iv) down-regulation of cell surface CD46. Recent evidence indicates that CD46 signaling can link innate and acquired immune function. The molecular mechanisms for these processes and the importance of intracellular trafficking of the receptor have not yet been elucidated. We demonstrate here that, in nonlymphoid cells, CD46 is constitutively internalized via clathrin-coated pits, traffics to multivesicular bodies, and is recycled to the cell surface. However, cross-linking of CD46 at the cell surface, by either multivalent antibody or by measles virus, induces pseudopodia that engulf the ligand in a process similar to macropinocytosis, and leads to the degradation of cell surface CD46. Thus, we have elucidated two pathways for CD46 internalization, which are regulated by the valence of cross-linking of CD46 and which utilize either clathrin-coated pits or pseudopodial extension. This has important implications for CD46 signaling, antigen presentation, CD46 down-regulation, and engulfment of pathogens.

Nicotinamide Phosphoribosyltransferase/Visfatin Does Not Catalyze Nicotinamide Mononucleotide Formation in Blood Plasma

Nicotinamide (Nam) phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme in mammalian NAD synthesis, catalyzing nicotinamide mononucleotide (NMN) formation from Nam and 5-phosphoribosyl 1-pyrophosphate (PRPP). NAMPT has also been described as an adipocytokine visfatin with a variety of actions, although physiological significance of this protein remains unclear. It has been proposed that possible actions of visfatin are mediated through the extracellular formation of NMN. However, we did not detect NMN in mouse blood plasma, even with a highly specific and sensitive liquid chromatography/tandem mass spectrometry. Furthermore, there is no or little ATP, the activator of NAMPT, in extracellular spaces. We thus questioned whether visfatin catalyzes the in situ formation of NMN under such extracellular milieus. To address this question, we here determined K(m) values for the substrates Nam and PRPP in the NAMPT reaction without or with ATP using a recombinant human enzyme and found that 1 mM ATP dramatically decreases K(m) values for the substrates, in particular PRPP to its intracellular concentration. Consistent with the kinetic data, only when ATP is present at millimolar levels, NAMPT efficiently catalyzed the NMN formation at the intracellular concentrations of the substrates. Much lower concentrations of Nam and almost the absence of PRPP and ATP in the blood plasma suggest that NAMPT should not efficiently catalyze its reaction under the extracellular milieu. Indeed, NAMPT did not form NMN in the blood plasma. From these kinetic analyses of the enzyme and quantitative determination of its substrates, activator, and product, we conclude that visfatin does not participate in NMN formation under the extracellular milieus. Together with the absence of NMN in the blood plasma, our conclusion does not support the concept of "NAMPT-mediated systemic NAD biosynthesis. " Our study would advance current understanding of visfatin physiology.

Dual roles of plcA in Listeria monocytogenes pathogenesis.

The plcA gene of Listeria monocytogenes encodes a secreted phosphatidylinositol-specific phospholipase C (Pl-PLC). Recent studies have established that transposon mutations within plcA result in avirulence for mice and pleiotropic effects when examined in tissue-culture models of infection. Genetic analysis reveals that many of the effects of the transposon insertions are due to loss of readthrough transcription from plcA into the downstream gene prfA, which encodes an essential transcription factor of numerous L. monocytogenes virulence genes. Construction of an in-frame deletion within plcA had no effect on expression of prfA thus allowing direct assignment of a role of the Pl-PLC in pathogenesis. Pl-PLC was shown to play a significant role in mediating escape of L. monocytogenes from phagosomes of primary murine macrophages. Interestingly, this defect manifested itself in vivo in the liver but not in the spleen of infected mice.

S-phase-promoting cyclin-dependent kinases prevent re-replication by inhibiting the transition of replication origins to a pre-replicative state

BACKGROUND DNA replication and mitosis are triggered by activation of kinase complexes, each made up of a cyclin and a cyclin-dependent kinase (Cdk). It had seemed possible that the association of Cdks with different classes of cyclins specifies whether S phase (replication) or M phase (mitosis) will occur. The recent finding that individual B-type cyclins (encoded by the genes CLB1-CLB6) can have functions in both processes in the budding yeast Saccharomyces cerevisiae casts doubt on this notion. RESULTS S. cerevisiae strains lacking C1b1-C1b4 undergo DNA replication once but fail to enter mitosis. We have isolated mutations in two genes, SIM1 and SIM2 (SIM2 is identical to SEC72), which allow such cells to undergo an extra round of DNA replication without mitosis. The Clb5 kinase, which promotes S phase, remains active during the G2-phase arrest of cells of the parental strain, but its activity declines rapidly in sim mutants. Increased expression of the CLB5 gene prevents re-replication. Thus, a cyclin B-kinase that promotes DNA replication in G1-phase cells can prevent re-replication in G2-phase cells. Inactivation of C1b kinases by expression of the specific C1b-Cdk1 inhibitor p40SIC1 is sufficient to induce a prereplicative state at origins of replication in cells blocked in G2/M phase by nocodazole. Re-activation of C1b-Cdk1 kinases induces a second round of DNA replication. CONCLUSIONS We propose that S-phase-promoting cyclin B--Cdk complexes prevent re-replication during S, G2 and M phases by inhibiting the transition of replication origins to a pre-replicative state. This model can explain both why origins 'fire' only once per S phase and why S phase is dependent on completion of the preceding M phase.

SvpA, a novel surface virulence-associated protein required for intracellular survival of Listeria monocytogenes.

A previously unknown protein, designated SvpA (surface virulence-associated protein) and implicated in the virulence of the intracellular pathogen Listeria monocytogenes, was identified. This 64 kDa protein, encoded by svpA, is both secreted in culture supernatants and surface-exposed, as shown by immunogold labelling of whole bacteria with an anti-SvpA antibody. Analysis of the peptide sequence revealed that SvpA contains a leader peptide, a predicted C-terminal transmembrane region and a positively charged tail resembling that of the surface protein ActA, suggesting that SvpA might partially reassociate with the bacterial surface by its C-terminal membrane anchor. An allelic mutant was constructed by disrupting svpA in the wild-type strain LO28. The virulence of this mutant was strongly attenuated in the mouse, with a 2 log decrease in the LD50 and restricted bacterial growth in organs as compared to the wild-type strain. This reduced virulence was not related either to a loss of adherence or to a lower expression of known virulence factors, which remained unaffected in the svpA mutant. It was caused by a restriction of intracellular growth of mutant bacteria. By following the intracellular behaviour of bacteria within bone-marrow-derived macrophages by confocal and electron microscopy studies, it was found that most svpA mutant bacteria remained confined within phagosomes, in contrast to wild-type bacteria which rapidly escaped to the cytoplasm. The regulation of svpA was independent of PrfA, the transcriptional activator of virulence genes in L. monocytogenes. In fact, SvpA was down-regulated by MecA, ClpC and ClpP, which are highly homologous to proteins of Bacillus subtilis forming a regulatory complex controlling the competence state of this saprophyte. The results indicate that: (i) SvpA is a novel factor involved in the virulence of L. monocytogenes, promoting bacterial escape from phagosomes of macrophages; (ii) SvpA is, at least partially, associated with the surface of bacteria; and (iii) SvpA is PrfA-independent and controlled by a MecA-dependent regulatory network.

Temporal regulation of topoisomerase IV activity in E. coli.

We isolated a mutant allele of dnaX, encoding the tau and gamma subunits of the DNA polymerase III holoenzyme, that causes extreme cell filamentation but does not affect either cell growth or DNA replication. This phenotype results from a defect in daughter chromosome decatenation during rapid growth. In these cells, ParC, one subunit of topoisomerase IV, no longer associated with the replication factory, as occurs in wild-type cells, and was instead distributed uniformly on the nucleoid; the distribution of ParE, the other subunit of topoisomerase IV, was unaffected. In addition, the majority of topoisomerase IV activity in synchronized cell populations was restricted to late in the cell cycle, when replication was essentially complete. These observations suggest that topoisomerase IV activity in vivo might be dependent on release of ParC from the replication factory.

Conditional Kif3a ablation causes abnormal hedgehog signaling topography, growth plate dysfunction, and excessive bone and cartilage formation during mouse skeletogenesis.

The motor protein Kif3a and primary cilia regulate important developmental processes, but their roles in skeletogenesis remain ill-defined. Here we created mice deficient in Kif3a in cartilage and focused on the cranial base and synchondroses. Kif3a deficiency caused cranial base growth retardation and dysmorphogenesis, which were evident in neonatal animals by anatomical and micro-computed tomography (microCT) inspection. Kif3a deficiency also changed synchondrosis growth plate organization and function, and the severity of these changes increased over time. By postnatal day (P)7, mutant growth plates lacked typical zones of chondrocyte proliferation and hypertrophy, and were instead composed of chondrocytes with an unusual phenotype characterized by strong collagen II (Col2a1) gene expression but barely detectable expression of Indian hedgehog (Ihh), collagen X (Col10a1), Vegf (Vegfa), MMP-13 (Mmp13) and osterix (Sp7). Concurrently, unexpected developmental events occurred in perichondrial tissues, including excessive intramembranous ossification all along the perichondrial border and the formation of ectopic cartilage masses. Looking for possible culprits for these latter processes, we analyzed hedgehog signalling topography and intensity by monitoring the expression of the hedgehog effectors Patched 1 and Gli1, and of the hedgehog-binding cell-surface component syndecan 3. Compared with controls, hedgehog signaling was quite feeble within mutant growth plates as early as P0, but was actually higher and was widespread all along mutant perichondrial tissues. Lastly, we studied postnatal mice deficient in Ihh in cartilage; their cranial base defects only minimally resembled those in Kif3a-deficient mice. In summary, Kif3a and primary cilia make unique contributions to cranial base development and synchondrosis growth plate function. Their deficiency causes abnormal topography of hedgehog signaling, growth plate dysfunction, and un-physiologic responses and processes in perichondrial tissues, including ectopic cartilage formation and excessive intramembranous ossification.

Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases

Realizing the full potential of human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) requires efficient methods for genetic modification. However, techniques to generate cell type–specific lineage reporters, as well as reliable tools to disrupt, repair or overexpress genes by gene targeting, are inefficient at best and thus are not routinely used. Here we report the highly efficient targeting of three genes in human pluripotent cells using zinc-finger nuclease (ZFN)–mediated genome editing. First, using ZFNs specific for the OCT4 (POU5F1) locus, we generated OCT4-eGFP reporter cells to monitor the pluripotent state of hESCs. Second, we inserted a transgene into the AAVS1 locus to generate a robust drug-inducible overexpression system in hESCs. Finally, we targeted the PITX3 gene, demonstrating that ZFNs can be used to generate reporter cells by targeting non-expressed genes in hESCs and hiPSCs.

The Germ Cell Determinant Blimp1 Is Not Required for Derivation of Pluripotent Stem Cells

Blimp1 (Prdm1), the key determinant of primordial germ cells (PGCs), plays a combinatorial role with Prdm14 during PGC specification from postimplantation epiblast cells. They together initiate epigenetic reprogramming in early germ cells toward an underlying pluripotent state, which is equivalent to embryonic stem cells (ESCs). Whereas Prdm14 alone can promote reprogramming and is important for the propagation of the pluripotent state, it is not known whether Blimp1 is similarly involved. By using a genetic approach, we demonstrate that Blimp1 is dispensable for the derivation and maintenance of ESCs and postimplantation epiblast stem cells (epiSCs). Notably, Blimp1 is also dispensable for reprogramming epiSCs to ESCs. Thus, although Blimp1 is obligatory for PGC specification, it is not required for the reversion of epiSCs to ESCs and for their maintenance thereafter. This study suggests that reprogramming, including that of somatic cells to ESCs, may not entail an obligatory route through a Blimp1-positive PGC-like state.

The mammalian target of rapamycin signaling pathway mediates epileptogenesis in a model of temporal lobe epilepsy.

Understanding molecular mechanisms mediating epileptogenesis is critical for developing more effective therapies for epilepsy. We recently found that the mammalian target of rapamycin (mTOR) signaling pathway is involved in epileptogenesis, and mTOR inhibitors prevent epilepsy in a mouse model of tuberous sclerosis complex. Here, we investigated the potential role of mTOR in a rat model of temporal lobe epilepsy initiated by status epilepticus. Acute kainate-induced seizures resulted in biphasic activation of the mTOR pathway, as evident by an increase in phospho-S6 (P-S6) expression. An initial rise in P-S6 expression started approximately 1 h after seizure onset, peaked at 3-6 h, and returned to baseline by 24 h in both hippocampus and neocortex, reflecting widespread stimulation of mTOR signaling by acute seizure activity. After resolution of status epilepticus, a second increase in P-S6 was observed in hippocampus only, which started at 3 d, peaked 5-10 d, and persisted for several weeks after kainate injection, correlating with the development of chronic epileptogenesis within hippocampus. The mTOR inhibitor rapamycin, administered before kainate, blocked both the acute and chronic phases of seizure-induced mTOR activation and decreased kainate-induced neuronal cell death, neurogenesis, mossy fiber sprouting, and the development of spontaneous epilepsy. Late rapamycin treatment, after termination of status epilepticus, blocked the chronic phase of mTOR activation and reduced mossy fiber sprouting and epilepsy but not neurogenesis or neuronal death. These findings indicate that mTOR signaling mediates mechanisms of epileptogenesis in the kainate rat model and that mTOR inhibitors have potential antiepileptogenic effects in this model.

Highly efficient miRNA-mediated reprogramming of mouse and human somatic cells to pluripotency.

Transcription factor-based cellular reprogramming has opened the way to converting somatic cells to a pluripotent state, but has faced limitations resulting from the requirement for transcription factors and the relative inefficiency of the process. We show here that expression of the miR302/367 cluster rapidly and efficiently reprograms mouse and human somatic cells to an iPSC state without a requirement for exogenous transcription factors. This miRNA-based reprogramming approach is two orders of magnitude more efficient than standard Oct4/Sox2/Klf4/Myc-mediated methods. Mouse and human miR302/367 iPSCs display similar characteristics to Oct4/Sox2/Klf4/Myc-iPSCs, including pluripotency marker expression, teratoma formation, and, for mouse cells, chimera contribution and germline contribution. We found that miR367 expression is required for miR302/367-mediated reprogramming and activates Oct4 gene expression, and that suppression of Hdac2 is also required. Thus, our data show that miRNA and Hdac-mediated pathways can cooperate in a powerful way to reprogram somatic cells to pluripotency.

Engraftment of engineered ES cell–derived cardiomyocytes but not BM cells restores contractile function to the infarcted myocardium

Cellular cardiomyoplasty is an attractive option for the treatment of severe heart failure. It is, however, still unclear and controversial which is the most promising cell source. Therefore, we investigated and examined the fate and functional impact of bone marrow (BM) cells and embryonic stem cell (ES cell)–derived cardiomyocytes after transplantation into the infarcted mouse heart. This proved particularly challenging for the ES cells, as their enrichment into cardiomyocytes and their long-term engraftment and tumorigenicity are still poorly understood. We generated transgenic ES cells expressing puromycin resistance and enhanced green fluorescent protein cassettes under control of a cardiac-specific promoter. Puromycin selection resulted in a highly purified (>99%) cardiomyocyte population, and the yield of cardiomyocytes increased 6–10-fold because of induction of proliferation on purification. Long-term engraftment (4–5 months) was observed when co-transplanting selected ES cell–derived cardiomyocytes and fibroblasts into the injured heart of syngeneic mice, and no teratoma formation was found (n = 60). Although transplantation of ES cell–derived cardiomyocytes improved heart function, BM cells had no positive effects. Furthermore, no contribution of BM cells to cardiac, endothelial, or smooth muscle neogenesis was detected. Hence, our results demonstrate that ES-based cell therapy is a promising approach for the treatment of impaired myocardial function and provides better results than BM-derived cells.

Tumor necrosis factor signaling mediates resistance to mycobacteria by inhibiting bacterial growth and macrophage death.

Tumor necrosis factor (TNF), a key effector in controlling tuberculosis, is thought to exert protection by directing formation of granulomas, organized aggregates of macrophages and other immune cells. Loss of TNF signaling causes progression of tuberculosis in humans, and the increased mortality of Mycobacterium tuberculosis-infected mice is associated with disorganized necrotic granulomas, although the precise roles of TNF signaling preceding this endpoint remain undefined. We monitored transparent Mycobacterium marinum-infected zebrafish live to conduct a stepwise dissection of how TNF signaling operates in mycobacterial pathogenesis. We found that loss of TNF signaling caused increased mortality even when only innate immunity was operant. In the absence of TNF, intracellular bacterial growth and granuloma formation were accelerated and was followed by necrotic death of overladen macrophages and granuloma breakdown. Thus, TNF is not required for tuberculous granuloma formation, but maintains granuloma integrity indirectly by restricting mycobacterial growth within macrophages and preventing their necrosis.

Linking Splicing to Pol II Transcription Stabilizes Pre-mRNAs and Influences Splicing Patterns

RNA processing is carried out in close proximity to the site of transcription, suggesting a regulatory link between transcription and pre-mRNA splicing. Using an in vitro transcription/splicing assay, we demonstrate that an association of RNA polymerase II (Pol II) transcription and pre-mRNA splicing is required for efficient gene expression. Pol II-synthesized RNAs containing functional splice sites are protected from nuclear degradation, presumably because the local concentration of the splicing machinery is sufficiently high to ensure its association over interactions with nucleases. Furthermore, the process of transcription influences alternative splicing of newly synthesized pre-mRNAs. Because other RNA polymerases do not provide similar protection from nucleases, and their RNA products display altered splicing patterns, the link between transcription and RNA processing is RNA Pol II-specific. We propose that the connection between transcription by Pol II and pre-mRNA splicing guarantees an extended half-life and proper processing of nascent pre-mRNAs.

Immunogenicity of induced pluripotent stem cells

Induced pluripotent stem cells (iPSCs), reprogrammed from somatic cells with defined factors, hold great promise for regenerative medicine as the renewable source of autologous cells. Whereas it has been generally assumed that these autologous cells should be immune-tolerated by the recipient from whom the iPSCs are derived, their immunogenicity has not been vigorously examined. We show here that, whereas embryonic stem cells (ESCs) derived from inbred C57BL/6 (B6) mice can efficiently form teratomas in B6 mice without any evident immune rejection, the allogeneic ESCs from 129/SvJ mice fail to form teratomas in B6 mice due to rapid rejection by recipients. B6 mouse embryonic fibroblasts (MEFs) were reprogrammed into iPSCs by either retroviral approach (ViPSCs) or a novel episomal approach (EiPSCs) that causes no genomic integration. In contrast to B6 ESCs, teratomas formed by B6 ViPSCs were mostly immune-rejected by B6 recipients. In addition, the majority of teratomas formed by B6 EiPSCs were immunogenic in B6 mice with T cell infiltration, and apparent tissue damage and regression were observed in a small fraction of teratomas. Global gene expression analysis of teratomas formed by B6 ESCs and EiPSCs revealed a number of genes frequently overexpressed in teratomas derived from EiPSCs, and several such gene products were shown to contribute directly to the immunogenicity of the B6 EiPSC-derived cells in B6 mice. These findings indicate that, in contrast to derivatives of ESCs, abnormal gene expression in some cells differentiated from iPSCs can induce T-cell-dependent immune response in syngeneic recipients. Therefore, the immunogenicity of therapeutically valuable cells derived from patient-specific iPSCs should be evaluated before any clinic application of these autologous cells into the patients.

A Tunable Diffusion‐Consumption Mechanism of Cytokine Propagation Enables Plasticity in Cell‐to‐Cell Communication in the Immune System

&NA; Immune cells communicate by exchanging cytokines to achieve a context‐appropriate response, but the distances over which such communication happens are not known. Here, we used theoretical considerations and experimental models of immune responses in vitro and in vivo to quantify the spatial extent of cytokine communications in dense tissues. We established that competition between cytokine diffusion and consumption generated spatial niches of high cytokine concentrations with sharp boundaries. The size of these self‐assembled niches scaled with the density of cytokine‐consuming cells, a parameter that gets tuned during immune responses. In vivo, we measured interactions on length scales of 80–120 &mgr;m, which resulted in a high degree of cell‐to‐cell variance in cytokine exposure. Such heterogeneous distributions of cytokines were a source of non‐genetic cell‐to‐cell variability that is often overlooked in single‐cell studies. Our findings thus provide a basis for understanding variability in the patterning of immune responses by diffusible factors. Graphical Abstract Figure. No caption available. HighlightsCytokine penetration in tissues is governed by a diffusion‐consumption mechanismSpherical cytokine niches are generated around cytokine‐producing cellsThe characteristic niche size depends on the density of cytokine consumersCytokine niches are a source of variability in otherwise identical cells &NA; Cytokine‐mediated communication allows immune cells to achieve a context‐appropriate response, but the distance over which this communication happens is unclear. Oyler‐Yaniv et al. (2017) show that a simple diffusion‐consumption mechanism quantitatively describes the spatial spread of cytokines in vivo and results in localized niches of high cytokine concentrations that contribute to cell‐to‐cell variability.

CENP-B Controls Centromere Formation Depending on the Chromatin Context

The centromere is a chromatin region that serves as the spindle attachment point and directs accurate inheritance of eukaryotic chromosomes during cell divisions. However, the mechanism by which the centromere assembles and stabilizes at a specific genomic region is not clear. The de novo formation of a human/mammalian artificial chromosome (HAC/MAC) with a functional centromere assembly requires the presence of alpha-satellite DNA containing binding motifs for the centromeric CENP-B protein. We demonstrate here that de novo centromere assembly on HAC/MAC is dependent on CENP-B. In contrast, centromere formation is suppressed in cells expressing CENP-B when alpha-satellite DNA was integrated into a chromosomal site. Remarkably, on those integration sites CENP-B enhances histone H3-K9 trimethylation and DNA methylation, thereby stimulating heterochromatin formation. Thus, we propose that CENP-B plays a dual role in centromere formation, ensuring de novo formation on DNA lacking a functional centromere but preventing the formation of excess centromeres on chromosomes.

Evidence for the presence of urease apoprotein complexes containing UreD, UreF, and UreG in cells that are competent for in vivo enzyme activation.

In vivo activation of Klebsiella aerogenes urease, a nickel-containing enzyme, requires the presence of functional UreD, UreF, and UreG accessory proteins and is further facilitated by UreE. These accessory proteins are proposed to be involved in metallocenter assembly (M. H. Lee, S. B. Mulrooney, M. J. Renner, Y. Markowicz, and R. P. Hausinger, J. Bacteriol. 174:4324-4330, 1992). A series of three UreD-urease apoprotein complexes are present in cells that express ureD at high levels, and these complexes are thought to be essential for in vivo activation of the enzyme (I.-S. Park, M. B. Carr, and R. P. Hausinger, Proc. Natl. Acad. Sci. USA 91:3233-3237, 1994). In this study, we describe the effect of accessory gene deletions on urease complex formation. The ureE, ureF, and ureG gene products were found not to be required for formation of the UreD-urease complexes; however, the complexes from the ureF deletion mutant exhibited delayed elution during size exclusion chromatography. Because these last complexes were of typical UreD-urease sizes according to native gel electrophoretic analysis, we propose that UreF alters the conformation of the UreD-urease complexes. The same studies revealed the presence of an additional series of urease apoprotein complexes present only in cells containing ureD, ureF, and ureG, along with the urease subunit genes. These new complexes were shown to contain urease, UreD, UreF, and UreG. We propose that the UreD-UreF-UreG-urease apoprotein complexes represent the activation-competent form of urease apoprotein in the cell.

Induction of stem-like cells with malignant properties by chronic exposure of human lung epithelial cells to single-walled carbon nanotubes

BACKGROUND Carbon nanotubes (CNT) hold great promise to create new and better products for commercial and biomedical applications, but their long-term adverse health effects are a major concern. The objective of this study was to address human lung cancer risks associated with chronic pulmonary exposure to single-walled (SW) CNT through the fundamental understanding of cellular and molecular processes leading to carcinogenesis. We hypothesized that the acquisition of cancer stem cells (CSC), a subpopulation that drive tumor initiation and progression, may contribute to CNT carcinogenesis. METHODS Non-tumorigenic human lung epithelial cells were chronically exposed to well-dispersed SWCNT for a period of 6 months at the physiologically relevant concentration of 0.02 μg/cm2 surface area dose. Chronic SWCNT-exposed cells were evaluated for the presence of CSC-like cells under CSC-selective conditions of tumor spheres and side population (SP). CSC-like cells were isolated using fluorescence-activated cell sorting and were assessed for aggressive behaviors, including acquired apoptosis resistance and increased cell migration and invasion in vitro, and tumor-initiating capability in vivo. Non-small cell lung cancer cells served as a positive control. RESULTS We demonstrated for the first time the existence of CSC-like cells in all clones of chronic SWCNT-exposed lung epithelial cells. These CSC-like cells, in contrary to their non-CSC counterpart, possessed all biological features of lung CSC that are central to irreversible malignant transformation, self-renewal, aggressive cancer behaviors, and in vivo tumorigenesis. These cells also displayed aberrant stem cell markers, notably Nanog, SOX-2, SOX-17 and E-cadherin. Restored expression of tumor suppressor p53 abrogated CSC properties of CSC-like cells. Furthermore, we identified specific stem cell surface markers CD24low and CD133high that are associated with SWCNT-induced CSC formation and tumorigenesis. CONCLUSIONS Our findings provide new and compelling evidence for the acquisition of CSC-like cells induced by chronic SWCNT exposure, which are likely to be a major driving force for SWCNT tumorigenesis. Thus, our study supports prudent adoption of prevention strategies and implementation of exposure control for SWCNT. We also suggest that the detection of CSC and associated surface markers may provide an effective screening tool for prediction of the carcinogenic potential of SWCNT and related nanoparticles.

Distinction and relationship between elongation rate and processivity of RNA polymerase II in vivo.

A number of proteins and drugs have been implicated in the process of transcriptional elongation by RNA polymerase (Pol) II, but the factors that govern the elongation rate (nucleotide additions per min) and processivity (nucleotide additions per initiation event) in vivo are poorly understood. Here, we show that a mutation in the Rpb2 subunit of Pol II reduces both the elongation rate and processivity in vivo. In contrast, none of the putative elongation factors tested affect the elongation rate, although mutations in the THO complex and in Spt4 significantly reduce processivity. The drugs 6-azauracil and mycophenolic acid reduce both the elongation rate and processivity, and this processivity defect is aggravated by mutations in Spt4, TFIIS, and CTDK-1. Our results suggest that, in vivo, a reduced rate of Pol II elongation leads to premature dissociation along the chromatin template and that Pol II processivity can be uncoupled from elongation rate.

Cyclin A2 is an RNA binding protein that controls Mre11 mRNA translation

Cyclin A2 activates the cyclin-dependent kinases Cdk1 and Cdk2 and is expressed at elevated levels from S phase until early mitosis. We found that mutant mice that cannot elevate cyclin A2 are chromosomally unstable and tumor-prone. Underlying the chromosomal instability is a failure to up-regulate the meiotic recombination 11 (Mre11) nuclease in S phase, which leads to impaired resolution of stalled replication forks, insufficient repair of double-stranded DNA breaks, and improper segregation of sister chromosomes. Unexpectedly, cyclin A2 controlled Mre11 abundance through a C-terminal RNA binding domain that selectively and directly binds Mre11 transcripts to mediate polysome loading and translation. These data reveal cyclin A2 as a mechanistically diverse regulator of DNA replication combining multifaceted kinase-dependent functions with a kinase-independent, RNA binding–dependent role that ensures adequate repair of common replication errors.

Efficient T cell activation requires an optimal dwell-time of interaction between the TCR and the pMHC complex

Cytotoxic T cell (CTL) activation by antigen requires the specific detection of peptide–major histo-compatibility class I (pMHC) molecules on the target-cell surface by the T cell receptor (TCR). We examined the effect of mutations in the antigen-binding site of a Kb-restricted TCR on T cell activation, antigen binding and dissociation from antigen. These parameters were also examined for variants derived from a Kd-restricted peptide that was recognized by a CTL clone. Using these two independent systems, we show that T cell activation can be impaired by mutations that either decrease or increase the binding half-life of the TCR-pMHC interaction. Our data indicate that efficient T cell activation occurs within an optimal dwell-time range of TCR-pMHC interaction. This restricted dwell-time range is consistent with the exclusion of either extremely low or high affinity T cells from the expanded population during immune responses.