Dynactin is an essential cofactor for the microtubule motor cytoplasmic dynein-1. We report the structure of the 23 subunit dynactin complex by cryo-electron microscopy to 4.0Å. Our reconstruction reveals how dynactin is built around a filament containing eight copies of the actin related protein Arp1 and one of β-actin. Capped at each end by distinct protein complexes, the length of the filament is defined by elongated peptides that emerge from the α-helical shoulder domain. A further 8.2Å structure of the complex between dynein, dynactin and the motility inducing cargo adaptor Bicaudal-D2 shows how the translational symmetry of the dynein tail matches that of the dynactin filament. The Bicaudal-D2 coiled coil runs between dynein and dynactin to stabilize the mutually dependent interactions between all three components.Dynactin works with the cytoplasmic dynein-1 motor (dynein) to transport cargos along the microtubule cytoskeleton (1-3). They maintain the cell's spatial organization, return components from the cell's periphery and assist with cellular division (4). Mutations in either complex cause neurodegeneration (5) and both can be co-opted by viruses that travel to the nucleus (6). Dynein and dynactin are similar in size and complexity. Dynein contains two copies of 6 different proteins and has a mass of 1.4 MDa. Dynactin, at about 1.0 MDa, contains more than 20 subunits, corresponding to 12 different proteins. Dynactin is built around a filament of actin related protein 1 (Arp1). In analogy to actin, the filament has a barbed and a pointed end; each capped by a different protein complex. On top sits the shoulder domain (7) from which emerges a long projection, corresponding to dynactin's largest subunit p150 Glued (DCTN1) (8).Despite the presence of a dynein binding site in p150 Glued (9-11), purified dynein and dynactin only form a stable complex in the presence of the cargo adaptor Bicaudal D2 † To whom correspondence should be addressed: cartera@mrc-lmb.cam.ac.uk. Author contributions L.U. prepared dynactin and determined the TDB structure. K.Z. determined the structure of dynactin. A.G.D. and M.Y. determined the DHC N-terminus crystal structure. C.M. and M.A.S. prepared the dynein tail complex. N.A.P. and C.V.R performed mass spectrometry. A.P.C. initiated the project and designed the experiments.

SUMMARY Embryonic development is characterized by dynamic changes in gene expression, yet the role of chromatin remodeling in these cellular transitions remains elusive. To address this question, we profiled the transcriptome and select chromatin modifications at defined stages during pancreatic endocrine differentiation of human embryonic stem cells. We identify removal of Polycomb group (PcG)-mediated repression on stage-specific genes as a key mechanism for the induction of developmental regulators. Furthermore, we discover that silencing of transitory genes during lineage progression associates with reinstatement of PcG-dependent repression. Significantly, in vivo-, but not in vitro-differentiated endocrine cells exhibit close similarity to primary human islets in regard to transcriptome and chromatin structure. We further demonstrate that endocrine cells produced in vitro, do not fully eliminate PcG-mediated repression on endocrine-specific genes, likely contributing to their malfunction. These studies reveal dynamic chromatin remodeling during developmental lineage progression and identify possible strategies for improving cell differentiation in culture.

Summary Embryonic development relies on the capacity of progenitor cells to appropriately respond to inductive cues; a cellular property known as developmental competence. Here we report that epigenetic priming of enhancers signifies developmental competence during endodermal lineage diversification. Chromatin mapping during pancreatic and hepatic differentiation of human embryonic stem cells revealed the en masse acquisition of a poised chromatin state at enhancers specific to endoderm-derived cell lineages in gut tube intermediates. Experimentally, the acquisition of this poised enhancer state predicts the ability of endodermal intermediates to respond to inductive signals. Furthermore, these enhancers are first recognized by the pioneer transcription factors FOXA1 and FOXA2 when competence is acquired, while subsequent recruitment of lineage-inductive transcription factors, such as PDX1, leads to enhancer and target gene activation. Together, our results identify the acquisition of a poised chromatin state at enhancers as a mechanism by which progenitor cells acquire developmental competence.

Flavin-containing monooxygenases (FMOs) are one of the most important monooxygenase systems in Eukaryotes and have many important physiological functions. FMOs have also been found in bacteria; however, their physiological function is not known. Here, we report the identification and characterization of trimethylamine (TMA) monooxygenase, termed Tmm, from Methylocella silvestris, using a combination of proteomic, biochemical, and genetic approaches. This bacterial FMO contains the FMO sequence motif (FXGXXXHXXXF/Y) and typical flavin adenine dinucleotide and nicotinamide adenine dinucleotide phosphate-binding domains. The enzyme was highly expressed in TMA-grown M. silvestris and absent during growth on methanol. The gene, tmm, was expressed in Escherichia coli, and the purified recombinant protein had high Tmm activity. Mutagenesis of this gene abolished the ability of M. silvestris to grow on TMA as a sole carbon and energy source. Close homologs of tmm occur in many Alphaproteobacteria, in particular Rhodobacteraceae (marine Roseobacter clade, MRC) and the marine SAR11 clade (Pelagibacter ubique). We show that the ability of MRC to use TMA as a sole carbon and/or nitrogen source is directly linked to the presence of tmm in the genomes, and purified Tmm of MRC and SAR11 from recombinant E. coli showed Tmm activities. The tmm gene is highly abundant in the metagenomes of the Global Ocean Sampling expedition, and we estimate that 20% of the bacteria in the surface ocean contain tmm. Taken together, our results suggest that Tmm, a bacterial FMO, plays an important yet overlooked role in the global carbon and nitrogen cycles.

We have previously shown the transcription factor SOX9 to be required for the maintenance of multipotential pancreatic progenitor cells in the early embryonic pancreas. However, the association of pancreatic endocrine defects with the Sox9-haploinsufficiency syndrome campomelic dysplasia (CD) implies additional later roles for Sox9 in endocrine development. Using short-term lineage tracing in mice, we demonstrate here that SOX9 marks a pool of multipotential pancreatic progenitors throughout the window of major cell differentiation. During mid-pancreogenesis, both endocrine and exocrine cells simultaneously arise from the SOX9+ epithelial cords. Our analysis of mice with 50%-reduced Sox9 gene dosage in pancreatic progenitors reveals endocrine-specific defects phenocopying CD. By birth, these mice display a specific reduction in endocrine cell mass, while their exocrine compartment and total organ size is normal. The decrease in endocrine cells is caused by reduced generation of endocrine progenitors from the SOX9+ epithelium. Conversely, formation of exocrine progenitors is insensitive to reduced Sox9 gene dosage, thus explaining the normal organ size at birth. Our results show that not only is SOX9 required for the maintenance of early pancreatic progenitors, but also governs their adoption of an endocrine fate. Our findings therefore suggest that defective endocrine specification might underlie the pancreatic phenotype of individuals with CD.

Background and purpose: M 1 muscarinic ACh receptors (mAChRs) represent an attractive drug target for the treatment of cognitive deficits associated with diseases such as Alzheimer's disease and schizophrenia. However, the discovery of subtypeselective mAChR agonists has been hampered by the high degree of conservation of the orthosteric ACh-binding site among mAChR subtypes. The advent of functional screening assays has enabled the identification of agonists such as AC-42 (4-nbutyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl]-piperidine), which bind to an allosteric site and selectively activate the M 1 mAChR subtype. However, studies with this compound have been limited to recombinantly expressed mAChRs. Experimental approach: In this study, we have compared the pharmacological profile of AC-42 and a close structural analogue, 77-LH-28-1 (1-[3-(4-butyl-1-piperidinyl)propyl]-3,4-dihydro-2(1H)-quinolinone) at human recombinant, and rat native, mAChRs by calcium mobilization, inositol phosphate accumulation and both in vitro and in vivo electrophysiology. Key results: Calcium mobilization and inositol phosphate accumulation assays revealed that both AC-42 and 77-LH-28-1 display high selectivity to activate the M 1 mAChR over other mAChR subtypes. Furthermore, 77-LH-28-1, but not AC-42, acted as an agonist at rat hippocampal M 1 receptors, as demonstrated by its ability to increase cell firing and initiate gamma frequency network oscillations. Finally, 77-LH-28-1 stimulated cell firing in the rat hippocampus in vivo following subcutaneous administration. Conclusions and implications: These data suggest that 77-LH-28-1 is a potent, selective, bioavailable and brain-penetrant agonist at the M 1 mAChR and therefore that it represents a better tool than AC-42, with which to study the pharmacology of the M 1 mAChR. (2008) 154, 1104-1115 doi:10.1038/bjp.2008 published online 5 May 2008 Keywords: muscarinic receptors; selective agonist; allosteric; AC-42; 77-LH-28-1; calcium mobilization; inositol phosphate; cell firing; network oscillations There is a wide array of pharmacological tools with which to study mAChRs. For example, N-methyl scopolamine, quinuclidinylbenzilate, pirenzepine and darifenacin are among numerous mAChR antagonists, and ACh and oxotremorine-M among mAChR agonists, which have been used in unlabelled and radiolabelled forms to characterize the localization, pharmacology and function of mAChRs. Unfortunately, most of these pharmacological tools exhibit poor selectivity between mAChR subtypes (Caulfield and Birdsall, 1998;Ellis, 2002). Those agents that do display high degrees of mAChR subtype selectivity are few in number and when discovered are often shown to interact with an allosteric, rather than the orthosteric, site as exemplified by the highly selective M 1 receptor peptide antagonist MT-7 (muscarinic toxin 7; Olianas et al., 2000). British Journal of PharmacologyTherefore, the identification of selective M 1 mAChR agonists would represent a significant advance in mAChR pharmacology and could offer t...

SUMMARYAll mature pancreatic cell types arise from organ-specific multipotent progenitor cells. Although previous studies have identified cell-intrinsic and -extrinsic cues for progenitor cell expansion, it is unclear how these cues are integrated within the niche of the developing organ. Here, we present genetic evidence in mice that the transcription factor Sox9 forms the centerpiece of a gene regulatory network that is crucial for proper organ growth and maintenance of organ identity. We show that pancreatic progenitorspecific ablation of Sox9 during early pancreas development causes pancreas-to-liver cell fate conversion. Sox9 deficiency results in cell-autonomous loss of the fibroblast growth factor receptor (Fgfr) 2b, which is required for transducing mesenchymal Fgf10 signals. Likewise, Fgf10 is required to maintain expression of Sox9 and Fgfr2 in epithelial progenitors, showing that Sox9, Fgfr2 and Fgf10 form a feed-forward expression loop in the early pancreatic organ niche. Mirroring Sox9 deficiency, perturbation of Fgfr signaling in pancreatic explants or genetic inactivation of Fgf10 also result in hepatic cell fate conversion. Combined with previous findings that Fgfr2b or Fgf10 are necessary for pancreatic progenitor cell proliferation, our results demonstrate that organ fate commitment and progenitor cell expansion are coordinately controlled by the activity of a Sox9/Fgf10/Fgfr2b feed-forward loop in the pancreatic niche. This self-promoting Sox9/Fgf10/Fgfr2b loop may regulate cell identity and organ size in a broad spectrum of developmental and regenerative contexts.

SummaryProtein folding in cells is regulated by networks of chaperones, including the heat shock protein 70 (Hsp70) system, which consists of the Hsp40 cochaperone and a nucleotide exchange factor. Hsp40 mediates complex formation between Hsp70 and client proteins prior to interaction with Hsp90. We used mass spectrometry (MS) to monitor assemblies formed between eukaryotic Hsp90/Hsp70/Hsp40, Hop, p23, and a client protein, a fragment of the glucocorticoid receptor (GR). We found that Hsp40 promotes interactions between the client and Hsp70, and facilitates dimerization of monomeric Hsp70. This dimerization is antiparallel, stabilized by post-translational modifications (PTMs), and maintained in the stable heterohexameric client-loading complex Hsp902Hsp702HopGR identified here. Addition of p23 to this client-loading complex induces transfer of GR onto Hsp90 and leads to expulsion of Hop and Hsp70. Based on these results, we propose that Hsp70 antiparallel dimerization, stabilized by PTMs, positions the client for transfer from Hsp70 to Hsp90.