Hot off the presses! Nov 12 Cell

The Nov 12 issue of the Cell is now up on Pubget (About Cell): if you're at a subscribing institution, just click the link in the latest link at the home page. (Note you'll only be able to get all the PDFs in the issue if your institution subscribes to Pubget.)

Latest Articles Include:

• In This Issue
  - cell 143(4):487, 489 (2010)
  
• Select: Manipulating Cellular Machinery
  - cell 143(4):491, 493 (2010)
  Cells can be manipulated for any number of purposes, owing to the emergence of synthetic biology and bioengineering tools. Recent biotechnological advances have generated improved therapeutics, sophisticated biosensors, and new energy sources. The findings presented in this Select highlight innovative ways to both dissect and reconstruct various cellular machineries and components.
• Modeling Rett Syndrome with Stem Cells
  - cell 143(4):499-500 (2010)
  The discovery that somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) raised the exciting possibility of modeling diseases with patient-specific cells. Marchetto et al. (2010) now use iPSC technology to generate, characterize, and treat an in vitro model for the autism spectrum disorder Rett syndrome.
• Translation by Remote Control
  - cell 143(4):501-502 (2010)
  Efficient and accurate gene expression requires the coordination of multiple steps along the pathway of mRNA and protein synthesis. Now, Harel-Sharvit et al. (2010) show that transcriptional imprinting of mRNAs with two subunits of RNA polymerase II, Rbp4p and Rpb7p, guides transcripts to the translation apparatus.
• Shining a Light on Germinal Center B Cells
  - cell 143(4):503-505 (2010)
  The mechanisms of B cell selection in lymphoid tissues are poorly understood. In this issue, Victora et al. (2010) use imaging of photoactivatable green fluorescent protein to define the movements of B cells in germinal centers and provide evidence that antibody affinity maturation is driven by competition for T cell help.
• A Straightjacket for Pain?
  - cell 143(4):505-507 (2010)
  Perception of pain involves both the peripheral and central nervous systems. Starting with a whole-genome RNA interference screen in Drosophila, Neely et al. (2010) identify a mammalian gene that is required not only for efficient transfer of pain signals between brain centers, but also for the suppression of inappropriate signaling between other sensory systems.
• Pluripotency and Cellular Reprogramming: Facts, Hypotheses, Unresolved Issues
  - cell 143(4):508-525 (2010)
  Direct reprogramming of somatic cells to induced pluripotent stem cells by ectopic expression of defined transcription factors has raised fundamental questions regarding the epigenetic stability of the differentiated cell state. In addition, evidence has accumulated that distinct states of pluripotency can interconvert through the modulation of both cell-intrinsic and exogenous factors. To fully realize the potential of in vitro reprogrammed cells, we need to understand the molecular and epigenetic determinants that convert one cell type into another. Here we review recent advances in this rapidly moving field and emphasize unresolved and controversial questions.
• A Model for Neural Development and Treatment of Rett Syndrome Using Human Induced Pluripotent Stem Cells
  - cell 143(4):527-539 (2010)
  Autism spectrum disorders (ASD) are complex neurodevelopmental diseases in which different combinations of genetic mutations may contribute to the phenotype. Using Rett syndrome (RTT) as an ASD genetic model, we developed a culture system using induced pluripotent stem cells (iPSCs) from RTT patients' fibroblasts. RTT patients' iPSCs are able to undergo X-inactivation and generate functional neurons. Neurons derived from RTT-iPSCs had fewer synapses, reduced spine density, smaller soma size, altered calcium signaling and electrophysiological defects when compared to controls. Our data uncovered early alterations in developing human RTT neurons. Finally, we used RTT neurons to test the effects of drugs in rescuing synaptic defects. Our data provide evidence of an unexplored developmental window, before disease onset, in RTT syndrome where potential therapies could be successfully employed. Our model recapitulates early stages of a human neurodevelopmental disease and re! presents a promising cellular tool for drug screening, diagnosis and personalized treatment.
• Pausing of RNA Polymerase II Disrupts DNA-Specified Nucleosome Organization to Enable Precise Gene Regulation
  - cell 143(4):540-551 (2010)
  Metazoan transcription is controlled through either coordinated recruitment of transcription machinery to the gene promoter or regulated pausing of RNA polymerase II (Pol II) in early elongation. We report that a striking difference between genes that use these distinct regulatory strategies lies in the "default" chromatin architecture specified by their DNA sequences. Pol II pausing is prominent at highly regulated genes whose sequences inherently disfavor nucleosome formation within the gene but favor occlusion of the promoter by nucleosomes. In contrast, housekeeping genes that lack pronounced Pol II pausing show higher nucleosome occupancy downstream, but their promoters are deprived of nucleosomes regardless of polymerase binding. Our results indicate that a key role of paused Pol II is to compete with nucleosomes for occupancy of highly regulated promoters, thereby preventing the formation of repressive chromatin architecture to facilitate further or future g! ene activation.
• RNA Polymerase II Subunits Link Transcription and mRNA Decay to Translation
  - cell 143(4):552-563 (2010)
  Little is known about crosstalk between the eukaryotic transcription and translation machineries that operate in different cell compartments. The yeast proteins Rpb4p and Rpb7p represent one such link as they form a heterodimer that shuttles between the nucleus, where it functions in transcription, and the cytoplasm, where it functions in the major mRNA decay pathways. Here we show that the Rpb4/7 heterodimer interacts physically and functionally with components of the translation initiation factor 3 (eIF3), and is required for efficient translation initiation. Efficient translation in the cytoplasm depends on association of Rpb4/7 with RNA polymerase II (Pol II) in the nucleus, leading to a model in which Pol II remotely controls translation. Hence, like in prokaryotes, the eukaryotic translation is coupled to transcription. We propose that Rpb4/7, through its interactions at each step in the mRNA lifecycle, represents a class of factors, "mRNA coordinators," whic! h integrate the various stages of gene expression into a system.
• A Family of Protein-Deglutamylating Enzymes Associated with Neurodegeneration
  - cell 143(4):564-578 (2010)
  Polyglutamylation is a posttranslational modification that generates glutamate side chains on tubulins and other proteins. Although this modification has been shown to be reversible, little is known about the enzymes catalyzing deglutamylation. Here we describe the enzymatic mechanism of protein deglutamylation by members of the cytosolic carboxypeptidase (CCP) family. Three enzymes (CCP1, CCP4, and CCP6) catalyze the shortening of polyglutamate chains and a fourth (CCP5) specifically removes the branching point glutamates. In addition, CCP1, CCP4, and CCP6 also remove gene-encoded glutamates from the carboxyl termini of proteins. Accordingly, we show that these enzymes convert detyrosinated tubulin into Δ2-tubulin and also modify other substrates, including myosin light chain kinase 1. We further analyze Purkinje cell degeneration (pcd) mice that lack functional CCP1 and show that microtubule hyperglutamylation is directly linked to neurodegeneration. Taken together,! our results reveal that controlling the length of the polyglutamate side chains on tubulin is critical for neuronal survival.
• Retrotranslocation of a Misfolded Luminal ER Protein by the Ubiquitin-Ligase Hrd1p
  - cell 143(4):579-591 (2010)
  Misfolded, luminal endoplasmic reticulum (ER) proteins are retrotranslocated into the cytosol and degraded by the ubiquitin/proteasome system. This ERAD-L pathway requires a protein complex consisting of the ubiquitin ligase Hrd1p, which spans the ER membrane multiple times, and the membrane proteins Hrd3p, Usa1p, and Der1p. Here, we show that Hrd1p is the central membrane component in ERAD-L; its overexpression bypasses the need for the other components of the Hrd1p complex. Hrd1p function requires its oligomerization, which in wild-type cells is facilitated by Usa1p. Site-specific photocrosslinking indicates that, at early stages of retrotranslocation, Hrd1p interacts with a substrate segment close to the degradation signal. This interaction follows the delivery of substrate through other ERAD components, requires the presence of transmembrane segments of Hrd1p, and depends on both the ubiquitin ligase activity of Hrd1p and the function of the Cdc48p ATPase complex. ! Our results suggest a model for how Hrd1p promotes polypeptide movement through the ER membrane.
• Germinal Center Dynamics Revealed by Multiphoton Microscopy with a Photoactivatable Fluorescent Reporter
  - cell 143(4):592-605 (2010)
  The germinal center (GC) reaction produces high-affinity antibodies by random mutation and selective clonal expansion of B cells with high-affinity receptors. The mechanism by which B cells are selected remains unclear, as does the role of the two anatomically defined areas of the GC, light zone (LZ) and dark zone (DZ). We combined a transgenic photoactivatable fluorescent protein tracer with multiphoton laser-scanning microscopy and flow cytometry to examine anatomically defined LZ and DZ B cells and GC selection. We find that B cell division is restricted to the DZ, with a net vector of B cell movement from the DZ to the LZ. The decision to return to the DZ and undergo clonal expansion is controlled by T helper cells in the GC LZ, which discern between LZ B cells based on the amount of antigen captured and presented. Thus, T cell help, and not direct competition for antigen, is the limiting factor in GC selection. PaperClip To listen to this audio, enable JavaScript on your browser. However, you can download and play the audio by clicking on the icon below Download this Audio (2922 K)
• Transcriptional Regulation of ROS Controls Transition from Proliferation to Differentiation in the Root
  - cell 143(4):606-616 (2010)
  The balance between cellular proliferation and differentiation is a key aspect of development in multicellular organisms. Using high-resolution expression data from the Arabidopsis root, we identified a transcription factor, UPBEAT1 (UPB1), that regulates this balance. Genomewide expression profiling coupled with ChIP-chip analysis revealed that UPB1 directly regulates the expression of a set of peroxidases that modulate the balance of reactive oxygen species (ROS) between the zones of cell proliferation and the zone of cell elongation where differentiation begins. Disruption of UPB1 activity alters this ROS balance, leading to a delay in the onset of differentiation. Modulation of either ROS balance or peroxidase activity through chemical reagents affects the onset of differentiation in a manner consistent with the postulated UPB1 function. This pathway functions independently of auxin and cytokinin plant hormonal signaling. Comparison to ROS-regulated growth control ! in animals suggests that a similar mechanism is used in plants and animals.
• Epiblast Stem Cell Subpopulations Represent Mouse Embryos of Distinct Pregastrulation Stages
  - cell 143(4):617-627 (2010)
  Embryonic stem cells (ESCs) comprise at least two populations of cells with divergent states of pluripotency. Here, we show that epiblast stem cells (EpiSCs) also comprise two distinct cell populations that can be distinguished by the expression of a specific Oct4-GFP marker. These two subpopulations, Oct4-GFP positive and negative EpiSCs, are capable of converting into each other in vitro. Oct4-GFP positive and negative EpiSCs are distinct from ESCs with respect to global gene expression pattern, epigenetic profile, and Oct4 enhancer utilization. Oct4-GFP negative cells share features with cells of the late mouse epiblast and cannot form chimeras. However, Oct4-GFP positive EpiSCs, which only represent a minor EpiSC fraction, resemble cells of the early epiblast and can readily contribute to chimeras. Our findings suggest that the rare ability of EpiSCs to contribute to chimeras is due to the presence of the minor EpiSC fraction representing the early epiblast.
• A Genome-wide Drosophila Screen for Heat Nociception Identifies α2δ3 as an Evolutionarily Conserved Pain Gene
  - cell 143(4):628-638 (2010)
  Worldwide, acute, and chronic pain affects 20% of the adult population and represents an enormous financial and emotional burden. Using genome-wide neuronal-specific RNAi knockdown in Drosophila, we report a global screen for an innate behavior and identify hundreds of genes implicated in heat nociception, including the α2δ family calcium channel subunit straightjacket (stj). Mice mutant for the stj ortholog CACNA2D3 (α2δ3) also exhibit impaired behavioral heat pain sensitivity. In addition, in humans, α2δ3 SNP variants associate with reduced sensitivity to acute noxious heat and chronic back pain. Functional imaging in α2δ3 mutant mice revealed impaired transmission of thermal pain-evoked signals from the thalamus to higher-order pain centers. Intriguingly, in α2δ3 mutant mice, thermal pain and tactile stimulation triggered strong cross-activation, or synesthesia, of brain regions involved in vision, olfaction, and hearing.
• Extensive In Vivo Metabolite-Protein Interactions Revealed by Large-Scale Systematic Analyses
  - cell 143(4):639-650 (2010)
  Natural small compounds comprise most cellular molecules and bind proteins as substrates, products, cofactors, and ligands. However, a large-scale investigation of in vivo protein-small metabolite interactions has not been performed. We developed a mass spectrometry assay for the large-scale identification of in vivo protein-hydrophobic small metabolite interactions in yeast and analyzed compounds that bind ergosterol biosynthetic proteins and protein kinases. Many of these proteins bind small metabolites; a few interactions were previously known, but the vast majority are new. Importantly, many key regulatory proteins such as protein kinases bind metabolites. Ergosterol was found to bind many proteins and may function as a general regulator. It is required for the activity of Ypk1, a mammalian AKT/SGK kinase homolog. Our study defines potential key regulatory steps in lipid biosynthetic pathways and suggests that small metabolites may play a more general role as regul! ators of protein activity and function than previously appreciated.
• Retraction Notice to: Population-Level Transcription Cycles Derive from Stochastic Timing of Single-Cell Transcription
  - cell 143(4):651 (2010)
  
• Enhanced SnapShot: Macromolecular Machines
  - cell 143(4):652-652.e1 (2010)