Akselrod, G. M., Timp, W., Mirsaidov, U., Zhao, Q., Li, C., Timp, R., Timp, K., Matsudaira, P. and Timp, G. (2006) Laser-guided assembly of heterotypic three-dimensional living cell microarrays. Biophysical Journal. 91 3465-3473. We have assembled three-dimensional heterotypic networks of living cells in hydrogel without loss of viability using arrays of time-multiplexed, holographic optical traps. The hierarchical control of the cell positions is achieved with, to our knowledge, unprecedented submicron precision, resulting in arrays with an intercell separation < 400 nm. In particular, we have assembled networks of Swiss 3T3. broblasts surrounded by a ring of bacteria. We have also demonstrated the ability to manipulate hundreds of Pseudomonas aeruginosa simultaneously into two- and three-dimensional arrays with a time-averaged power < 2 mW per trap. This is the first time to our knowledge that living cell arrays of such complexity have been synthesized, and it represents a milestone in synthetic biology and tissue engineering.Full Text

ArtavanisTsakonas, K., Love, J. C., Ploegh, H. L. and Vyas, J. M. (2006) Recruitment of CD63 to Cryptococcus neoformans phagosomes requires acidification. Proc Natl Acad Sci U S A.Oct 24;103(43):15945-50 The subcellular localization of the cluster of differentiation 63 (CD63) tetraspanin and its interaction with the class II MHC antigen presentation pathway were examined in the context of phagocytosis by live cell imaging, by using monomeric red fluorescent protein-tagged mouse CD63 expressed in primary bone marrow-derived cell cultures. Upon phagocytosis of Cryptococcus neoformans and polystyrene beads, CD63 was recruited selectively to C. neoformans-containing phagosomes in a MyD88-independent acidification-dependent manner. Bead-containing phagosomes, within a C. neoformans-containing cell, acidified to a lesser extent and failed to recruit CD63 to a level detectable by microscopy. CD63 recruitment to yeast phagosomes occurred independently of class II MHC and LAMP-1. These observations indicate that the composition of distinct phagosomal compartments within the same cell is determined by phagosomal cargo and may affect the outcome of antigen processing and presentation.Full Text

ArtavanisTsakonas, K., Misaghi, S., Comeaux, C. A., Catic, A., Spooner, E., Duraisingh, M. T. and Ploegh, H. L. (2006) Identification by functional proteomics of a deubiquitinating/deNeddylating enzyme in Plasmodium falciparum. Mol Microbiol. 61 1187-95. Ubiquitination is a post-translational modification implicated in a variety of cellular functions, including transcriptional regulation, protein degradation and membrane protein trafficking. Ubiquitin and the enzymes that act on it, although conserved and essential in eukaryotes, have not been well studied in parasites, despite sequencing of several parasite genomes. Several putative ubiquitin hydrolases have been identified in Plasmodium falciparum based on sequence homology alone, with no evidence of expression or function. Here we identify the first deubiquitinating enzyme in P. falciparum, PfUCH54, by its activity. We show that PfUCH54 also has deNeddylating activity, as assayed by a mammalian Nedd8-based probe. This activity is absent from mammalian homologues of PfUCH54. Given the importance of parasitic membrane protein trafficking as well as protein degradation in the virulence of this parasite, this family of enzymes may represent a target for pharmacological intervention with this disease Full Text

Axtell, M. J., Jan, C., Rajagopalan, R. and Bartel, D. P. (2006) A Two-Hit Trigger for siRNA Biogenesis in Plants. Cell. 127 565-77. In Arabidopsis, microRNA-directed cleavage can define one end of RNAs that then generate phased siRNAs. However, most miRNA-targeted RNAs do not spawn siRNAs, suggesting the existence of additional determinants within those that do. We find that in moss, phased siRNAs arise from regions flanked by dual miR390 cleavage sites. AtTAS3, an siRNA locus important for development and conserved among higher plants, also has dual miR390 complementary sites. Both sites bind miR390 in vitro and are functionally required in Arabidopsis, but cleavage is undetectable at the 5' site-demonstrating that noncleavable sites can be functional in plants. Phased siRNAs also emanate from the bounded regions of every Arabidopsis gene with two known microRNA/siRNA complementary sites, but only rarely from genes with single sites. Therefore, two "hits,"-often, but not always, two cleavage events-constitute a conserved trigger for siRNA biogenesis, a finding with implications for recognition and silencing of aberrant RNA.Full Text

Bailey, S. N., Ali, S. M., Carpenter, A. E., Higgins, C. O. and Sabatini, D. M. (2006) Microarrays of lentiviruses for gene function screens in immortalized and primary cells. Nat Methods. 3 117-22. Here we describe lentivirus-infected cell microarrays for the high-throughput screening of gene function in mammalian cells. To create these arrays, we cultured mammalian cells on glass slides 'printed' with lentiviruses pseudotyped as vesicular stomatitis virus glycoprotein, which encode short hairpin RNA or cDNA. Cells that land on the printed 'features' become infected with lentivirus, creating a living array of stably transduced cell clusters within a monolayer of uninfected cells. The small size of the features of the microarrays (300 mum in diameter) allows high-density spotting of lentivirus, permitting thousands of distinct parallel infections on a single glass slide. Because lentiviruses have a wide cellular tropism, including primary cells, lentivirus-infected cell microarrays can be used as a platform for high-throughput screening in a variety of cell types. Full Text

Ballabio, A., Nelson, D. and Rozen, S. (2006) The sex chromosomes and human disease. Current Opinion in Genetics & Development. 16 209-212. Full Text

Bard, K. A., Todd, B. K., Bernier, C., Love, J. and Leavens, D. A. (2006) Self-awareness in human and chimpanzee infants: What is measured and what is meant by the mark and mirror test? Infancy. 9 191-219. The objective study of self-recognition, with a mirror and a mark applied to the face, was conducted independently by Gallup (1970) for use with chimpanzees and monkeys, and by Amsterdam (1972) for use with infant humans. Comparative psychologists have followed the model (and assumptions) set by Gallup, whereas developmental psychologists have followed a different model (e.g., Lewis & Brooks-Gunn, 1979). This article explores the assumptions in the definitions and methods of self-recognition assessments in the 30 years since these initial studies, and charts the divergence between the developmental mark test and the comparative mark test. Two new studies, 1 with infant chimpanzees and I with infant humans, illustrate a reconciliation of the 2 approaches. Overt application of the mark, or other procedures related to how the mark is discovered, did not enhance mirror self-recognition. In contrast, maternal scaffolding appears to enhance performance, perhaps by eliciting well-rehearsed verbal responses (i.e., naming self). When comparable testing procedures and assessment criteria are used, chimpanzee and human infants perform comparably. A combined developmental comparative approach allows us to suggest that mirror self-recognition may be based on a specific aspect of mental representation, the cognitive ability to symbolize.

Baltus, A. E., Menke, D. B., Hu, Y. C., Goodheart, M. L., Carpenter, A. E., de Rooij, D. G. and Page, D. C. (2006) In germ cells of mouse embryonic ovaries, the decision to enter meiosis precedes premeiotic DNA replication. Nature Genetics 38 (12) The transition from mitosis to meiosis is a defining juncture in the life cycle of sexually reproducing organisms. In yeast, the decision to enter meiosis is made before the single round of DNA replication that precedes the two meiotic divisions. We present genetic evidence of an analogous decision point in the germ line of a multicellular organism. The mouse Stra8 gene is expressed in germ cells of embryonic ovaries, where meiosis is initiated, but not in those of embryonic testes, where meiosis does not begin until after birth. Here we report that in female embryos lacking Stra8 gene function, the early, mitotic development of germ cells is normal, but these cells then fail to undergo premeiotic DNA replication, meiotic chromosome condensation, cohesion, synapsis and recombination. Combined with previous findings, these genetic data suggest that active differentiation of ovarian germ cells commences at a regulatory point upstream of premeiotic DNA replication.Full Text

Beard, C., Hochedlinger, K., Plath, K., Wutz, A. and Jaenisch, R. (2006) Efficient method to generate single-copy transgenic mice by site-specific integration in embryonic stem cells. Genesis. 44 23-28. Transgenic and gene-targeted mutant mice provide powerful tools for analysis of the cellular processes involved in early development and in the pathogenesis of many diseases. Here we describe a transgene integration strategy mediated by site-specific recombination that allows establishment of multiple embryonic stem (ES) cell lines carrying tetracycline-inducible genes targeted to a specific locus to assure predictable temporal and spatial expression in ES cells and mice. Using homologous recombination we inserted an frt homing site into which tetracycline-inducible transgenes can be integrated efficiently in the presence of FLPe recombinase. This strategy and the vectors described here are generally applicable to any locus in ES cells and should allow for the rapid production of mice with transgenes efficiently targeted to a defined site. Full Text.

Bell, G. W. and Lewitter, F. (2006) Visualizing networks. Methods Enzymol. 411 408-21. An interrelated set of genes or proteins can be represented effectively as a network that describes physical interactions, regulatory relationships, or metabolic pathways. Visualizing a network can be a helpful method to extract biological meaning and to generate testable hypotheses about large-scale biological data. This chapter describes some potential rationales for visualizing networks of microarray and other data types, which can be integrated and filtered to show potentially significant relationships. It also presents a practical introduction to Osprey and Cytoscape, two software platforms that are powerful tools for visualizing, integrating, and manipulating networks.Full Text

Bernstein, B. E., Mikkelsen, T. S., Xie, X., Kamal, M., Huebert, D. J., Cuff, J., Fry, B., Meissner, A., Wernig, M., Plath, K., Jaenisch, R., Wagschal, A., Feil, R., Schreiber, S. L. and Lander, E. S. (2006) A bivalent chromatin structure marks key developmental genes in embryonic stem cells. Cell. 125 315-26. The most highly conserved noncoding elements (HCNEs) in mammalian genomes cluster within regions enriched for genes encoding developmentally important transcription factors (TFs). This suggests that HCNE-rich regions may contain key regulatory controls involved in development. We explored this by examining histone methylation in mouse embryonic stem (ES) cells across 56 large HCNE-rich loci. We identified a specific modification pattern, termed "bivalent domains," consisting of large regions of H3 lysine 27 methylation harboring smaller regions of H3 lysine 4 methylation. Bivalent domains tend to coincide with TF genes expressed at low levels. We propose that bivalent domains silence developmental genes in ES cells while keeping them poised for activation. We also found striking correspondences between genome sequence and histone methylation in ES cells, which become notably weaker in differentiated cells. These results highlight the importance of DNA sequence in defining the initial epigenetic landscape and suggest a novel chromatin-based mechanism for maintaining pluripotency.Full Text

Blelloch, R., Wang, Z., Meissner, A., Pollard, S., Smith, A. and Jaenisch, R. (2006) Reprogramming Efficiency following Somatic Cell Nuclear Transfer is Influenced by the Differentiation and Methylation State of the Donor Nucleus. Stem Cells.May 18;. [Epub ahead of print] Reprogramming of a differentiated cell nucleus by somatic cell nuclear transplantation is an inefficient process. Following nuclear transfer, the donor nucleus often fails to express early embryonic genes and establish a normal embryonic pattern of chromatin modifications. These defects correlate with the low number of cloned embryos able to produce embryonic stem cells or develop into adult animals. Here, we show that the differentiation and methylation state of the donor cell influence the efficiency of genomic reprogramming. First, neural stem cells, when used as donors for nuclear transplantation, produce embryonic stem cells at a higher efficiency than blastocysts derived from terminally differentiated neuronal donor cells demonstrating a correlation between the state of differentiation and cloning efficiency. Second, using a hypomorphic allele of DNA methlytransferase-I, we found that global hypomethylation of a differentiated cell genome improved cloning efficiency. Our results provide functional evidence that the differentiation and epigenetic state of the donor nucleus influences reprogramming efficiency.PDF

Boyer, L. A., Mathur, D. and Jaenisch, R. (2006) Molecular control of pluripotency. Curr Opin Genet Dev Aug 18; [Epub ahead of print] . Transcriptional regulators and epigenetic modifiers play crucial roles throughout development to ensure that proper gene expression patterns are established and maintained in any given cell type. Recent genome-wide studies have begun to unravel how genetic and epigenetic factors maintain the undifferentiated state of embryonic stem cells while allowing these cells to remain poised to differentiate into somatic cells in response to developmental cues. These studies provide a conceptual framework for understanding pluripotency and lineage-specification at the molecular level. Full Text

Boyer, L. A., Plath, K., Zeitlinger, J., Brambrink, T., Medeiros, L. A., Lee, T. I., Levine, S. S., Wernig, M., Tajonar, A., Ray, M. K., Bell, G. W., Otte, A. P., Vidal, M., Gifford, D. K., Young, R. A. and Jaenisch, R. (2006) Polycomb complexes repress developmental regulators in murine embryonic stem cells. Nature Apr 19; [Epub ahead of print] . The mechanisms by which embryonic stem (ES) cells self-renew while maintaining the ability to differentiate into virtually all adult cell types are not well understood. Polycomb group (PcG) proteins are transcriptional repressors that help to maintain cellular identity during metazoan development by epigenetic modification of chromatin structure. PcG proteins have essential roles in early embryonic development and have been implicated in ES cell pluripotency, but few of their target genes are known in mammals. Here we show that PcG proteins directly repress a large cohort of developmental regulators in murine ES cells, the expression of which would otherwise promote differentiation. Using genome-wide location analysis in murine ES cells, we found that the Polycomb repressive complexes PRC1 and PRC2 co-occupied 512 genes, many of which encode transcription factors with important roles in development. All of the co-occupied genes contained modified nucleosomes (trimethylated Lys 27 on histone H3). Consistent with a causal role in gene silencing in ES cells, PcG target genes were de-repressed in cells deficient for the PRC2 component Eed, and were preferentially activated on induction of differentiation. Our results indicate that dynamic repression of developmental pathways by Polycomb complexes may be required for maintaining ES cell pluripotency and plasticity during embryonic development.Full Text

Brambrink, T., Hochedlinger, K., Bell, G. and Jaenisch, R. (2006) ES cells derived from cloned and fertilized blastocysts are transcriptionally and functionally indistinguishable. PNAS Jan 24;103(4):933-8.Reproductive cloning is uniformly rejected as a valid technology in humans because of the severely abnormal phenotypes seen in cloned animals. Gene expression aberrations observed in tissues of cloned animals have also raised concerns regarding the therapeutic application of "customized" embryonic stem (ES) cells derived by nuclear transplantation (NT) from a patient's somatic cells. Although previous experiments in mice have demonstrated that the developmental potential of ES cells derived from cloned blastocysts (NT-ES cells) is identical to that of ES cells derived from fertilized blastocysts, a systematic molecular characterization of NT-ES cell lines is lacking. To investigate whether transcriptional aberrations, similar to those observed in tissues of cloned mice, also occur in NT-ES cells, we have compared transcriptional profiles of 10 mouse NT- and fertilization-derived-ES cell lines. We report here that the ES cell lines derived from cloned and fertilized mouse blastocysts are indistinguishable based on their transcriptional profiles, consistent with their normal developmental potential. Our results indicate that, in contrast to embryonic and fetal development of clones, the process of NT-ES cell derivation rigorously selects for those immortal cells that have erased the "epigenetic memory" of the donor nucleus and, thus, become functionally equivalent. Our findings support the notion that ES cell lines derived from cloned or fertilized blastocysts have an identical therapeutic potential PDF

Brinkmann, M. M. and Schulz, T. F. (2006) Regulation of intracellular signalling by the terminal membrane proteins of members of the Gammaherpesvirinae. Journal of General Virology. 87 1047-1074. The human gamma(1)-herpesvirus Epstein-Barr virus (EBV) and the gamma(2)-herpesviruses Kaposi's sarcoma-associated herpesvirus (KSHV), rhesus rhadinovirus (RRV), herpesvirus saimiri (HVS) and herpesvirus ateles (HVA) all contain genes located adjacent to the terminal-repeat region of their genomes, encoding membrane proteins involved in signal transduction. Designated 'terminal membrane proteins' (TMPs) because of their localization in the viral genome, they interact with a variety of cellular signalling molecules, such as non-receptor protein tyrosine kinases, tumour-necrosis factor receptor-associated factors, Ras and Janus kinase (JAK), thereby initiating further downstream signalling cascades, such as the MAPK, PI3K/Akt, NF-kappa B and JAK/STAT pathways. In the case of TMPs expressed during latent persistence of EBV and HVS (LMP1, LMP2A, Stp and Tip), their modulation of intracellular signalling pathways has been linked to the provision of survival signals to latently infected cells and, hence, a contribution to occasional cellular transformation. In contrast, activation of similar pathways by TMPs of KSHV (K1 and K15) and RRV (R1), expressed during lytic replication, may extend the lifespan of virus-producing cells, alter their migration and/or modulate antiviral immune responses. Whether R1 and K1 contribute to the oncogenic properties of KSHV and RRV has not been established satisfactorily, despite their transforming qualities in experimental settings.Full Text

Brummelkamp, T., Fabius, A., Mullenders, J., Madiredjo, M., Velds, A., Kerkhoven, R., Bernards, R. and Beijersbergen, R. (2006) An shRNA barcode screen provides insight into cancer cell vulnerability to MDM2 inhibitors. Nat Chem Biol. 2006 Feb 13; [Epub ahead of print] . The identification of the cellular targets of small molecules with anticancer activity is crucial to their further development as drug candidates. Here, we present the application of a large-scale RNA interference-based short hairpin RNA (shRNA) barcode screen to gain insight in the mechanism of action of nutlin-3 (1). Nutlin-3 is a small-molecule inhibitor of MDM2, which can activate the p53 pathway. Nutlin-3 shows strong antitumor effects in mice, with surprisingly few side effects on normal tissues 1 . Aside from p53, we here identify 53BP1 as a critical mediator of nutlin-3-induced cytotoxicity. 53BP1 is part of a signaling network induced by DNA damage that is frequently activated in cancer but not in healthy tissues 2. Our results suggest that nutlin-3's tumor specificity may result from its ability to turn a cancer cell-specific property (activated DNA damage signaling 3) into a weakness that can be exploited therapeutically.Full Text

Burdick, M. M., Chu, J. T., Godar, S. and Sackstein, R. (2006) HCELL is the major E- and L-selectin ligand expressed on LS174T colon carcinoma cells. Journal of Biological Chemistry. 281 13899-13905. Engagement of vascular E-selectin and leukocyte L-selectin with relevant counter-receptors expressed on tumor cells contributes to the hematogenous spread of colon carcinoma. We recently demonstrated that the LS174T colon carcinoma cell line expresses the CD44 glycoform known as hematopoietic cell E-/L-selectin ligand (HCELL), which functions as a high affinity E- and L-selectin ligand on these cells. To define the contribution of HCELL to selectin-mediated adhesion on intact tumor cells, we measured the binding of LS174T cells transduced with CD44 short interfering RNA (siRNA) or with vector alone to 6-h interleukin-1 beta-stimulated human umbilical vein endothelial cells (HUVEC) and to human peripheral blood mononuclear cells (PBMC) under physiological flow conditions. LS174T cell attachment to HUVEC was entirely E-selectin-dependent, and PBMC tethering to tumor cell monolayers was completely L-selectin-dependent. At physiological shear stress, CD44 siRNA transduction led to an similar to 50% decrease in the number of LS174T cells binding to stimulated HUVEC relative to vector alone-transduced cells. CD44 siRNA-transduced cells also rolled significantly faster than vector-transduced cells on HUVEC, indicating prominent HCELL participation in stabilizing tumor cell-endothelial adhesive interactions against fluid shear. Furthermore, HCELL was identified as the principal L-selectin ligand on LS174T cells, as PBMC binding to CD44 siRNA-transduced tumor cells was reduced similar to 80% relative to vector-transduced cells. These data indicate that expression of HCELL confers robust and predominant tumor cell binding to E- and L-selectin, highlighting a central role for HCELL in promoting shear-resistant adhesive interactions essential for hematogenous cancer dissemination. Full Text

Camargo, F. D. and Goodell, M. A. (2006) The Hoechst low-fluorescent profile of the side population: clonogenicity versus dye retention - Response. Blood. 108 1774-1775. Full Text

Camargo, F. D., Chambers, S. M., Drew, E., McNagny, K. M. and Goodell, M. A. (2006) Hematopoietic stem cells do not engraft with absolute efficiencies. Blood. 107 501-7. Hematopoietic stem cells (HSCs) can be isolated from murine bone marrow by their ability to efflux the Hoechst 33342 dye. This method defines an extremely small and hematopoietically potent subset of cells known as the side population (SP). Recent studies suggest that transplanted single SP cells are capable of lymphohematopoietic repopulation at near absolute efficiencies. Here, we carefully reevaluate the hematopoietic potential of individual SP cells and find substantially lower rates of reconstitution. Our strategy involved the cotransplantation of single SP cells along with different populations of competitor cells that varied in their self-renewal capacity. Even with minimized HSC competition, SP cells were only able to reconstitute up to 35% of recipient mice. Furthermore, through immunophenotyping and clonal in vitro assays we find that SP cells are virtually homogeneous. Isolation of HSCs on the basis of Hoechst exclusion and a single cell-surface marker allows enrichment levels similar to that obtained with complex multicolor strategies. Altogether, our results indicate that even an extremely homogeneous HSC population, based on phenotype and dye efflux, cannot reconstitute mice at absolute efficiencies.Full Text

Cao, Y., Kumar, R. M., Penn, B. H., Berkes, C. A., Kooperberg, C., Boyer, L. A., Young, R. A. and Tapscott, S. J. (2006) Global and gene-specific analyses show distinct roles for Myod and Myog at a common set of promoters. Embo Journal. 25 502-511. We used a combination of genome-wide and promoter-specific DNA binding and expression analyses to assess the functional roles of Myod and Myog in regulating the program of skeletal muscle gene expression. Our findings indicate that Myod and Myog have distinct regulatory roles at a similar set of target genes. At genes expressed throughout the program of myogenic differentiation, Myod can bind and recruit histone acetyltransferases. At early targets, Myod is sufficient for near full expression, whereas, at late expressed genes, Myod initiates regional histone modification but is not sufficient for gene expression. At these late genes, Myog does not bind efficiently without Myod; however, transcriptional activation requires the combined activity of Myod and Myog. Therefore, the role of Myog in mediating terminal differentiation is, in part, to enhance expression of a subset of genes previously initiated by Myod. Full Text

Carpenter, A. E., Jones, T. R., Lamprecht, M. R., Clarke, C., Kang, I. H., Friman, O., Guertin, D. A., Chang, J. H., Lindquist, R. A., Moffat, J., Golland, P. and Sabatini, D. M. (2006) CellProfiler: image analysis software for identifying and quantifying cell phenotypes. Genome Biol. 7 R100. Biologists can now prepare and image thousands of samples per day using automation, enabling chemical screens and functional genomics (for example, using RNA interference). Here we describe the first free, open-source system designed for flexible, high-throughput cell image analysis, CellProfiler. CellProfiler can address a variety of biological questions quantitatively, including standard assays (for example, cell count, size, per-cell protein levels) and complex morphological assays (for example, cell/organelle shape or subcellular patterns of DNA or protein staining) Full Text

Chang, Q., Khare, G., Dani, V., Nelson, S. and Jaenisch, R. (2006) The disease progression of mecp2 mutant mice is affected by the level of BDNF expression. Neuron. 49 341-8. Mutations in the MECP2 gene cause Rett syndrome (RTT). Bdnf is a MeCP2 target gene; however, its role in RTT pathogenesis is unknown. We examined Bdnf conditional mutant mice for RTT-relevant pathologies and observed that loss of BDNF caused smaller brain size, smaller CA2 neurons, smaller glomerulus size, and a characteristic hindlimb-clasping phenotype. BDNF protein level was reduced in Mecp2 mutant mice, and deletion of Bdnf in Mecp2 mutants caused an earlier onset of RTT-like symptoms. To assess whether this interaction was functional and potentially therapeutically relevant, we increased BDNF expression in the Mecp2 mutant brain with a conditional Bdnf transgene. BDNF overexpression extended the lifespan, rescued a locomotor defect, and reversed an electrophysiological deficit observed in Mecp2 mutants. Our results provide in vivo evidence for a functional interaction between Mecp2 and Bdnf and demonstrate the physiological significance of altered BDNF expression/signaling in RTT disease progression.Full Text

Chappell, C., Beard, C., Altman, J., Jaenisch, R. and Jacob, J. (2006) DNA methylation by DNA methyltransferase 1 is critical for effector CD8 T cell expansion. Journal of Immunology. 176 4562-4572. Transcriptional silencing mediated by DNA methylation is a critical component of epigenetic regulation during early embryonic development in animals. However, the requirement for DNA methylation during activation and differentiation of mature CD8(+) T cells into effector and memory cells is not clear. Using cre-mediated deletion of DNA methyltransferase 1 (Dnmt1)at the time of CD8(+) T cell activation, we investigated the obligation for maintaining patterns of DNA methylation during the generation of Ag-specific effector and memory CD8(+) T cells in response to acute viral infection of mice with lymphocytic choriomeningitis virus. Dnmt1(-/-) CD8(+) T cells failed to undergo the massive CD8(+) T cell expansion characteristic of lymphocytic choriomeningitis virus infection, leading to > 80% reductions in Ag-specific effector CD8(+) T cells at the height of the response. Despite this, Dnmt1(-/-)CD8(+) T cells efficiently controlled the viral infection. Interestingly, the number of Ag-specific Dnmt1(-/-) memory CD8(+) T cells was moderately reduced compared with the reductions seen at day 8 postinfection. Our data suggest that ablation of Dnmt1 and subsequent DNA methylation affect the finite proliferative potential of Ag-specific CD8(+) T cells with moderate effects on their differentiation to effector and memory CD8(+) T cells. Full Text

Chen, D., Steele, A., Lindquist, S. and Guarente, L. (2006) Smaller, hungrier mice - Response. Science. 311 1553-1554. Full Text

Chen, H. and Fink, G. R. (2006) Feedback control of morphogenesis in fungi by aromatic alcohols. Genes Dev Apr 17; [Epub ahead of print] Many fungi undergo a developmental transition from a unicellular yeast form to an invasive filamentous form in response to environmental cues. Here we describe a quorum signaling pathway that links environmental sensing to morphogenesis in Saccharomyces cerevisiae. Saccharomyces cells secrete aromatic alcohols that stimulate morphogenesis by inducing the expression of FLO11 through a Tpk2p-dependent mechanism. Mutants defective in synthesis of these alcohols show reduced filamentous growth, which is partially suppressed by the addition of these aromatic alcohols. The production of these autosignaling alcohols is regulated by nitrogen: High ammonia restricts it by repressing the expression of their biosynthetic pathway, whereas nitrogen-poor conditions activate it. Moreover, the production of these aromatic alcohols is controlled by cell density and subjected to positive feedback regulation, which requires the transcription factor Aro80p. These interactions define a quorum-sensing circuit that allows Saccharomyces to respond to both cell density and the nutritional state of the environment. These same autoregulatory molecules do not evoke the morphological switch in Candida albicans, suggesting that these molecular signals are species-specific.PDF

Chu, F, Nusinow, D. A., Chalkely, R. J., Plath, K., Panning, B. and Burlingame, A. L. (2006) Mapping post-translational modifications of the histone variant macroH2A1 using tandem mass spectrometry. Molecular & Cellular Proteomics. 5 194-203. Post-translational histone modifications modulate chromatin-templated processes and therefore affect cellular proliferation, growth, and development. Although posttranslational modifications on the core histones have been under intense investigation for several years, the modifications on variant histones are poorly understood. We used tandem mass spectrometry to identify covalent modifications on a histone H2A variant, macroH2A1.2. MacroH2A1.2 can be monoubiquitinated; however, the site of monoubiquitination has not been documented. In this study we used green fluorescent protein-tagged macroH2A1.2 to determine that Lys(115) is a site of ubiquitination. In addition, we found that this variant H2A is methylated on the epsilon amino group of lysine residues Lys(17), Lys(122), and Lys(238) and phosphorylated on Thr(128). Three of these modifications were also found to be present in the endogenous protein by mass spectrometric analysis. These results provide the first direct evidence that multiple post-translational modifications are imposed on macroH2A1.2, suggesting that, like canonical H2A, this variant H2A is subject to regulation by combinatorial use of covalent modifications.Full Text

Clarke, A. and Orr-Weaver, T. L. (2006) Sister Chromatid Cohesion at the Centromere: Confrontation between Kinases and Phosphatases? Dev Cell. 10 544-7. Accurate chromosome segregation in mitosis and meiosis requires that the cohesin complex be protected at the centromere by the Shugoshin/MEI-S332 protein family. Recent studies show that Sgo directly binds the phosphatase PP2A, tethering it to the centromere where it can protect cohesin subunits from phosphorylation, and that localization of Sgo/MEI-S332 itself is regulated by phosphorylation. Full Text

Cooper, A. A., Gitler, A. D., Cashikar, A., Haynes, C. M., Hill, K. J., Bhullar, B., Liu, K., Xu, K., Strathearn, K. E., Liu, F., Cao, S., Caldwell, K. A., Caldwell, G. A., Marsischky, G., Kolodner, R. D., Labaer, J., Rochet, J. C., Bonini, N. M. and Lindquist, S. (2006) {alpha}-Synuclein Blocks ER-Golgi Traffic and Rab1 Rescues Neuron Loss in Parkinson's Models. Science.Published online 22 June.Alpha-synuclein (alphaSyn) misfolding is associated with several devastating neurodegenerative disorders including Parkinson's Disease (PD). In yeast cells and in neurons alphaSyn accumulation is cytotoxic, but little is known about its normal function or pathobiology. The earliest defect following alphaSyn expression in yeast was a block in endoplasmic reticulum (ER) to Golgi vesicular trafficking. In a genome-wide screen, the largest class of toxicity modifiers were proteins functioning at this same step, including the Rab GTPase Ypt1p, which associated with cytoplasmic alphaSyn inclusions. Elevated expression of Rab1, the mammalian YPT1 homolog, protected against alphaSyn-induced dopaminergic neuron loss in animal models of PD. Thus synucleinopathies may result from disruptions in basic cellular functions that interface with the unique biology of particular neurons to make them especially vulnerable.PDF

Cowen, L. E., Carpenter, A. E., Matangkasombut, O., Fink, G. R. and Lindquist, S. (2006) Genetic Architecture of Hsp90-Dependent Drug Resistance. Eukaryot Cell. Oct 20; [Epub ahead of print] Hsp90 potentiates the evolution of azole resistance in the model yeast Saccharomyces cerevisiae and the opportunistic pathogen Candida albicans via calcineurin. Here, we explored effectors downstream of calcineurin regulating this Hsp90-dependent trait. Using S. cerevisiae erg3 mutants as a model, we determined that both Crz1 and Hph1 modulate azole resistance.PDF

Cullinan, S. B. and Whitesell, L. (2006) Heat shock protein 90: a unique chemotherapeutic target. Semin Oncol. 33 457-65. A large body of work spanning the past decade has identified the molecular chaperone heat shock protein 90 (Hsp90) as a critical modulator of an extensive network of cellular signaling pathways. Many of the processes overseen by Hsp90 are deregulated in tumor cells, including cell cycle control, gene transcription, and apoptotic signaling. Hsp90 inhibition offers the potential of accomplishing what most molecularly targeted anticancer therapies do not-the simultaneous disruption of multiple signaling events critical to tumor cell growth and survival. Indeed, small molecule inhibitors of Hsp90 function are actively being evaluated in the clinic as anticancer agents. In this review, we highlight the current understanding of Hsp90 biology as it relates to cancer and discuss the discovery, development, and clinical status of Hsp90 inhibitors as anticancer drugs.

DiBacco, A., Ouyang, J., Lee, H. Y., Catic, A., Ploegh, H. and Gill, G. (2006) The SUMO-specific protease SENP5 is required for cell division. Molecular and Cellular Biology. 26 4489-4498. Posttranslational modification of substrates by the small ubiquitin-like modifier, SUMO, regulates diverse biological processes, including transcription, DNA repair, nucleocytoplasmic trafficking, and chromosome segregation. SUMOylation is reversible, and several mammalian homollogs of the yeast SUMO-specific protease Ulp1, termed SENPs, have been identified. We demonstrate here that SENP5, a previously uncharacterized Ulp1 homolog, has SUMO C-terminal hydrollase and SUMO isopeptidase activities. In contrast to other SENPs, the C-terminall catalytic domain of SENP5 preferentially processed SUMO-3 compared to SUMO-1 precursors and preferentially removed SUMO-2 and SUMO-3 from SUNIO-modified RanGAP1 in vitro. In cotransfection assays, SENP5 preferentially reduced high-molecular-weight conjugates of SUNIO-2 compared to SUMO-1 in vivo. Full-length SENP5 localized to the nucleolus. Deletion of the noncatalytic N-terminal domain led to loss of nucleolar localization and increased de-SUMOylation activity in vivo. Knockdown of SENP5 by RNA interference resulted in increased levels of SUMO-1 and SUMO-2/3 conjugates, inhibition of cell proliferation, defects in nuclear morphology, and appearance of binucleate cells, revealing an essential role for SENP5 in mitosis and/or cytokinesis. These findings establish SENP5 as a SUMO-specific protease required for cell division and suggest that mechanisms involving both the catalytic and noncatalytic domains determine the distinct substrate specificities of the mammalian SUMO-specific proteases. Full Text

Dickinson, A. J. and Sive, H. (2006) Development of the primary mouth in Xenopus laevis. Dev Biol.Apr 6; [Epub ahead of print] The initial opening between the gut and the outside of the deuterostome embryo breaks through at the extreme anterior. This region is unique in that ectoderm and endoderm are directly juxtaposed, without intervening mesoderm. This opening has been called the stomodeum, buccopharyngeal membrane or oral cavity at various stages of its formation, however, in order to clarify its function, we have termed this the "primary mouth". In vertebrates, the neural crest grows around the primary mouth to form the face and a "secondary mouth" forms. The primary mouth then becomes the pharyngeal opening. In order to establish a molecular understanding of primary mouth formation, we have begun to examine this process during Xenopus laevis development. An early step during this process occurs at tailbud and involves dissolution of the basement membrane between the ectoderm and endoderm. This is followed by ectodermal invagination to create the stomodeum. A subsequent step involves localized cell death in the ectoderm, which may lead to ectodermal thinning. Subsequently, ectoderm and endoderm apparently intercalate to generate one to two cell layers. The final step is perforation, where (after hatching) the primary mouth opens. Fate mapping has defined the ectodermal and endodermal regions that will form the primary mouth. Extirpations and transplants of these and adjacent regions indicate that, at tailbud, the oral ectoderm is not specifically required for primary mouth formation. In contrast, underlying endoderm and surrounding regions are crucial, presumably sources of necessary signals. This study indicates the complexity of primary mouth formation, and lays the groundwork for future molecular analyses of this important structure. Full Text

Doroquez, D. B. and Rebay, I. (2006) Signal Integration During Development: Mechanisms of EGFR and Notch Pathway Function and Cross-Talk. Crit Rev Biochem Mol Biol. 41 339-385. Metazoan development relies on a highly regulated network of interactions between conserved signal transduction pathways to coordinate all aspects of cell fate specification, differentiation, and growth. In this review, we discuss the intricate interplay between the epidermal growth factor receptor (EGFR; Drosophila EGFR/DER) and the Notch signaling pathways as a paradigm for signal integration during development. First, we describe the current state of understanding of the molecular architecture of the EGFR and Notch signaling pathways that has resulted from synergistic studies in vertebrate, invertebrate, and cultured cell model systems. Then, focusing specifically on the Drosophila eye, we discuss how cooperative, sequential, and antagonistic relationships between these pathways mediate the spatially and temporally regulated processes that generate this sensory organ. The common themes underlying the coordination of the EGFR and Notch pathways appear to be broadly conserved and should, therefore, be directly applicable to elucidating mechanisms of information integration and signaling specificity in vertebrate systems.Full Text

Duennwald, M. L., Jagadish, S., Muchowski, P. J. and Lindquist, S. (2006) Flanking sequences profoundly alter polyglutamine toxicity in yeast. Proc Natl Acad Sci U S A. 103 11045-50. Protein misfolding is the molecular basis for several human diseases. How the primary amino acid sequence triggers misfolding and determines the benign or toxic character of the misfolded protein remains largely obscure. Among proteins that misfold, polyglutamine (polyQ) expansion proteins provide an interesting case: Each causes a distinct neurodegenerative disease that selectively affects different neurons. However, all are broadly expressed and most become toxic when the glutamine expansion exceeds approximately 39 glutamine residues. The disease-causing polyQ expansion proteins differ profoundly in the amino acids flanking the polyQ region. We therefore hypothesized that these flanking sequences influence the specific toxic character of each polyQ expansion protein. Using a yeast model, we find that sequences flanking the polyQ region of human huntingtin exon I can convert a benign protein to a toxic species and vice versa. Further, we observe that flanking sequences can direct polyQ misfolding to at least two morphologically distinct types of polyQ aggregates. Very tight aggregates always are benign, whereas amorphous aggregates can be toxic. We thereby establish a previously undescribed systematic characterization of the influence of flanking amino acid sequences on polyQ toxicity.Full Text

Duennwald, M. L., Jagadish, S., Giorgini, F., Muchowski, P. J. and Lindquist, S. (2006) A network of protein interactions determines polyglutamine toxicity. Proc Natl Acad Sci Jul 18;103(29):. Several neurodegenerative diseases are associated with the toxicity of misfolded proteins. This toxicity must arise from a combination of the amino acid sequences of the misfolded proteins and their interactions with other factors in their environment. A particularly compelling example of how profoundly these intramolecular and intermolecular factors can modulate the toxicity of a misfolded protein is provided by the polyglutamine (polyQ) diseases. All of these disorders are caused by glutamine expansions in proteins that are broadly expressed, yet the nature of the proteins that harbor the glutamine expansions and the particular pathologies they produce are very different. We find, using a yeast model, that amino acid sequences that modulate polyQ toxicity in cis can also do so in trans. Furthermore, the prion conformation of the yeast protein Rnq1 and the level of expression of a suite of other glutamine-rich proteins profoundly affect polyQ toxicity. They can convert polyQ expansion proteins from toxic to benign and vice versa. Our work presents a paradigm for how a complex, dynamic interplay between intramolecular features of polyQ proteins and intermolecular factors in the cellular environment might determine the unique pathobiologies of polyQ expansion proteins.Full Text

Ehrnhoefer, D. E., Duennwald, M., Markovic, P., Wacker, J. L., Engemann, S., Roark, M., Legleiter, J., Marsh, J. L., Thompson, L. M., Lindquist, S., Muchowski, P. J. and Wanker, E. E. (2006) Green tea (-)-epigallocatechin-gallate modulates early events in huntingtin misfolding and reduces toxicity in Huntington's disease models. Human Molecular Genetics. 15 2743-2751. Huntington's disease (HD) is a progressive neurodegenerative disorder for which only symptomatic treatments of limited effectiveness are available. Preventing early misfolding steps and thereby aggregation of the polyglutamine (polyQ)-containing protein huntingtin (htt) in neurons of patients may represent an attractive therapeutic strategy to postpone the onset and progression of HD. Here, we demonstrate that the green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) potently inhibits the aggregation of mutant htt exon 1 protein in a dose-dependent manner. Dot-blot assays and atomic force microscopy studies revealed that EGCG modulates misfolding and oligomerization of mutant htt exon 1 protein in vitro, indicating that it interferes with very early events in the aggregation process. Also, EGCG significantly reduced polyQ-mediated htt protein aggregation and cytotoxicity in an yeast model of HD. When EGCG was fed to transgenic HD flies overexpressing a pathogenic htt exon 1 protein, photoreceptor degeneration and motor function improved. These results indicate that modulators of htt exon 1 misfolding and oligomerization like EGCG are likely to reduce polyQ-mediated toxicity in vivo. Our studies may provide the basis for the development of a novel pharmacotherapy for HD and related polyQ disorders.Full Text

ElDifrawy, S. A., Srivastava, A., Gismondi, E. A., McKenna, B. K. and Ehrlich, D. J. (2006) Numerical model for DNA loading in microdevices: Stacking and autogating effects. Electrophoresis.Sep 7; [Epub ahead of print] Many electrophoresis-based DNA sequencing and genotyping microdevices rely on field-driven effects to load and preconcentrate the sample. A quantitative model is developed for a broad class of electrophoresis-based microfabricated sample injectors. Quantitative predictions of DNA preconcentration are compared with experimental data and are shown to qualitatively reproduce the detailed time-evolving sample distribution in the injector. The model provides practical guidance on device and protocol design, in order to optimize this critical aspect of microfluidic devices. PDF

Elliott, S., Busse, L., Bass, M. B., Lu, H., Sarosi, I., Sinclair, A. M., Spahr, C., Um, M., Van, G. and Begley, C. G. (2006) Anti-Epo receptor antibodies do not predict Epo receptor expression. Blood. 107 1892-1895. Investigators using anti-EpoR antibodies for immunoblotting and immunostaining have reported erythropoietin receptor (EpoR) expression in nonhematopoietic tissues including human tumors. However, these antibodies detected proteins of 66 to 78 kDa, significantly larger than the predicted molecular weight of EpoR (56-57 kDa). We investigated the specificity of these antibodies and showed that they all detected non-EpoR proteins. C-20 detected 3 proteins in tumor cell lines (35, 66, and 100 kDa). Sequences obtained from preparative gels had similarity to the C-20-immunizing peptide. The 66-kDa protein was a heat shock protein (HSP70) to which antibody binding was abrogated in peptide competition experiments. Antibody M-20 readily identified a 59-kDa EpoR protein. However, neither M-20 nor C-20 was suitable for detection of EpoR using immunohistochemical methods. We concluded that these antibodies have limited utility for detecting EpoR. Thus, reports of EpoR expression in tumor cells using these antibodies should be viewed with caution. Full Text

Evans, J. G. and Matsudaira, P. (2006) Structure and dynamics of macrophage podosomes. Eur J Cell Biol. 85 145-9. Podosomes are short-lived, dynamic actin-rich adhesions classically formed in macrophages, osteoclasts and Src-transformed fibroblasts. Though related to the more commonly studied focal adhesions, sharing several structural and regulatory proteins, podosomes have distinct functional, structural, and dynamic characteristics. Here, we discuss current understanding of the function of podosomes in disparate cell types and how this relates to their structure and dynamics.Full Text

Frias, M. A., Thoreen, C. C., Jaffe, J. D., Schroder, W., Sculley, T., Carr, S. A. and Sabatini, D. M. (2006) mSin1 Is Necessary for Akt/PKB Phosphorylation, and Its Isoforms Define Three Distinct mTORC2s. Curr Biol. Aug 15; [Epub ahead of print] The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that participates in at least two distinct multiprotein complexes, mTORC1 and mTORC2 . These complexes play important roles in the regulation of cell growth, proliferation, survival, and metabolism. mTORC2 is a hydrophobic motif kinase for the cell-survival protein Akt/PKB and, here, we identify mSin1 as a component of mTORC2 but not mTORC1. mSin1 is necessary for the assembly of mTORC2 and for its capacity to phosphorylate Akt/PKB. Alternative splicing generates at least five isoforms of the mSin1 protein , three of which assemble into mTORC2 to generate three distinct mTORC2s. Even though all mTORC2s can phosphorylate Akt/PKB in vitro, insulin regulates the activity of only two of them. Thus, we propose that cells contain several mTORC2 flavors that may phosphorylate Akt/PKB in response to different signals.Full Text

Ge, H., Player, C. M. and Zou, L. (2006) Toward a global picture of development: Lessons from genome-scale analysis in Caenorhabditis elegans embryonic development. Developmental Dynamics Early View June 15. Development is the result of complex events, including cascades of transcriptional programs and numerous molecular interactions. Traditionally, research focus has been given to the characterization of individual mutants, regulators, or interactions. With the availability of complete genome sequences and high-throughput (HT) experimental techniques, probing development on a system level has become feasible. Pioneering work initiated in invertebrate model systems such as Caenorhabditis elegans has provided first drafts of catalogs of essential components, transcriptional regulatory diagrams and molecular interaction networks underlying developmental processes. Integrating these drafts approximates a system-level picture of development and provides local models for protein/gene functions. Here we summarize the progress toward elucidating developmental processes on a system level, including the applications of genomic technologies and computational analyses. We discuss C. elegans embryonic development in case studies to illustrate how various HT approaches can be integrated and how biological insights can be gained from these approaches.Full Text

Gemelli, T., Berton, O., Nelson, E. D., Perrotti, L. I., Jaenisch, R. and Monteggia, L. M. (2006) Postnatal loss of methyl-CpG binding protein 2 in the Forebrain is sufficient to mediate behavioral aspects of Rett syndrome in mice. Biological Psychiatry. 59 468-476. Mutations in the methyl-CpG binding protein 2 (MeCP2) gene cause Rett syndrome (RTT), a neurodevelopmental disorder that is accompanied by a broad array of behavioral phenotypes, mainly affecting females. Methyl-CpG binding protein 2 is a transcriptional repressor that is widely expressed in all tissues. To investigate whether the postnatal loss of MeCP2 in the forebrain is sufficient to produce the behavioral phenotypes observed in RTT, we have generated conditional MeCP2 knockout mice. These mice display behavioral abnormailities similar to RTT phenotypes, including hindlimb clasping, impaired motor coordination, increased anxiety, and abnormal social behavior with other mice. These mice, however, have normal locomotor activity and unimpaired context-dependent fear conditioning, suggesting that the behavioral deficits observed are the result of loss of MeCP2 function in postnatal forebrain and not the result of generalized global deficits. These data highlight the important role of MeCP2 in the forebrain and suggest that even partial loss of MeCP2 expression in these brain regions is sufficient to recapitulate features of RTT. Full Text

Ghaffari, S., Kitidis, C., Zhao, W., Marinkovic, D., Fleming, M. D., Luo, B., Marszalek, J. and Lodish, H. F. (2006) AKT induces erythroid-cell maturation of JAK2-deficient fetal liver progenitor cells and is required for Epo regulation of erythroid-cell differentiation. Blood. 107 1888-1891. AKT serine threonine kinase of the protein kinase B (PKB) family plays essential roles in cell survival, growth, metabolism, and differentiation. In the erythroid system, AKT is known to be rapidly phosphorylated and activated in response to erythropoletin (Epo) engagement of Epo receptor (EpoR) and to sustain survival signals in cultured erythroid cells. Here we demonstrate that activated AKT complements EpoR signaling and supports erythroid-cell differentiation in wild-type and JAK2-deficient fetal liver cells. We show that erythroid maturation of AKT-transduced cells is not solely dependent on AKT-induced cell survival or proliferation signals, suggesting that AKT transduces also a differentiation-specific signal downstream of EpoR in erythroid cells. Downregulation of expression of AKT kinase by RNA interference, or AKT activity by expression of dominant negative forms, inhibits significantly fetal liver-derived erythroid-cell colony formation and gene expression, demonstrating that AKT is required for Epo regulation of erythroid-cell maturation. Full Text

Goedecke, N., McKenna, B., El-Difrawy, S., Gismondi, E., Swenson, A., Carey, L., Matsudaira, P. and Ehrlich, D. J. (2006) Microdevice DNA forensics by the simple tandem repeat method. J Chromatogr A. 1111 206-13. We review recent experiments on DNA forensics by the simple tandem repeat (STR) method using a 16-lane micromachined device as the active separation element. Separations by linear polyacrylamide matrices show very high data quality metrics when evaluated with statistically significant data sets. Full 16-locus multiplexes are verified on the multilane system. Multi-donor mixed samples are studied in the context of the limits of the laser-induced fluorescence detector and data-reduction software. The microdevice appears to be posed to outperform current capillary arrays in terms of stability and, through specialized sample loading, in the interpretation of complex mixtures.Full Text

Gross, A. W. and Lodish, H. F. (2006) Cellular trafficking and degradation of erythropoietin and novel erythropoiesis stimulating protein (NESP). Journal of Biological Chemistry. 281 2024-2032. Erythropoietin (Epo) is essential for the production of mature red blood cells, and recombinant Epo is commonly used to treat anemia, but how Epo is degraded and cleared from the body is not understood. Glycosylation of Epo is required for its in vivo bioactivity, although not for invitro receptor binding or stimulation of Epo-dependent celllines; Epoglycosylation actually reduces the affinity of Epo for the Epo receptor (EpoR). Interestingly, a hyperglycosylated analog of Epo, called novel erythropoiesis-stimulating protein (NESP), has a lower affinity than Epo for the EpoR but has greater in vivo activity and a longer serum half-life than Epo. We hypothesize that a major mechanism for degradation of Epo in the body occurs in cells expressing the Eporeceptor, through receptor-mediated endocytosis of Epo followed by degradation in lysosomes, and therefore investigated the trafficking and degradation of Epo and NESP by EpoR-expressing cells. We show that Epo and NESP are degraded only by cultured cells that express the EpoR, and their receptor binding, dissociation, and trafficking properties determine their rates of intracellular degradation. Epo binds surface EpoR faster than NESP (k(on) approximate to 5.0 x 10(8) M-1 min(-1) versus 1.1 x 10(8) M-1 min(-1)) but dissociates slower (koff = 0.029 min(-1) versus 0.042 min(-1)). Surface-bound Epo and NESP are internalized at the same rate (k(in) = 0.06 min(-1)), and after internalization 60% of each ligand is resecreted intact and 40% degraded. Our kinetic model of Epo and NESP receptor binding, intracellular trafficking, and degradation explains why Epo is degraded faster than NESP at the cellular level. Full Text.

Guertin, D. A., Stevens, D. M., Thoreen, C. C., Burds, A. A., Kalaany, N. Y., Moffat, J., Brown, M., Fitzgerald, K. J. and Sabatini, D. M. (2006) Ablation in Mice of the mTORC Components raptor, rictor, or mLST8 Reveals that mTORC2 Is Required for Signaling to Akt-FOXO and PKCalpha, but Not S6K1. Dev Cell. 11 859-71. The mTOR kinase controls cell growth, proliferation, and survival through two distinct multiprotein complexes, mTORC1 and mTORC2. mTOR and mLST8 are in both complexes, while raptor and rictor are part of only mTORC1 and mTORC2, respectively. To investigate mTORC1 and mTORC2 function in vivo, we generated mice deficient for raptor, rictor, or mLST8. Like mice null for mTOR, those lacking raptor die early in development. However, mLST8 null embryos survive until e10.5 and resemble embryos missing rictor. mLST8 is necessary to maintain the rictor-mTOR, but not the raptor-mTOR, interaction, and both mLST8 and rictor are required for the hydrophobic motif phosphorylation of Akt/PKB and PKCalpha, but not S6K1. Furthermore, insulin signaling to FOXO3, but not to TSC2 or GSK3beta, requires mLST8 and rictor. Thus, mTORC1 function is essential in early development, mLST8 is required only for mTORC2 signaling, and mTORC2 is a necessary component of the Akt-FOXO and PKCalpha pathways.Full Text

Guertin, D. A., Guntur, K. V., Bell, G. W., Thoreen, C. C. and Sabatini, D. M. (2006) Functional Genomics Identifies TOR-Regulated Genes that Control Growth and Division. Curr Biol. 16 958-70. BACKGROUND: The TOR (target of rapamycin) ser/thr protein kinase is the central component of a eukaryotic signaling pathway that regulates growth and is the direct target of the clinically useful drug rapamycin. Recent efforts have identified at least two multiprotein complexes that contain TOR, but little is known in higher eukaryotes about the genes downstream of TOR that control growth. RESULTS: By combining the use of a small molecule inhibitor (rapamycin), transcriptional profiling, and RNA interference in Drosophila tissue culture cells, we identified genes whose expression responds to Drosophila TOR (dTOR) inhibition and that regulate cell size. Several of the dTOR-regulated genes that function in cell size control have additional roles in cell division. Most of these genes are conserved in mammals and several are linked to human disease. This set of genes is highly enriched for regulators of ribosome biogenesis, which emphasizes the importance of TOR-dependent transcription in building the protein synthesis machinery in higher eukaryotes. In addition, we identify two dTOR-regulated genes, CG3071 and CG6677, whose human orthologs, SAW and ASH2L, are also under TOR-dependent transcriptional control and encode proteins with conserved functional roles in growth. CONCLUSIONS: We conclude that combining RNA interference with genomic analysis approaches, such as transcriptional profiling, is an effective way to identify genes functioning in a particular biological process. Moreover, this strategy, if applied in model systems with simpler genomes, can identify genes with conserved functions in mammals. Full Text

Handwerger, K. E. and Gall, J. G. (2006) Subnuclear organelles: new insights into form and function. Trends in Cell Biology. 16 19-26. The cell nucleus is a complex and highly dynamic environment with many functionally specialized regions of substructure that form and maintain themselves in the absence of membranes. Relatively little is known about the basic physical properties of the nuclear interior or how domains within the nucleus are structurally and functionally organized and interrelated. Here, we summarize recent data that shed light on the structural and functional properties of three prominent subnuclear organelles - nucleoli, Cajal bodies (CBs) and speckles. We discuss how these findings impact our understanding of the guiding principles of nuclear organization and various types of human disease. Full Text

Hang, H. C., Loureiro, J., Spooner, E., van der Velden, A. W., Kim, Y. M., Pollington, A. M., Maehr, R., Starnbach, M. N. and Ploegh, H. L. (2006) Mechanism-based probe for the analysis of cathepsin cysteine proteases in living cells. ACS Chem Biol. 1 713-23. Mechanism-based probes are providing new tools to evaluate the enzymatic activities of protein families in complex mixtures and to assign protein function. The application of these chemical probes for the visualization of protein labeling in cells and proteomic analysis is still challenging. As a consequence, imaging and proteomic analysis often require different sets of chemical probes. Here we describe a mechanism-based probe, azido-E-64, that can be used for both imaging and proteomics. Azido-E-64 covalently modifies active Cathepsin (Cat) B in living cells, an abundant cysteine protease involved in microbial infections, apoptosis, and cancer. Furthermore, azido-E-64 contains an azide chemical handle that can be selectively derivatized with phosphine reagents via the Staudinger ligation, which enables the imaging and proteomic analysis of Cat B. We have utilized azido-E-64 to visualize active Cat B during infection of primary macrophages with Salmonella typhimurium , an facultative intracellular bacterial pathogen. These studies demonstrated that active Cat B is specifically excluded from Salmonella -containing vacuoles, which suggests that inhibition of protease activity within bacteria-containing vacuoles may contribute to bacterial virulence.Full Text

Hartwell, K. A., Muir, B., Reinhardt, F., Carpenter, A. E., Sgroi, D. C. and Weinberg, R. A. (2006) The Spemann organizer gene, Goosecoid, promotes tumor metastasis. Proc Natl Acad Sci U S A. Dec 1; [Epub ahead of print] The process of invasion and metastasis during tumor progression is often reminiscent of cell migration events occurring during embryonic development. We hypothesized that genes controlling cellular changes in the Spemann organizer at gastrulation might be reactivated in tumors. The Goosecoid homeobox transcription factor is a known executer of cell migration from the Spemann organizer. We found that indeed Goosecoid is overexpressed in a majority of human breast tumors. Ectopic expression of Goosecoid in human breast cells generated invasion-associated cellular changes, including an epithelial-mesenchymal transition. TGF-beta signaling, known to promote metastasis, induced Goosecoid expression in human breast cells. Moreover, Goosecoid significantly enhanced the ability of breast cancer cells to form pulmonary metastases in mice. These results demonstrate that Goosecoid promotes tumor cell malignancy and suggest that other conserved organizer genes may function similarly in human cancer.PDF

Hill, A. E., Lander, E. S. and Nadeau, J. H. (2006) Chromosome substitution strains: a new way to study genetically complex traits. Methods Mol Med. 128 153-72. Many biological traits and heritable diseases are multifactorial, involving combinations of genetic variants and environmental factors. To dissect the genetic basis for these traits and to characterize their functional consequences, mouse models are widely used, not only because of their genetic and physiological similarity to humans, but also because an extraordinary variety of genetic resources enable rigorous functional studies. Chromosome substitution strains (CSSs) are a powerful complement to existing resources for studying multigenic traits. By partitioning the genome into a panel of new inbred strains with single chromosome substitutions, one strain for each of the autosomes, the X and Y chromosome, and the mitochondria, unique experimental designs and considerable statistical power are possible. Multigenic trait genes (or quantitative trait loci [QTLs]) with weak effects are easily detected, linkage and congenic crosses can be quickly made, gene interactions are readily characterized, and discovery of QTLs is greatly accelerated. Several published studies demonstrate the considerable utility of these strains and new applications for CSSs continue to be discovered.

Hochedlinger, K. and Jaenisch, R. (2006) Nuclear reprogramming and pluripotency. Nature. 441 1061-1067. The cloning of mammals from differentiated donor cells has refuted the old dogma that development is an irreversible process. It has demonstrated that the oocyte can reprogramme an adult nucleus into an embryonic state that can direct development of a new organism. The prospect of deriving patient-specific embryonic stem cells by nuclear transfer underscores the potential use of this technology in regenerative medicine. The future challenge will be to study alternatives to nuclear transfer in order to recapitulate reprogramming in a Petri dish without the use of oocytes.Full Text

Hongay, C. F., Grisafi, P. L., Galitski, T. and Fink, G. R. (2006) Antisense Transcription Controls Cell Fate in Saccharomyces cerevisiae. Cell. 127 735-745. Entry into meiosis is a key developmental decision. We show here that meiotic entry in Saccharomyces cerevisiae is controlled by antisense-mediated regulation of IME4, a gene required for initiating meiosis. In MAT a/alpha diploids the antisense IME4 transcript is repressed by binding of the a1/alpha2 heterodimer at a conserved site located downstream of the IME4 coding sequence. MAT a/alpha diploids that produce IME4 antisense transcript have diminished sense transcription and fail to initiate meiosis. Haploids that produce the sense transcript have diminished antisense transcription and manifest several diploid phenotypes. Our data are consistent with transcription interference as a regulatory mechanism at the IME4 locus that determines cell fate Full Text

Houstis, N., Rosen, E. D. and Lander, E. S. (2006) Reactive oxygen species have a causal role in multiple forms of insulin resistance. Nature. 440 944-8. Insulin resistance is a cardinal feature of type 2 diabetes and is characteristic of a wide range of other clinical and experimental settings. Little is known about why insulin resistance occurs in so many contexts. Do the various insults that trigger insulin resistance act through a common mechanism? Or, as has been suggested, do they use distinct cellular pathways? Here we report a genomic analysis of two cellular models of insulin resistance, one induced by treatment with the cytokine tumour-necrosis factor-alpha and the other with the glucocorticoid dexamethasone. Gene expression analysis suggests that reactive oxygen species (ROS) levels are increased in both models, and we confirmed this through measures of cellular redox state. ROS have previously been proposed to be involved in insulin resistance, although evidence for a causal role has been scant. We tested this hypothesis in cell culture using six treatments designed to alter ROS levels, including two small molecules and four transgenes; all ameliorated insulin resistance to varying degrees. One of these treatments was tested in obese, insulin-resistant mice and was shown to improve insulin sensitivity and glucose homeostasis. Together, our findings suggest that increased ROS levels are an important trigger for insulin resistance in numerous settings.Full Text

Hughes, J. F., Skaletsky, H., Rozen, S., Wilson, R. K. and Page, D. C. (2006) Has the chimpanzee Y chromosome been sequenced? Nat Genet. 38 853-4.Full Text

Ivanovska, I. and Orr-Weaver, T. L. (2006) Histone Modifications and the Chromatin Scaffold for Meiotic Chromosome Architecture. Cell Cycle.Sep 15;5(18) [Epub ahead of print] 5 Chromosomes are capable of remarkable structural adaptability that enables their diverse functions. Histone modifications play pivotal roles in conferring structural diversity to chromosomes by influencing the compactness of chromatin. Several multi-protein complexes bind to chromatin and affect chromosome dynamics, including cohesin, condensin, the chromosome passenger complex, and the synaptonemal complex. The roles of these complexes in promoting chromosome functions include cohesion, condensation and synapsis. It is now crucial to define the relationship between the protein complexes that affect chromosome architecture and the underlying state of the chromatin. During meiosis chromosomes undergo striking morphological changes, including alignment of homologous chromosomes, double-strand break formation and repair, and establishment of meiosis-specific chromosome structures. These dynamic chromosome arrangements are accompanied by the recruitment and expulsion of multi-protein complexes from chromatin. Meiotic chromosome dynamics ensure proper chromosome segregation and production of healthy gametes. Meiosis thus affords an excellent opportunity to determine how histone modifications impact higher order chromosome dynamics by affecting localization and function of chromosome protein complexes. A meiotic mutation in the Drosophila histone kinase, NHK-1, uncovered a critical requirement for histone modifications in chromosome architecture, underscoring the power of this approach.

Jaenisch, R., Meissner, A. and Solter, D. (2006) Politically correct human embryonic stem cells? N Engl J Med. 354 1208-9. Full Text

JonesRhoades, M. W., Bartel, D. P. and Bartel, B. (2006) MicroRNAs and Their Regulatory Roles in Plants. Annu Rev Plant Biol. 57: 19-53 MicroRNAs (miRNAs) are small, endogenous RNAs that regulate gene expression in plants and animals. In plants, these approximately 21-nucleotide RNAs are processed from stem-loop regions of long primary transcripts by a Dicer-like enzyme and are loaded into silencing complexes, where they generally direct cleavage of complementary mRNAs. Although plant miRNAs have some conserved functions extending beyond development, the importance of miRNA-directed gene regulation during plant development is now particularly clear. Identified in plants less than four years ago, miRNAs are already known to play numerous crucial roles at each major stage of development-typically at the cores of gene regulatory networks, targeting genes that that are themselves regulators, such as those encoding transcription factors and F-box proteins.Full Text

Kalaany, N. Y. and Mangelsdorf, D. J. (2006) LXRs AND FXR: The Yin and Yang of cholesterol and fat metabolism. Annual Review of Physiology. 68 159-191. Liver X receptors (LXRs) and farnesoid X receptor (FXR) are nuclear receptors that function as intracellular sensors for sterols and bile acids, respectively. In response to their ligands, these receptors induce transcriptional responses that maintain a balanced, finely tuned regulation of cholesterol and bile acid metabolism. LXRs also permit the efficient storage of carbohydrate- and fat-derived energy, whereas FXR activation results in an overall decrease in triglyceride levels and modulation of glucose metabolism. The elegant, dual interplay between these two receptor systems Suggests that they coevolved to Constitute a highly sensitive and efficient system for the maintenance of total body fat and cholesterol homeostasis. Emerging evidence suggests that the tissue-specific action of these receptors is also Crucial for the proper function of the cardiovascular, immune, reproductive, endocrine pancreas, renal, and central nervous systems. Together, LXRs and FXR represent potential therapeutic targets for the treatment and prevention of numerous metabolic and lipid-related diseases Full Text

Kassir, Y., Rubin-Bejerano, I. and Mandel-Gutfreund, Y. (2006) The Saccharomyces cerevisiae GSK-3 beta homologs. Current Drug Targets. 7 1455-1465. Yeast cells carry four homologs of GSK-3 beta, RIM11, MCK1, MRK1 and YGK3. The significant homologs are RIM11 and MCK1 that presumably arose from a recent genome duplication followed by a rapid divergence. Accordingly, these homologs phosphorylate specific substrates. Rim I I is essential for entry into meiosis, whereas Mck1 is essential for growth at elevated and low temperatures. Both kinases transmit nutrient signals, but Mck1 transmits additional signals including stress signals such as, temperature, osmotic shock and Ca2+. Consequently, Mck1 plays a role in multiple functions, including cell wall integrity, meiosis and centromere function. The other two homologs, MRK1 and YGK3 that belong to the RIM11 and MCK1 phylogenetic trees, respectively, show no distinct phenotype. These paralogs posses redundant roles, though less important, with Rim11 and Mck1 functions. This review summarizes the cellular roles of these kinases, their mode of regulation, and the signals that they transmit.

Kim, Y. M., Pan, J. Y., Korbel, G. A., Peperzak, V., Boes, M. and Ploegh, H. L. (2006) Monovalent ligation of the B cell receptor induces receptor activation but fails to promote antigen presentation. Proc Natl Acad Sci U S A. Published online before print February 21, 2006, We explored the role of antigen valency in B cell receptor (BCR) activation and rearrangement of intracellular MHC class II compartments as factors that contribute to the efficacy of antigen presentation. Using primary B cells that express a hen egg lysozyme (HEL)-specific BCR, we found that oligomeric HEL more efficiently promoted both BCR activation and internalization than did monovalent HEL, although monovalent HEL, unlike monovalent Fab fragments of anti-Ig, readily triggered the BCR. Nonetheless, oligovalent ligation positions the BCR in a membrane microdomain that is distinct from one engaged in the course of monovalent ligation, as judged by detergent extraction of the BCR. Furthermore, oligovalent HEL induced more pronounced rearrangement of MHC class II-containing antigen-processing compartments. Using videomicroscopy we observed in real time the rearrangement of MHC class II compartments as well as delivery of antigen in primary B cells. The observed increase in rearrangement of MHC class II-positive compartments and the disposition of antigen-bound BCRs therein correlates with improved presentation of a HEL-derived epitope. Although monomeric HEL efficiently engages the BCR, presentation of HEL-derived epitopes is impaired compared to oligovalent antigens. This trait may help explain the known ability of soluble, disaggregated antigen to induce a state of B cell tolerance.Full Text.

Koch, K. S., Son, K. H., Maehr, R., Pellicciotta, I., Ploegh, H. L., Zanetti, M., Sell, S. and Leffert, H. L. (2006) Immune-privileged embryonic Swiss mouse STO and STO cell-derived progenitor cells: major histocompatibility complex and cell differentiation antigen expression patterns resemble those of human embryonic stem cell lines. Immunology. 119 98-115. Embryonic mouse STO (S, SIM; T, 6-thioguanine resistant; O, ouabain resistant) and 3(8)21-enhanced green fluorescent protein (EGFP) cell lines exhibit long-term survival and hepatic progenitor cell behaviour after xenogeneic engraftment in non-immunosuppressed inbred rats, and were previously designated major histocompatibility complex (MHC) class I- and class II-negative lines. To determine the molecular basis for undetectable MHC determinants, the expression and haplotype of H-2K, H-2D, H-2L and I-A proteins were reassessed by reverse transcriptase-polymerase chain reaction (RT-PCR), cDNA sequencing, RNA hybridization, immunoblotting, quantitative RT-PCR (QPCR), immunocytochemistry and flow cytometry. To detect cell differentiation (CD) surface antigens characteristic of stem cells, apoptotic regulation or adaptive immunity that might facilitate progenitor cell status or immune privilege, flow cytometry was also used to screen untreated and cytokine [interferon (IFN)-gamma]-treated cultures. Despite prior PCR genotyping analyses suggestive of H-2(q) haplotypes in STO, 3(8)21-EGFP and parental 3(8)21 cells, all three lines expressed H-2K cDNA sequences identical to those of d-haplotype BALB/c mice, as well as constitutive and cytokine-inducible H-2K(d) determinants. In contrast, apart from H-2L(d[LOW]) display in 3(8)21 cells, H-2D(d), H-2L(d) and I-A(d) determinants were undetectable. All three lines expressed constitutive and cytokine-inducible CD34; however, except for inducible CD117([LOW]) expression in 3(8)21 cells, no expression of CD45, CD117, CD62L, CD80, CD86, CD90.1 or CD95L/CD178 was observed. Constitutive and cytokine-inducible CD95([LOW]) expression was detected in STO and 3(8)21 cells, but not in 3(8)21-EGFP cells. MHC (class I+[LOW]/class II-) and CD (CD34(+)/CD80(-)/CD86(-)/CD95L(-)) expression patterns in STO and STO cell-derived progenitor cells resemble patterns reported for human embryonic stem cell lines. Whether these patterns reflect associations with mechanisms that are regulatory of immune privilege or functional tissue-specific plasticity is unknown.Full Text

Koubova, J., Menke, D. B., Zhou, Q., Capel, B., Griswold, M. D. and Page, D. C. (2006) Retinoic acid regulates sex-specific timing of meiotic initiation in mice. Proc Natl Acad Sci U S A.Feb 6; [Epub ahead of print] This contribution is part of the special series of Inaugural Articles by members of the National Academy of Sciences elected on May 3, 2005. In mammals, meiosis is initiated at different time points in males and females, but the mechanism underlying this difference is unknown. Female germ cells begin meiosis during embryogenesis. In males, embryonic germ cells undergo G0/G1 mitotic cell cycle arrest, and meiosis begins after birth. In mice, the Stimulated by Retinoic Acid Gene 8 (Stra8) has been found to be required for the transition into meiosis in both female and male germ cells. Stra8 is expressed in embryonic ovaries just before meiotic initiation, whereas its expression in testes is first detected after birth. Here we examine the mechanism underlying the sex-specific timing of Stra8 expression and meiotic initiation in mice. Our work shows that signaling by retinoic acid (RA), an active derivative of vitamin A, is required for Stra8 expression and thereby meiotic initiation in embryonic ovaries. We also discovered that RA is sufficient to induce Stra8 expression in embryonic testes and in vitamin A-deficient adult testes in vivo. Finally, our results show that cytochrome p450 (CYP)-mediated RA metabolism prevents premature Stra8 expression in embryonic testes. Treatment with an inhibitor specific to RA-metabolizing enzymes indicates that a cytochrome p450 from the 26 family (CYP26) is responsible for delaying Stra8 expression in embryonic testes. Sex-specific regulation of RA signaling thus plays an essential role in meiotic initiation in embryonic ovaries and precludes its occurrence in embryonic testes. Because RA signaling regulates Stra8 expression in both embryonic ovaries and adult testes, this portion of the meiotic initiation pathway may be identical in both sexes.PDF

Kritzer, J. A. (2006) When undergraduates ask "why," chemical biology answers. ACS Chem Biol. 1 411-3.PDF

Lau, N. C., Seto, A. G., Kim, J., Kuramochi-Miyagawa, S., Nakano, T., Bartel, D. P. and Kingston, R. E. (2006) Characterization of the piRNA complex from rat testes. Science. 313 363-367. Small noncoding RNAs regulate processes essential for cell growth and development, including mRNA degradation, translational repression, and transcriptional gene silencing (TGS). During a search for candidate mammalian factors for TGS, we purified a complex that contains small RNAs and Riwi, the rat homolog to human Piwi. The RNAs, frequently 29 to 30 nucleotides in length, are called Piwi-interacting RNAs (piRNAs), 94% of which map to 100 defined (<= 101 kb) genomic regions. Within these regions, the piRNAs generally distribute across only one genomic strand or distribute on two strands but in a divergent, nonoverlapping manner. Preparations of piRNA complex ( piRC) contain rRecQ1, which is homologous to qde-3 from Neurospora, a gene implicated in silencing pathways. Piwi has been genetically linked to TGS in flies, and slicer activity cofractionates with the purified complex. These results are consistent with a gene-silencing role for piRC in mammals Full Text

Lee, T. I., Jenner, R. G., Boyer, L. A., Guenther, M. G., Levine, S. S., Kumar, R. M., Chevalier, B., Johnstone, S. E., Cole, M. F., Isono, K., Koseki, H., Fuchikami, T., Abe, K., Murray, H. L., Zucker, J. P., Yuan, B., Bell, G. W., Herbolsheimer, E., Hannett, N. M., Sun, K., Odom, D. T., Otte, A. P., Volkert, T. L., Bartel, D. P., Melton, D. A., Gifford, D. K., Jaenisch, R. and Young, R. A. (2006) Control of developmental regulators by polycomb in human embryonic stem cells. Cell. 125 301-13. Polycomb group proteins are essential for early development in metazoans, but their contributions to human development are not well understood. We have mapped the Polycomb Repressive Complex 2 (PRC2) subunit SUZ12 across the entire nonrepeat portion of the genome in human embryonic stem (ES) cells. We found that SUZ12 is distributed across large portions of over two hundred genes encoding key developmental regulators. These genes are occupied by nucleosomes trimethylated at histone H3K27, are transcriptionally repressed, and contain some of the most highly conserved noncoding elements in the genome. We found that PRC2 target genes are preferentially activated during ES cell differentiation and that the ES cell regulators OCT4, SOX2, and NANOG cooccupy a significant subset of these genes. These results indicate that PRC2 occupies a special set of developmental genes in ES cells that must be repressed to maintain pluripotency and that are poised for activation during ES cell differentiation. Full Text

Lin, H., Yamada, Y., Nguyen, S., Linhart, H., Jackson-Grusby, L., Meissner, A., Meletis, K., Lo, G. and Jaenisch, R. (2006) Suppression of intestinal neoplasia by deletion of dnmt3b. Mol Cell Biol. 26 2976-83. Aberrant gene silencing accompanied by DNA methylation is associated with neoplastic progression in many tumors that also show global loss of DNA methylation. Using conditional inactivation of de novo methyltransferase Dnmt3b in Apc(Min/+) mice, we demonstrate that the loss of Dnmt3b has no impact on microadenoma formation, which is considered the earliest stage of intestinal tumor formation. Nevertheless, we observed a significant decrease in the formation of macroscopic colonic adenomas. Interestingly, many large adenomas showed regions with Dnmt3b inactivation, indicating that Dnmt3b is required for initial outgrowth of macroscopic adenomas but is not required for their maintenance. These results support a role for Dnmt3b in the transition stage between microadenoma formation and macroscopic colonic tumor growth and further suggest that Dnmt3b, and by extension de novo methylation, is not required for maintaining tumor growth after this transition stage has occurred.Full Text

Loureiro, J. and Ploegh, H. L. (2006) Antigen presentation and the ubiquitin-proteasome system in host-pathogen interactions. Adv Immunol. 92 225-305. Relatively small genomes and high replication rates allow viruses and bacteria to accumulate mutations. This continuously presents the host immune system with new challenges. On the other side of the trenches, an increasingly well-adjusted host immune response, shaped by coevolutionary history, makes a pathogen's life a rather complicated endeavor. It is, therefore, no surprise that pathogens either escape detection or modulate the host immune response, often by redirecting normal cellular pathways to their advantage. For the purpose of this chapter, we focus mainly on the manipulation of the class I and class II major histocompatibility complex (MHC) antigen presentation pathways and the ubiquitin (Ub)-proteasome system by both viral and bacterial pathogens. First, we describe the general features of antigen presentation pathways and the Ub-proteasome system and then address how they are manipulated by pathogens. We discuss the many human cytomegalovirus (HCMV)-encoded immunomodulatory genes that interfere with antigen presentation (immunoevasins) and focus on the HCMV immunoevasins US2 and US11, which induce the degradation of class I MHC heavy chains by the proteasome by catalyzing their export from the endoplasmic reticulum (ER)-membrane into the cytosol, a process termed ER dislocation. US2- and US11-mediated subversion of ER dislocation ensures proteasomal degradation of class I MHC molecules and presumably allows HCMV to avoid recognition by cytotoxic T cells, whilst providing insight into general aspects of ER-associated degradation (ERAD) which is used by eukaryotic cells to purge their ER of defective proteins. We discuss the similarities and differences between the distinct pathways co-opted by US2 and US11 for dislocation and degradation of human class I MHC molecules and also a putatively distinct pathway utilized by the murine herpes virus (MHV)-68 mK3 immunoevasin for ER dislocation of murine class I MHC. We speculate on the implications of the three pathogen-exploited dislocation pathways to cellular ER quality control. Moreover, we discuss the ubiquitin (Ub)-proteasome system and its position at the core of antigen presentation as proteolysis and intracellular trafficking rely heavily on Ub-dependent processes. We add a few examples of manipulation of the Ub-proteasome system by pathogens in the context of the immune system and such diverse aspects of the host-pathogen relationship as virus budding, bacterial chromosome integration, and programmed cell death, to name a few. Finally, we speculate on newly found pathogen-encoded deubiquitinating enzymes (DUBs) and their putative roles in modulation of host-pathogen interactions.

Loureiro, J., Lilley, B. N., Spooner, E., Noriega, V., Tortorella, D. and Ploegh, H. L. (2006) Signal peptide peptidase is required for dislocation from the endoplasmic reticulum. Nature.(advance online publication 31 May 2006) Human cytomegalovirus (HCMV) prevents the display of class I major histocompatibility complex (MHC) peptide complexes at the surface of infected cells as a means of escaping immune detection. Two HCMV-encoded immunoevasins, US2 and US11, induce the dislocation of class I MHC heavy chains from the endoplasmic reticulum membrane and target them for proteasomal degradation in the cytosol. Although the outcome of the dislocation reactions catalysed is similar, US2 and US11 operate differently: Derlin-1 is a key component of the US11 but not the US2 pathway. So far, proteins essential for US2-dependent dislocation have not been identified. Here we compare interacting partners of wild-type US2 with those of a dislocation-incompetent US2 mutant, and identify signal peptide peptidase (SPP) as a partner for the active form of US2. We show that a decrease in SPP levels by RNA-mediated interference inhibits heavy-chain dislocation by US2 but not by US11. Our data implicate SPP in the US2 pathway and indicate the possibility of a previously unknown function for this intramembrane-cleaving aspartic protease in dislocation from the endoplasmic reticulum.Full Text

Love, J. C., Ronan, J. L., Grotenbreg, G. M., van der Veen, A. G. and Ploegh, H. L. (2006) A microengraving method for rapid selection of single cells producing antigen-specific antibodies. Nature Biotechnology .Published online: 14 May 2006 Monoclonal antibodies that recognize specific antigens of interest are used as therapeutic agents and as tools for biomedical research. Discovering a single monoclonal antibody requires retrieval of an individual hybridoma from polyclonal mixtures of cells producing antibodies with a variety of specificities. The time required to isolate hybridomas by a limiting serial-dilution, however, has restricted the diversity and breadth of available antibodies. Here we present a soft lithographic method based on intaglio printing to generate microarrays comprising the secreted products of single cells. These engraved arrays enable a rapid (<12 h) and high-throughput (>100,000 individual cells) system for identification, recovery and clonal expansion of cells producing antigen-specific antibodies. This method can be adapted, in principle, to detect any secreted product in a multiplexed manner.Full Text

Lu, X., Gross, A. W. and Lodish, H. F. (2006) Active conformation of the erythropoietin receptor: Random and cysteine-scanning mutagenesis of the extracellular juxtamembrane and transmembrane domains. Journal of Biological Chemistry 281 (11): 7002-7011 MAR 17 2006. In the absence of erythropoietin (Epo) cell surface Epo receptors (EpoR) are dimeric; dimerization is mediated mainly by the transmembrane domain. Binding of Epo changes the orientation of the two receptor subunits. This conformational change is transmitted through the juxt