In Planta Single-Molecule Pull-Down Reveals Tetrameric Stoichiometry of HD-ZIPIII:LITTLE ZIPPER Complexes

Deciphering complex biological processes markedly benefits from approaches that directly assess the underlying biomolecular interactions. Most commonly used approaches to monitor protein-protein interactions typically provide nonquantitative readouts that lack statistical power and do not yield information on the heterogeneity or stoichiometry of protein complexes. Single-molecule pull-down (SiMPull) uses single-molecule fluorescence detection to mitigate these disadvantages and can quantitatively interrogate interactions between proteins and other compounds, such as nucleic acids, small molecule ligands, and lipids. Here, we establish SiMPull in plants using the HOMEODOMAIN LEUCINE ZIPPER III (HD-ZIPIII) and LITTLE ZIPPER (ZPR) interaction as proof-of-principle. Colocalization analysis of fluorophore-tagged HD-ZIPIII and ZPR proteins provides strong statistical evidence of complex formation. In addition, we use SiMPull to directly quantify YFP and mCherry maturation probabilities, showing these differ substantially from values obtained in mammalian systems. Leveraging these probabilities, in conjunction with fluorophore photobleaching assays on over 2000 individual complexes, we determined HD-ZIPIII:ZPR stoichiometry. Intriguingly, these complexes appear as heterotetramers, comprising two HD-ZIPIII and two ZPR molecules, rather than heterodimers as described in the current model. This surprising result raises new questions about the regulation of these key developmental factors and is illustrative of the unique contribution SiMPull is poised to make to in planta protein interaction studies.     

Transfection of nonmuscle beta- and gamma-actin genes into myoblasts elicits different feedback regulatory responses from endogenous actin genes

We have examined the role of feedback-regulation in the expression of the nonmuscle actin genes. C2 mouse myoblasts were transfected with the human beta- and gamma-actin genes. In gamma-actin transfectants we found that the total actin mRNA and protein pools remained unchanged. Increasing levels of human gamma-actin expression resulted in a progressive down-regulation of mouse beta- and gamma-actin mRNAs. Transfection of the beta-actin gene resulted in an increase in the total actin mRNA and protein pools and induced an increase in the levels of mouse beta-actin mRNA. In contrast, transfection of a beta-actin gene carrying a single-point mutation (beta sm) produced a feedback-regulatory response similar to that of the gamma-actin gene. Expression of a beta-actin gene encoding an unstable actin protein had no impact on the endogenous mouse actin genes. This suggests that the nature of the encoded actin protein determines the feedback-regulatory response of the mouse genes. The role of the actin cytoskeleton in mediating this feedback-regulation was evaluated by disruption of the actin network with Cytochalasin D. We found that treatment with Cytochalasin D abolished the down-regulation of mouse gamma-actin in both the gamma- and beta sm-actin transfectants. In contrast, a similar level of increase was observed for the mouse beta-actin mRNA in both control and transfected cells. These experiments suggest that the down-regulation of mouse gamma-actin mRNA is dependent on the organization of the actin cytoskeleton. In addition, the mechanism responsible for the down-regulation of beta-actin may be distinct from that governing gamma-actin. We conclude that actin feedback-regulation provides a biochemical assay for differences between the two nonmuscle actin genes.     

A hormonal regulatory module that provides flexibility to tropic responses

Plants orient their growth depending on directional stimuli such as light and gravity, in a process known as tropic response. Tropisms result from asymmetrical accumulation of auxin across the responding organ relative to the direction of the stimulus, which causes differential growth rates on both sides of the organ. Here, we show that gibberellins (GAs) attenuate the gravitropic reorientation of stimulated hypocotyls of dark-grown Arabidopsis (Arabidopsis thaliana) seedlings. We show that the modulation occurs through induction of the expression of the negative regulator of auxin signaling INDOLE-3-ACETIC ACID INDUCIBLE19/MASSUGU2. The biological significance of this regulatory mechanism involving GAs and auxin seems to be the maintenance of a high degree of flexibility in tropic responses. This notion is further supported by observations that GA-deficient seedlings showed a much lower variance in the response to gravity compared to wild-type seedlings and that the attenuation of gravitropism by GAs resulted in an increased phototropic response. This suggests that the interplay between auxin and GAs may be particularly important for plant orientation under competing tropic stimuli.     

A conserved Pbx-Wnt-p63-Irf6 regulatory module controls face morphogenesis by promoting epithelial apoptosis

Morphogenesis of mammalian facial processes requires coordination of cellular proliferation, migration, and apoptosis to develop intricate features. Cleft lip and/or palate (CL/P), the most frequent human craniofacial birth defect, can be caused by perturbation of any of these programs. Mutations of?WNT, P63, and IRF6 yield CL/P in humans and mice; however, how these genes are regulated remains elusive. We generated mouse lines lacking Pbx genes in cephalic ectoderm and demonstrated that they exhibit fully penetrant CL/P and perturbed Wnt signaling. We also characterized a midfacial regulatory element that Pbx proteins bind to control the expression of Wnt9b-Wnt3, which in turn regulates p63. Altogether, we establish a Pbx-dependent Wnt-p63-Irf6 regulatory module in midfacial ectoderm that is conserved within mammals. Dysregulation of this network leads to localized suppression of midfacial apoptosis and CL/P. Ectopic Wnt ectodermal expression in Pbx mutants rescues the clefting, opening avenues for tissue repair.     

中国稀见植物——草茱萸

草茱萸(拟)Chamaepericlymenum canadadense(Linn.)Ashers.etGraebn.F1.Nard.Flachl.(1898)539.——Cornus canadensis Linn.Sp.P1. (1753)117,属山茱萸科(Cornaceae)的多年生草本,全株伏生白色丁字毛。根茎木质化,长而细弱,匍匐生长,多分枝。     

Modelling periodic oscillation in gene regulatory networks by cyclic feedback systems

In this paper, we develop a new methodology to analyze and design periodic oscillators of biological networks, in particular gene regulatory networks with multiple genes, proteins and time delays, by using negative cyclic feedback systems. We show that negative cyclic feedback networks have no stable equilibria but stable periodic orbits when certain conditions are satisfied. Specifically, we first prove the basic properties of the biological networks composed of cyclic feedback loops, and then extend our results to general cyclic feedback network with less restriction, thereby making our theoretical analysis and design of oscillators easy to implement, even for large-scale systems. Finally, we use one circadian network formed by a period protein (PER) and per mRNA, and one biologically plausible synthetic gene network, to demonstrate the theoretical results. Since there is less restriction on the network structure, the results of this paper can be expected to apply to a wide variety of areas on modelling, analyzing and designing of biological systems.     

Developmental regulatory genes and echinoderm evolution

Modified interactions among developmental regulatory genes and changes in their expression domains are likely to be an important part of the developmental basis for evolutionary changes in morphology. Although developmental regulatory genes are now being studied in an increasing number of taxa, there has been little attempt to analyze the resulting data within an explicit phylogenetic context. Here we present comparative analyses of expression data from regulatory genes in the phylum Echinodermata, considering the implications for understanding both echinoderm evolution as well as the evolution of regulatory genes in general. Reconstructing the independent evolutionary histories of regulatory genes, their expression domains, their developmental roles, and the structures in which they are expressed reveals a number of distinct evolutionary patterns. A few of these patterns correspond to interpretations common in the literature, whereas others have received little prior mention. Together, the analyses indicate that the evolution of echinoderms involved: (1) the appearance of many apomorphic developmental roles and expression domains, some of which have plesiomorphic bilateral symmetry and others of which have apomorphic radial symmetry or left-right asymmetry; (2) the loss of some developmental roles and expression domains thought to be plesiomorphic for Bilateria; and (3) the retention of some developmental roles thought to be plesiomorphic for Bilateria, although with modification in expression domains. Some of the modifications within the Echinodermata concern adult structures; others, transient larval structures. Some changes apparently appeared early in echinoderm evolution (> 450 Ma), whereas others probably happened more recently (< 50 Ma). Cases of likely convergence in expression domains suggest caution when using developmental regulatory genes to make inferences about homology among morphological structures of distantly related taxa.     

A feedback regulatory loop between methyltransferase PRMT1 and orphan receptor TR3

PRMT1, an arginine methyltransferase, plays an important role in numerous cellular processes. In this study, we demonstrate a feedback regulatory loop between PRMT1 and the orphan receptor TR3. Unlike another orphan receptor HNF4, TR3 is not methylated by PRMT1 although they physically interact with each other. By delaying the TR3 protein degradation, PRMT1 binding leads to the elevation of TR3 cellular protein level, thereby enhances the DNA binding and transactivation activity of TR3 in a non-methyltransferase manner. Another coactivator SRC-2 acts synergistically with PRMT1 to regulate TR3 functions. In turn, TR3 binding to the catalytic domain of PRMT1 causes an inhibition of the PRMT1 methyltransferase activity. This repression results in the functional changes in some of PRMT1 substrates, including STAT3 and Sam68. The negative regulation of PRMT1 by TR3 was further confirmed in both TR3-knockdown cells and TR3-knockout mice with the use of an agonist for TR3. Taken together, our study not only identifies a regulatory role of PRMT1, independent on methyltransferase activity, in TR3 transactivation, but also characterizes a novel function of TR3 in the repression of PRMT1 methyltransferase activity.     

A local regulatory T cell feedback circuit maintains immune homeostasis by pruning self-activated T cells

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