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Primary cilia organize Hedgehog signaling and shape embryonic development, and their dysregulation is the unifying cause of ciliopathies. We conducted a functional genomic screen for Hedgehog signaling by engineering antibiotic-based selection of Hedgehog-responsive cells and applying genome-wide CRISPR-mediated gene disruption. The screen can robustly identify factors required for ciliary signaling with few false positives or false negatives. Characterization of hit genes uncovered novel components of several ciliary structures, including a protein complex that contains δ-tubulin and ε-tubulin and is required for centriole maintenance. The screen also provides an unbiased tool for classifying ciliopathies and showed that many congenital heart disorders are caused by loss of ciliary signaling. Collectively, our study enables a systematic analysis of ciliary function and of ciliopathies, and also defines a versatile platform for dissecting signaling pathways through CRISPR-based screening.

Cytoplasmic dyneins are motor proteins in the AAA+ superfamily that transport cellular cargos toward microtubule minus-ends. Recently, ciliobrevins were reported as selective cell-permeable inhibitors of cytoplasmic dyneins. As is often true for first-in-class inhibitors, the use of ciliobrevins has in part been limited by low potency. Moreover, suboptimal chemical properties, such as the potential to isomerize, have hindered efforts to improve ciliobrevins. Here, we characterized the structure of ciliobrevins and designed conformationally constrained isosteres. These studies identified dynapyrazoles, inhibitors more potent than ciliobrevins. At single-digit micromolar concentrations dynapyrazoles block intraflagellar transport in the cilium and lysosome motility in the cytoplasm, processes that depend on cytoplasmic dyneins. Further, we find that while ciliobrevins inhibit both dynein's microtubule-stimulated and basal ATPase activity, dynapyrazoles strongly block only microtubule-stimulated activity. Together, our studies suggest that chemical-structure-based analyses can lead to inhibitors with improved properties and distinct modes of inhibition.

Cytoplasmic dyneins 1 and 2 are related members of the AAA+ superfamily (ATPases associated with diverse cellular activities) that function as the predominant minus-end-directed microtubule motors in eukaryotic cells. Dynein 1 controls mitotic spindle assembly, organelle movement, axonal transport, and other cytosolic, microtubule-guided processes, whereas dynein 2 mediates retrograde trafficking within motile and primary cilia. Small-molecule inhibitors are important tools for investigating motor protein-dependent mechanisms, and ciliobrevins were recently discovered as the first dynein-specific chemical antagonists. Here, we demonstrate that ciliobrevins directly target the heavy chains of both dynein isoforms and explore the structure-activity landscape of these inhibitors in vitro and in cells. In addition to identifying chemical motifs that are essential for dynein blockade, we have discovered analogs with increased potency and dynein 2 selectivity. These antagonists effectively disrupt Hedgehog signaling, intraflagellar transport, and ciliogenesis, making them useful probes of these and other cytoplasmic dynein 2-dependent cellular processes.

Pancreatic ductal adenocarcinoma (PDA) is the most lethal of common human malignancies, with no truly effective therapies for advanced disease. Preclinical studies have suggested a therapeutic benefit of targeting the Hedgehog (Hh) signaling pathway, which is activated throughout the course of PDA progression by expression of Hh ligands in the neoplastic epithelium and paracrine response in the stromal fibroblasts. Clinical trials to test this possibility, however, have yielded disappointing results. To further investigate the role of Hh signaling in the formation of PDA and its precursor lesion, pancreatic intraepithelial neoplasia (PanIN), we examined the effects of genetic or pharmacologic inhibition of Hh pathway activity in three distinct genetically engineered mouse models and found that Hh pathway inhibition accelerates rather than delays progression of oncogenic Kras-driven disease. Notably, pharmacologic inhibition of Hh pathway activity affected the balance between epithelial and stromal elements, suppressing stromal desmoplasia but also causing accelerated growth of the PanIN epithelium. In striking contrast, pathway activation using a small molecule agonist caused stromal hyperplasia and reduced epithelial proliferation. These results indicate that stromal response to Hh signaling is protective against PDA and that pharmacologic activation of pathway response can slow tumorigenesis. Our results provide evidence for a restraining role of stroma in PDA progression, suggesting an explanation for the failure of Hh inhibitors in clinical trials and pointing to the possibility of a novel type of therapeutic intervention.

The recognition of distinctly modified histones by specialized 'effector' proteins constitutes a key mechanism for transducing molecular events at chromatin to biological outcomes. Effector proteins influence DNA-templated processes, including transcription, DNA recombination and DNA repair; however, no effector functions have yet been identified within the mammalian machinery that regulate DNA replication. Here we show that ORC1--a component of ORC (origin of replication complex), which mediates pre-DNA replication licensing--contains a bromo adjacent homology (BAH) domain that specifically recognizes histone H4 dimethylated at lysine 20 (H4K20me2). Recognition of H4K20me2 is a property common to BAH domains present within diverse metazoan ORC1 proteins. Structural studies reveal that the specificity of the BAH domain for H4K20me2 is mediated by a dynamic aromatic dimethyl-lysine-binding cage and multiple intermolecular contacts involving the bound peptide. H4K20me2 is enriched at replication origins, and abrogating ORC1 recognition of H4K20me2 in cells impairs ORC1 occupancy at replication origins, ORC chromatin loading and cell-cycle progression. Mutation of the ORC1 BAH domain has been implicated in the aetiology of Meier-Gorlin syndrome (MGS), a form of primordial dwarfism, and ORC1 depletion in zebrafish results in an MGS-like phenotype. We find that wild-type human ORC1, but not ORC1-H4K20me2-binding mutants, rescues the growth retardation of orc1 morphants. Moreover, zebrafish depleted of H4K20me2 have diminished body size, mirroring the phenotype of orc1 morphants. Together, our results identify the BAH domain as a novel methyl-lysine-binding module, thereby establishing the first direct link between histone methylation and the metazoan DNA replication machinery, and defining a pivotal aetiological role for the canonical H4K20me2 mark, via ORC1, in primordial dwarfism.

The conversion of chemical energy into mechanical force by AAA+ (ATPases associated with diverse cellular activities) ATPases is integral to cellular processes, including DNA replication, protein unfolding, cargo transport and [...]

Small-cell lung cancer (SCLC) is an aggressive neuroendocrine subtype of lung cancer for which there is no effective treatment (1,2). Using a mouse model in which deletion of Rbl and Trp53 in the lung epithelium of adult mice induces SCLC (3,4), we found that the Hedgehog signaling pathway is activated in SCLC cells independently of the lung microenvironment. Constitutive activation of the Hedgehog signaling molecule Smoothened (Smo) promoted the clonogenicity of human SCLC in vitro and the initiation and progression of mouse SCLC in vivo. Reciprocally, deletion of Smo in Rbl and Trp53-mutant lung epithelial cells strongly suppressed SCLC initiation and progression in mice. Furthermore, pharmacological blockade of Hedgehog signaling inhibited the growth of mouse and human SCLC, most notably following chemotherapy. These findings show a crucial cell-intrinsic role for Hedgehog signaling in the development and maintenance of SCLC and identify Hedgehog pathway inhibition as a therapeutic strategy to slow the progression of disease and delay cancer recurrence in individuals with SCLC., Activation of Hedgehog signaling has been reported in a subset of human SCLC cell lines and tumors (5-8) without changes in Hedgehog pathway gene copy numbers (9). Furthermore, we sequenced [...]

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2009.01.036 Byline: John K. Mich (a), Heiko Blaser (b), Natalie A. Thomas (c), Ari J. Firestone (d), Deborah Yelon (c), Erez Raz (b), James K. Chen (d) Keywords: Primordial germ cell; Zebrafish; Hedgehog signaling; Smoothened; Cyclopamine Abstract: Primordial germ cells (PGCs) are the progenitors of reproductive cells in metazoans and are an important model for the study of cell migration in vivo. Previous reports have suggested that Hedgehog (Hh) protein acts as a chemoattractant for PGC migration in the Drosophila embryo and that downstream signaling proteins such as Patched (Ptc) and Smoothened (Smo) are required for PGC localization to somatic gonadal precursors. Here we interrogate whether Hh signaling is required for PGC migration in vertebrates, using the zebrafish as a model system. We find that cyclopamine, an inhibitor of Hh signaling, causes strong defects in the migration of PGCs in the zebrafish embryo. However, these defects are not due to inhibition of Smoothened (Smo) by cyclopamine; rather, we find that neither maternal nor zygotic Smo is required for PGC migration in the zebrafish embryo. Cyclopamine instead acts independently of Smo to decrease the motility of zebrafish PGCs, in part by dysregulating cell adhesion and uncoupling cell polarization and translocation. These results demonstrate that Hh signaling is not required for zebrafish PGC migration, and underscore the importance of regulated cell-cell adhesion for cell migration in vivo. Author Affiliation: (a) Department of Biochemistry, Stanford University School of Medicine, Stanford, CA 94305, USA (b) Institute of Cell Biology, ZMBE, University of Munster, Von-Esmarch-Stra[beta]e 56, 48149 Munster, Germany (c) Developmental Genetics Program and Department of Cell Biology, Kimmel Center for Biology and Medicine at the Skirball Institute of Biomolecular Medicine, New York University School of Medicine, New York, NY 10016, USA (d) Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, CA 94305, USA Article History: Received 10 November 2008; Revised 27 January 2009; Accepted 27 January 2009

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2008.06.014 Byline: Wee-Chuang Low (a), Chengbing Wang (a), Yong Pan (a), Xin-Yun Huang (b), James K. Chen (d), Baolin Wang (a)(c) Keywords: Hedgehog; Smoothened; G protein; Gli3; Neural tube Abstract: The Hedgehog (Hh) signal is transmitted by two receptor molecules, Patched (Ptc) and Smoothened (Smo). Ptc suppresses Smo activity, while Hh binds Ptc and alleviates the suppression, which results in activation of Hh targets. Smo is a seven-transmembrane protein with a long carboxyl terminal tail. Vertebrate Smo has been previously shown to be coupled to G[alpha].sub.i proteins, but the biological significance of the coupling in Hh signal transduction is not clear. Here we show that although inhibition of G[alpha].sub.i protein activity appears to significantly reduce Hh pathway activity in Ptc.sup.-/- mouse embryonic fibroblasts and the NIH3T3-based Shh-light cells, it fails to derepress Shh- or a Smo-agonist-induced inhibition of Gli3 protein processing, a known in vivo indicator of Hh signaling activity. The inhibition of G[alpha].sub.i protein activity also cannot block the Sonic Hedgehog (Shh)-dependent specification of neural progenitor cells in the neural tube. Consistent with these results, overexpression of a constitutively active G[alpha].sub.i protein, G[alpha].sub.i2QL, cannot ectopically specify the neural cell types in the spinal cord, whereas an active Smo, SmoM2, can. Thus, our results indicate that the Smo-induced G[alpha].sub.i activity plays an insignificant role in the regulation of Gli3 processing and Shh-regulated neural tube patterning. Author Affiliation: (a) Department of Genetic Medicine, Weill Medical College of Cornell University, 1300 York Avenue, W404, New York, NY 10021, USA (b) Department of Physiology, Weill Medical College of Cornell University, 1300 York Avenue, W404, New York, NY 10021, USA (c) Department of Cell and Developmental Biology, Weill Medical College of Cornell University, 1300 York Avenue, W404, New York, NY 10021, USA (d) Department of Chemical and Systems Biology, Stanford University, Stanford, CA 94305, USA Article History: Received 24 October 2007; Revised 4 June 2008; Accepted 6 June 2008

Author(s): Jussi Taipale [1]; James K. Chen [1]; Michael K. Cooper [2, 1]; Baolin Wang [1]; Randall K. Mann [1]; Ljiljana Milenkovic [3]; Matthew P. Scott [3]; Philip A. Beachy [...]

The solution structure of phosphatidylinositol 3-kinase SH3 domain complexed to proline-rich ligand RKLPPRPSK was established. Protein-rich motifs having the left-handed type II polyproline helix conformation are recognized by SH3 domains.

This paper describes convenient preparations of heterogeneous multisectional polyacrylamide slab gels and the protocols that use these gels to measure protein--ligand binding constants [using bovine carbonic anhydrase B (CAB) as a model system]. Unlike procedures for affinity electrophoresis using tube gels, all binding information concerning protein--ligand interactions was encoded in a single multisectional gel: the procedure involving for measuring binding constants required no postelectrophoresis manipulation of gels. Use of these types of gels improves the accuracy of affinity gel electrophoresis (AGE) by providing reliable internal protein standards. Binding constants measured by AGE agree with those determined in homogeneous solution by spectrophotometric measurements. This technique has been used to investigate the influence of the length of the spacer separating the ligand and the polyacrylamide backbone on the binding constants. Dissociation constants obtained using the affinity gels approach the values measured in free solution, when the spacer is sufficiently long ([greater than or equal to]18 A); affinity ligands having short spacers give high apparent dissociation constants., The utilization for affinity drug design of affinity gel electrophoresis is discussed. This involves the measurement of protein-ligand binding constants by heterogenous multisectional polyacrylamide slab gels and protocols with bovine carbonic anhydrase B as a model system. It was found that utilization of these types of gels provide reliable internal protein standards that improve the accuracy of affinity gel electrophoresis.