Emergent Self-Organized Criticality in Gene Expression Dynamics: Temporal Development of Global Phase Transition Revealed in a Cancer Cell Line

Background

The underlying mechanism of dynamic control of the genome-wide expression is a fundamental issue in bioscience. We addressed it in terms of phase transition by a systemic approach based on both density analysis and characteristics of temporal fluctuation for the time-course mRNA expression in differentiating MCF-7 breast cancer cells.

Methodology

In a recent work, we suggested criticality as an essential aspect of dynamic control of genome-wide gene expression. Criticality was evident by a unimodal-bimodal transition through flattened unimodal expression profile. The flatness on the transition suggests the existence of a critical transition at which up- and down-regulated expression is balanced. Mean field (averaging) behavior of mRNAs based on the temporal expression changes reveals a sandpile type of transition in the flattened profile. Furthermore, around the transition, a self-similar unimodal-bimodal transition of the whole expression occurs in the density profile of an ensemble of mRNA expression. These singular and scaling behaviors identify the transition as the expression phase transition driven by self-organized criticality (SOC).

Principal Findings

Emergent properties of SOC through a mean field approach are revealed: i) SOC, as a form of genomic phase transition, consolidates distinct critical states of expression, ii) Coupling of coherent stochastic oscillations between critical states on different time-scales gives rise to SOC, and iii) Specific gene clusters (barcode genes) ranging in size from kbp to Mbp reveal similar SOC to genome-wide mRNA expression and ON-OFF synchronization to critical states. This suggests that the cooperative gene regulation of topological genome sub-units is mediated by the coherent phase transitions of megadomain-scaled conformations between compact and swollen chromatin states.

Conclusion and Significance

In summary, our study provides not only a systemic method to demonstrate SOC in whole-genome expression, but also introduces novel, physically grounded concepts for a breakthrough in the study of biological regulation.     

Loss-of-function and gain-of-function mutations in FAB1A/B impair endomembrane homeostasis, conferring pleiotropic developmental abnormalities in Arabidopsis

In eukaryotic cells, PtdIns 3,5-kinase, Fab1/PIKfyve produces PtdIns (3,5) P(2) from PtdIns 3-P, and functions in vacuole/lysosome homeostasis. Herein, we show that expression of Arabidopsis (Arabidopsis thaliana) FAB1A/B in fission yeast (Schizosaccharomyces pombe) fab1 knockout cells fully complements the vacuole morphology phenotype. Subcellular localizations of FAB1A and FAB1B fused with green fluorescent protein revealed that FAB1A/B-green fluorescent proteins localize to the endosomes in root epidermal cells of Arabidopsis. Furthermore, reduction in the expression levels of FAB1A/B by RNA interference impairs vacuolar acidification and endocytosis. These results indicate that Arabidopsis FAB1A/B functions as PtdIns 3,5-kinase in plants and in fission yeast. Conditional knockdown mutant shows various phenotypes including root growth inhibition, hyposensitivity to exogenous auxin, and disturbance of root gravitropism. These phenotypes are observed also in the overproducing mutants of FAB1A and FAB1B. The overproducing mutants reveal additional morphological phenotypes including dwarfism, male-gametophyte sterility, and abnormal floral organs. Taken together, this evidence indicates that imbalanced expression of FAB1A/B impairs endomembrane homeostasis including endocytosis, vacuole formation, and vacuolar acidification, which causes pleiotropic developmental phenotypes mostly related to the auxin signaling in Arabidopsis.     

Coalescence of phospholipid membranes as a possible origin of anticoagulant effect of serum proteins

Interactions between phospholipid membranes (made of palmitoyloleoylphosphatidylcholine, cardiolipin and cholesterol) after addition of beta2 glycoprotein I (beta2GPI) or anti-beta2GPI antibodies or a mixture of both were studied by observing giant phospholipid vesicles under the phase contrast microscope. Both, negatively charged and neutral vesicles coalesced into complexes and adhered to the bottom of the observation chamber in the presence of beta2GPI in solution. Anti-beta2GPIs alone or previously mixed with beta2GPI caused coalescence of charged but not neutral vesicles, i.e. for neutral membranes the effect of beta2GPI was abolished by the presence of anti-beta2GPIs. Since the presence of the above adhesion mediators can prevent fragmentation of the membrane we propose a (new) possible anticoagulant mechanism for some serum proteins by preventing the release of prothrombogenic microexovesicles into circulation.     

Phosphatidylinositol 3-Phosphate 5-Kinase,FAB1/PIKfyve Kinase Mediates Endosome Maturation to Establish Endosome-Cortical Microtubule Interaction in Arabidopsis

Phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P₂] is an important lipid in membrane trafficking in animal and yeast systems; however, its role is still largely obscure in plants. Here, we demonstrate that the phosphatidylinositol 3-phosphate 5-kinase, formation of aploid and binucleate cells1 (FAB1)/FYVE finger-containing phosphoinositide kinase (PIKfyve), and its product, PtdIns(3,5)P₂, are essential for the maturation process of endosomes to mediate cortical microtubule association of endosomes, thereby controlling proper PIN-FORMED protein trafficking in young cortical and stele cells of root. We found that FAB1 predominantly localizes on the Sorting Nexin1 (SNX1)-residing late endosomes, and a loss of FAB1 function causes the release of late endosomal proteins, Ara7, and SNX1 from the endosome membrane, indicating that FAB1, or its product PtdIns(3,5)P₂, mediates the maturation process of the late endosomes. We also found that loss of FAB1 function causes the release of endosomes from cortical microtubules and disturbs proper cortical microtubule organization.Phosphoinositides play an important role in various cellular processes, including determination of organelle identity and mediating signal transduction by recruiting effector molecules to various organelles (Balla, 2013). Among those, D3-phosphorylated phosphoinositides, phosphatidylinositol 3-phosphate (PtdIns3P) and phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P₂], play essential roles in the endosomal trafficking and the vacuolar sorting. PtdIns3P is produced from phosphatidylinositol by class III PI3-kinase, vacuolar protein sorting34 (VPS34). In animal cells, PtdIns3P predominantly localizes to the early endosomes and controls endosome maturation, recycling, and degradation of cargo proteins coordinated with Rab5 GTPases (Jean and Kiger, 2012). In Arabidopsis (Arabidopsis thaliana), PtdIns3P mainly resides on the late endosomes and the prevacuolar membrane (Vermeer et al., 2006; Simon et al., 2014). Dysfunction of AtVPS34 resulted in a defect in growth (Welters et al., 1994), root hair elongation (Lee et al., 2008a), and pollen development (Lee et al., 2008b), indicating an important role for AtVPS34 and its product PtdIns3P in plant development. VPS34-mediated PtdIns3P synthesis at the endosomes recruits phosphatidylinositol 3-phosphate 5-kinase formation of aploid and binucleate cells1 (FAB1)/FYVE finger-containing phosphoinositide kinase (PIKfyve), then FAB1/PIKfyve produces PtdIns(3,5)P₂ from PtdIns3P to mediate late endosome maturation in yeast (Saccharomyces cerevisiae) and animals (Ho et al., 2012; Jean and Kiger, 2012). PtdIns(3,5)P₂ has crucial roles in the maintenance of lysosome/vacuole morphology and acidification, membrane trafficking of proteins, autophagy, and signaling mediation in response to various stresses (Shisheva, 2008).FAB1 was discovered in yeast, where mutations were found to result in the formation of aploid and binucleate cells (hence its name FAB). In addition, a loss of Fab1p function causes defects in vacuole function and morphology, cell surface integrity, and cell growth (Yamamoto et al., 1995). In mammalian cells, this kinase is called PIKfyve (FYVE is a PI3P-binding domain). FAB1/PIKfyve forms a protein complex with an adaptor-like protein, Vacuole14 (Bonangelino et al., 1997) and PtdIns(3,5)P₂ 5-phosphatase (Fig. 4; Gary et al., 2002), indicating that the FAB1 complex catalyzes both PtdIns(3,5)P₂ synthesis and turnover simultaneously. In mammalian cells, interference of FAB1/PIKfyve function causes severe defects during embryogenesis, resulting in embryonic lethality in Drosophila spp., Caenorhabditis elegans, and mice (Nicot et al., 2006; Rusten et al., 2006; Ikonomov et al., 2011; Takasuga et al., 2013). Whereas most genomes from human to yeast contain a single-copy gene, the Arabidopsis genome codes for four FAB1 genes (FAB1A–D), of which only FAB1A and FAB1B contain a FYVE domain (Mueller-Roeber and Pical, 2002), and fab1a/fab1b double mutant reveals male gametophyte lethality phenotype in Arabidopsis (Whitley et al., 2009). The mutant pollen shows severe defects in vacuolar reorganization following the first mitotic division of development, suggesting an important role of FAB1 and PtdIns(3,5)P₂ in vacuolar rearrangement for pollen development (Whitley et al., 2009).Open in a separate windowFigure 4.Localization of endosomal markers upon down-regulation of FAB1A/B or inhibition of PtdIns(3,5)P₂ synthesis in young root cortical cells. Localization of mRFP-SYP43, mRFP-vesicle-associated membrane protein (VAMP727), mRFP-ARA7, and SNX1-mRFP without estradiol (A, E, I, and M) or with estradiol (B, F, J, and N) in the FAB1A/B-amiRNA line, or wild-type (WT) plants without YM201636 (C, G, K, and O) or with YM201636 (D, H, L, and P). Bar = 10 μm. Measurement of fluorescent dot structures (Q). Data represent fluorescent dots per cell (mean ± sd). *, P < 0.001 (Student’s t test).We previously developed a transgenic Arabidopsis line that is able to conditionally down-regulate FAB1A and FAB1B expression simultaneously, and demonstrated that a loss of FAB1 function causes various abnormal phenotypes, including growth inhibition, hypersensitivity to exogenous auxin, disturbance of root gravitropism, and floral organ abnormalities (Hirano et al., 2011). In addition, we found that down-regulation of FAB1A/B expression impaired endomembrane homeostasis, including endocytosis, vacuole formation, and vacuolar acidification, likely causing pleiotropic developmental phenotypes that mostly related to the auxin signaling in Arabidopsis (Hirano et al., 2011; Hirano and Sato, 2011). In plants, auxin is a crucial phytohormone that has a wide variety of physiological roles associated with growth, development, and tropic responses (Zhao, 2010). The polar cell-to-cell transport of auxin is mediated by auxin transporters localized on the plasma membrane (PM), such as PIN-FORMED (PIN) proteins (Vieten et al., 2007; Feraru and Friml, 2008). PINs are used as model molecules for polarity establishment on the PM in Arabidopsis. The establishment of PIN polarity is accomplished by the recycling of PINs between the PM and endosomal compartments comprising the trans-Golgi network/early endosomes (TGN/EEs) and the late endosomes (LEs)/prevacuolar compartments. The PIN-recycling pathway is mediated by multiple endosomal regulatory proteins, such as Rab family GTPases and Sorting Nexin (SNX; Jaillais et al., 2006; Park and Jürgens, 2011).Rab proteins function as molecular switches to regulate the tethering and fusion step of transport vesicles to target membranes. Rab5 members of the Rab GTPases have various functions in the endocytic pathway in eukaryotes. The maturation of the early-to-late endosomes is regulated by Rab5-to-Rab7 conversion, which is regulated by the Mon1/Sand-1-Ccz1 complex (Nordmann et al., 2010; Poteryaev et al., 2010). In plants, Rab5-family proteins, Ara6 and Ara7, and Rha1 play important roles in Rab5-mediated endosomal trafficking including the vacuolar trafficking pathway, thereby regulating of the polar transport of auxin and responses to environmental conditions (Ebine et al., 2011; Inoue et al., 2013).SNXs are composed of two conserved domains: the PHOX domain, involved in the interaction with the phosphoinositides, PtdIns3P and PtdIns (3,5)P₂, in the endosomal membrane in animals (Cozier et al., 2002), and the BAR domain, mediating dimerization and binding to curved membranes (Peter et al., 2004). Loss of SNX function disrupts the stable association of the retromer subcomplex, VPS26-Vps29-Vps35, with endosomal membranes, and thus results in retromer dysfunction, indicating that SNXs have a crucial role in the assembly and maintenance of the core retromer function (Teasdale et al., 2001; Cullen and Korswagen, 2012). The first plant SNX was identified as a protein that interacts with various receptor kinases in Brassica oleracea (Vanoosthuyse et al., 2003), and then three SNX genes (SNX1, SNX2a, and SNX2b) were identified in Arabidopsis. The snx1 null mutant exhibits a semidwarf phenotype with other subtle developmental defects (Pourcher et al., 2010). SNX1 is localized to the late endosome and is involved in PIN2 recycling between endosomes and the PM (Jaillais et al., 2006). SNX1 has been reported to interact with cortical microtubules via the microtubule-associated protein Cytoplasmic Linker Associated Protein (CLASP), and the clasp1 null mutant displays aberrant SNX1 endosomes and enhanced PIN2 degradation in the lytic vacuoles, suggesting that an association of SNX1 endosomes and CLASP is important for recycling of PIN transporters (Ambrose et al., 2013).Although many analyses of FAB1/PIKfyve, Rab5 family GTPases, SNXs, and microtubles have been reported, and there are significant similarities in endosomal trafficking, a functional relationship between them is still largely obscure.In this study, we demonstrate that FAB1 produced PtdIns(3,5)P₂ in Arabidopsis, and knockdown of FAB1 expression or inhibition of FAB1 activity with a FAB1/PIKfyve inhibitor, YM201636, decreased PtdIns(3,5)P₂ content. We also found that FAB1 and its product PtdIns(3,5)P₂ mediate the late endosome maturation by recruiting endosomal effector molecules, Ara7 and SNX1, onto endosomes to establish endosome-cortical microtubule interaction. Subsequently, the basal polarity of PIN2 in young cortical cells and PIN1 in stele cells is achieved.     

Microwave-assisted Boc-solid phase peptide synthesis of cyclic cysteine-rich peptides.

In this study we describe the first protocols for the synthesis of cystine-rich peptides in the presence of microwave radiation with Boc-solid phase peptide synthesis (SPPS). This method is exemplified for macrocyclic peptides known as cyclotides, which comprise approximately 30 amino acids and incorporate a cystine knot arrangement of their three disulfide bonds. However, the method is broadly applicable for a wide range of peptides using Boc-SPPS, especially for SPPS of large peptides via native chemical ligation. Microwave radiation produces peptides in high yield and with high purity, and we were able to reduce the time for the assembly of approximately 30 mer peptide chains to an overnight reaction in the automated microwave-assisted synthesis.     

Distribution and morphological changes of the Golgi apparatus during Drosophila spermatogenesis

In spermatogenesis, the Golgi apparatus is important for the formation of the acrosome, which is a sperm‐specific organelle essential for fertilization. Comprehensive examinations of the spatiotemporal distribution and morphological characterizations of the Golgi in various cells during spermatogenesis are necessary for functional analyses and mutant screenings in the model eukaryote Drosophila. Here, we examined the distribution and morphology of the Golgi during Drosophila spermatogenesis with immunofluorescence and electron microscopy. In pre‐meiotic germ cells, the Golgi apparatuses were distributed evenly in the cytoplasm. In contrast, they were located exclusively in two regions near the poles during the meiotic metaphase, where they were segregated prior to the chromosomes. In cells in anaphase to telophase, the Golgi were predominantly left behind in the equatorial region between the separating daughter nuclei. After completion of meiosis, the dispersed Golgi were assembled at the apical side of the spermatid nucleus to form the acrosome. Further investigation of the Golgi distribution in β2‐tubulin mutants showed aberrant and uneven distributions of the Golgi among sister cells in the meiotic spermatocytes and in the post‐meiotic spermatids. At the ultrastructural level, the Golgi apparatus in pre‐meiotic spermatocytes comprised a pair of stacks. The two stacks were situated adjacent to each other, as if they had duplicated before entering into meiotic division. These results highlight the dynamic nature of the Golgi during spermatogenesis and provide a framework for analyzing the correlations between the dynamics of the Golgi and its function in sperm development.     

Staphylococcus aureus DsbA does not have a destabilizing disulfide. A new paradigm for bacterial oxidative folding

In Gram-negative bacteria, the introduction of disulfide bonds into folding proteins occurs in the periplasm and is catalyzed by donation of an energetically unstable disulfide from DsbA, which is subsequently re-oxidized through interaction with DsbB. Gram-positive bacteria lack a classic periplasm but nonetheless encode Dsb-like proteins. Staphylococcus aureus encodes just one Dsb protein, a DsbA, and no DsbB. Here we report the crystal structure of S. aureus DsbA (SaDsbA), which incorporates a thioredoxin fold with an inserted helical domain, like its Escherichia coli counterpart EcDsbA, but it lacks the characteristic hydrophobic patch and has a truncated binding groove near the active site. These findings suggest that SaDsbA has a different substrate specificity than EcDsbA. Thermodynamic studies indicate that the oxidized and reduced forms of SaDsbA are energetically equivalent, in contrast to the energetically unstable disulfide form of EcDsbA. Further, the partial complementation of EcDsbA by SaDsbA is independent of EcDsbB and biochemical assays show that SaDsbA does not interact with EcDsbB. The identical stabilities of oxidized and reduced SaDsbA may facilitate direct re-oxidation of the protein by extracellular oxidants, without the need for DsbB.     

Graph-representation of oxidative folding pathways

Background  

The process of oxidative folding combines the formation of native disulfide bond with conformational folding resulting in the native three-dimensional fold. Oxidative folding pathways can be described in terms of disulfide intermediate species (DIS) which can also be isolated and characterized. Each DIS corresponds to a family of folding states (conformations) that the given DIS can adopt in three dimensions.     

Effect of cell electroporation on the conductivity of a cell suspension

An increased permeability of a cell membrane during the application of high-voltage pulses results in increased transmembrane transport of molecules that otherwise cannot enter the cell. Increased permeability of a cell membrane is accompanied by increased membrane conductivity; thus, by measuring electric conductivity the extent of permeabilized tissue could be monitored in real time. In this article the effect of cell electroporation caused by high-voltage pulses on the conductivity of a cell suspension was studied by current-voltage measurements during and impedance measurement before and after the pulse application. At the same time the percentage of permeabilized and survived cells was determined and the extent of osmotic swelling measured. For a train of eight pulses a transient increase in conductivity of a cell suspension was obtained above permeabilization threshold in low- and high-conductive medium with complete relaxation in <1 s. Total conductivity changes and impedance measurements showed substantial changes in conductivity due to the ion efflux in low-conductive medium and colloid-osmotic swelling in both media. Our results show that by measuring electric conductivity during the pulses we can detect limit permeabilization threshold but not directly permeabilization level, whereas impedance measurements in seconds after the pulse application are not suitable.