Synthesis and elaboration of N-methylpyrrolidone as an acetamide fragment substitute in bromodomain inhibition

N-Methylpyrrolidone is a solvent molecule which has been shown to compete with acetyl-lysine-containing peptides for binding to bromodomains. From crystallographic studies, it has also been shown to closely mimic the acetamide binding motif in several bromodomains, but has not yet been directly pursued as a fragment in bromodomain inhibition. In this paper, we report the elaboration of N-methylpyrrolidone as a potential lead in fragment-based drug design. Firstly, N-methylpyrrolidone was functionalised to provide points for chemical elaboration. Then, the moiety was incorporated into analogues of the reported bromodomain inhibitor, Olinone. X-ray crystallography revealed that the modified analogues showed comparable binding affinity and structural mimicry to Olinone in the bromodomain binding site.     

Development of the post-natal growth plate requires intraflagellar transport proteins

In the post-natal growth plate, chondrocytes are arranged in columns parallel to the long axis of the bone. Chondrocytes divide perpendicular to this axis and then move into position one on top of another in a process called "rotation" that maintains columnar organization. Primary cilia are non-motile microtubule base appendages extending from the surface of almost all vertebrate cells. Primary cilia were described on chondrocytes almost 40 years ago but the function of these structures in cartilage biology is not known. Intraflagellar transport (IFT) is the process by which primary cilia are generated and maintained. This study tested the hypothesis that IFT plays an important role in post-natal skeletal development. Kif3a, a subunit of the Kinesin II motor complex, that is required for intraflagellar transport and the formation of cilia, was deleted in mouse chondrocytes via Col2a-Cre-mediated recombination. Disruption of IFT resulted in subsequent depletion of cilia and post-natal dwarfism due to premature loss of the growth plate likely a result of reduced proliferation and accelerated hypertrophic differentiation of chondrocytes. Cell shape and columnar orientation in the growth plate were also disrupted suggesting a defect in the process of rotation. Alterations in chondrocyte rotation were accompanied by disruption of the actin cytoskeleton and alterations in the localization of activated FAK to focal adhesion-like structures on chondrocytes. This is the first report indicating a role for IFT and primary cilia in the development of the post-natal growth plate. The results suggest a model in which IFT/cilia act to maintain the columnar organization of the growth plate via the process of chondrocyte rotation.     

Beyond the epithelium: cadherin function in fibrous connective tissues

In fibrous connective tissues, fibroblasts are organized into syncytia, cellular networks that enable matrix remodeling and that are interconnected by intercellular adherens junctions (AJs). The AJs of fibroblasts are mediated by N-cadherin, a broadly expressed classical cadherin that is critically involved in developmental processes, wound healing and several diseases of mesenchymal tissues. In contrast to E-cadherin-dependent junctions of epithelia, the formation of AJs in fibrous connective tissues is relatively uncharacterized. Work over the last several years has documented an expanding list of molecules which function to regulate N-cadherin mediated junctions such as: Fer, PTP1B, cortactin, calcium, gelsolin, PIP5KIgamma, PIP2, and the Rho family of GTPases. We present an overview on the regulation of N-cadherin-mediated junction formation that highlights recent molecular advances in the field and rationalizes the roles of N-cadherin in connective tissue function.     

Somatostatin induces rapid contraction of neuroendocrine cells

The peptide hormone somatostatin, as well as the somatostatin analog octreotide, induces rapid morphological changes in neuroendocrine cells. The effect can be detected in less than 2 min: retraction fibers are formed, cells round up and cell-cell contacts are broken. Somatostatin-dependent cell contraction is inhibited by Y-27632, indicating that this effect is dependent on Rho kinase. In BON1 cells, the somatostatin-induced inhibition of forskolin-induced secretion of chromogranin A is not blocked by Y-27632. It is therefore concluded that the inhibitory effect of somatostatin in forskolin-stimulated cells is not dependent on cell contraction.     

Variable actin dynamics requirement for the exit of different cargo from the trans-Golgi network

Efficient post-Golgi trafficking depends on microtubules, but actin filaments and actin-associated proteins are also postulated. Here we examined, by inverse fluorescence recovery after photobleaching, the role of actin dynamics in the exit from the TGN of fluorescent-tagged apical or basolateral and raft or non-raft-associated cargoes. Either the actin-stabilizing jasplakinolide or the actin-depolymerising latrunculin B variably but significantly inhibited post-Golgi traffic of non-raft associated apical p75NTR and basolateral VSV-G cargoes. The TGN-exit of the apical-destined VSV-G mutant was impaired only by latrunculin B. Strikingly, the raft-associated GPI-anchor protein was not affected by either actin toxin. Results indicate that actin dynamics participates in the TGN egress of both apical- and basolateral-targeted proteins but is not needed for apical raft-associated cargo.     

Cryo-EM structures of cardiac thin filaments reveal the 3D architecture of troponin

Troponin is an essential component of striated muscle and it regulates the sliding of actomyosin system in a calcium-dependent manner. Despite its importance, the structure of troponin has been elusive due to its high structural heterogeneity. In this study, we analyzed the 3D structures of murine cardiac thin filaments using a cryo-electron microscope equipped with a Volta phase plate (VPP). Contrast enhancement by a VPP enabled us to reconstruct the entire repeat of the thin filament. We determined the orientation of troponin relative to F-actin and tropomyosin, and characterized the interactions between troponin and tropomyosin. This study provides a structural basis for understanding the molecular mechanism of actomyosin system.     

Divide and conquer: cytokinesis in plant cells

Plant cells divide in two by constructing a new cell wall (cell plate) between daughter nuclei after mitosis. Golgi-derived vesicles are transported to the equator of a cytoskeletal structure called a phragmoplast, where they fuse together to form the cell plate. Orientation of new cell walls involves actindependent guidance of phragmoplasts and associated cell plates to cortical sites established prior to mitosis. Recent work has provided new insights into how actin filaments and other proteins in the phragmoplast and cell plate contribute to cytokinesis. Newly discovered mutations have identified a variety of genes required for cytokinesis or its spatial regulation.     

RLIP mediates downstream signalling from RalB to the actin cytoskeleton during Xenopus early development

The Ras protein activates at least three different pathways during early development. Two of them regulate mesodermal gene expression and the third is thought to participate in the control of actin cytoskeleton dynamics via the Ral protein. From a yeast two-hybrid screen of a Xenopus maternal cDNA library, we identified the Xenopus orthologue of the Ral interacting protein (RLIP, RIP1 or RalBP1), a putative effector of small G protein Ral. Previously, we observed that a constitutively activated form of Ral GTPase (XralB G23V) induced bleaching of the animal hemisphere and disruption of the cortical actin cytoskeleton. To demonstrate that RLIP is the effector of RalB in early development, we show that the artificial targeting of RLIP to the membrane induces a similar phenotype to that of activated RalB. We show that overexpression of the Ral binding domain (RalBD) of XRLIP, which binds to the effector site of Ral, acts in competition with the endogenous effector of Ral and protects against the destructive effect of XralB G23V on the actin cytoskeleton. In contrast, the XRLIP has a synergistic effect on the activated form of XralB, which is dependent on the RalBD of RLIP. We provide evidence for the involvement of RLIP by way of its RalBD on the dynamics of the actin cytoskeleton and propose that signalling from Ral to RLIP is required for gastrulation.     

Evolutionarily conserved modules in actin nucleation: lessons from Dictyostelium discoideum and plants

Summary. The actin cytoskeleton plays a central part in the dynamic organization of eukaryotic cell structure. Nucleation of actin filaments is a crucial step in the establishment of new cytoskeletal structures or modification of existing ones, providing abundant targets for regulatory processes. A substantial part of our understanding of actin nucleation derives from studies on yeast and metazoan cells. However, recent advances in structural and functional genome analysis in less traditional models, such as plants or Dictyostelium discoideum, provide an emerging picture of an evolutionarily conserved core of at least two actin nucleation mechanisms, one mediated by the Arp2/3 complex and the other one by the formin-based module. A considerable degree of conservation is found also in the systems controlling the balance between filamentous and globular actin (profilin, actin-depolymerizing factor/cofilin) and even in certain regulatory aspects, such as the involvement of Rho-related small GTPases. Identification of such conserved elements provides a prerequisite for the characterization of evolutionarily variable aspects of actin regulation which may be responsible for the rich morphological diversity of eukaryotic cells.Correspondence and reprints: Department of Plant Physiology, Faculty of Sciences, Charles University, Vininá 5, 128 44 Praha 2, Czech Republic.