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Kinetic analysis of a signal-transduction pathway by time-resolved somatic complementation of mutants.

Sensory control of sporulation in Physarum polycephalum plasmodia is mediated by a branched signal-transduction pathway that integrates blue light, far-red light, heat shock and the starvation state. Mutants defective in the pathway were isolated and three phenotypes obtained: blue-blind, general-blind and light-independent sporulating. When plasmodia of the blue-blind mutant Blu1 were exposed to a pulse of blue light and subsequently fused to non-induced wild-type plasmodia, the resulting heterokaryons sporulated, indicating a functional blue- light photoreceptor in the mutant. When the general-blind mutant Nos1 was fused to a wild-type plasmodium which had been induced by light, sporulation of the heterokaryon was blocked. However, the dominant inhibition of sporulation by Nos1 was gradually lost with increasing time between induction by light and time of fusion, suggesting that Nos1 can be bypassed by the time-dependent formation of a downstream signal-transduction intermediate. Phenotype expression in constitutively sporulating (Cos) mutants depended on starvation. The Cos2 product was titrated by fusing mutant plasmodia of different sizes to wild-type plasmodia of constant size and analysing the sporulation probability of the resulting heterokaryon. The titration curve indicates that a small change in the amount of Cos2 product can cause sporulation. We conclude that somatic complementation analysis allows the time-resolved evaluation of the regulatory function of mutations in a signal-transduction pathway without prior cloning of the gene. This shortcut allows us to characterize many mutants quickly and to select those for molecular analysis that display a well-defined regulatory function.

Animals↗

The FAR1 locus encodes a novel nuclear protein specific to phytochrome A signaling.

The phytochrome family of photoreceptors has a well-defined role in regulating gene expression in response to informational light signals. Little is known, however, of the early steps of phytochrome signal transduction. Here we describe a new Arabidopsis mutant, far1 (far-red-impaired response), which has reduced responsiveness to continuous far-red light, but responds normally to other light wavelengths. This phenotype implies a specific requirement for FAR1 in phyA signal transduction. The far1 locus maps to the south arm of chromosome 4, and is not allelic to photomorphogenic loci identified previously. All five far1 alleles isolated have single nucleotide substitutions that introduce stop codons in a single ORF. The FAR1 gene encodes a protein with no significant sequence similarity to any proteins of known function. The FAR1 protein contains a predicted nuclear localization signal and is targeted to the nucleus in transient transfection assays. This result supports an emerging view that early steps in phytochrome signaling may be centered in the nucleus. The FAR1 gene defines a new multigene family, which consists of at least four genes in Arabidopsis. This observation raises the possibility of redundancy in the phyA-signaling pathway, which could account for the incomplete block of phyA signaling observed in the far1 mutant.

Alleles↗

Presence of ERK2 in rat retinal cells.

PURPOSE: Extracellular signal-regulated kinase 2 (ERK2) participates in the phosphorylation cascade that is activated in the an early intracellular response to various hormones and growth factors. We examined the expression and distribution of the ERK2 protein and mRNA in the rat retina before and after light exposure. METHODS: Rats were held on a 12 hr light/dark cycle and their retinas were removed and examined either just before or 2 or 30 min after light exposure. The tissue was processed for Western blotting to evaluate the presence of the protein for ERK2, and for in situ hybridization to evaluate the mRNA of ERK2. RESULTS: The Western blotting method showed a strong specific staining of a 42 kDa protein band in the retinal samples. This band corresponded to the expected size of p42 MAP kinase (ERK2). In situ hybridization histochemistry showed an intense localization of ERK2 mRNA in the outer nuclear layer (ONL), the inner nuclear layer (INL), and the ganglion cell layer (GCL) of the retina. The intensity and distribution of these signals did not differ among the animals, regardless of exposure to light. CONCLUSIONS: While ERK2 may be involved in the signal transduction system activated in retinal cells by light exposure, its precise role remains to be defined.

Animals↗

Retrograde plastid redox signals in the expression of nuclear genes for chloroplast proteins of Arabidopsis thaliana.

Excitation imbalances between photosystem I and II generate redox signals in the thylakoid membrane of higher plants which induce acclimatory changes in the structure of the photosynthetic apparatus. They affect the accumulation of reaction center and light-harvesting proteins as well as chlorophylls a and b. In Arabidopsis thaliana the re-adjustment of photosystem stoichiometry is mainly mediated by changes in the number of photosystem I complexes, which are accompanied by corresponding changes in transcripts for plastid reaction center genes. Because chloroplast protein complexes contain also many nuclear encoded components we analyzed the impact of such photosynthetic redox signals on nuclear genes. Light shift experiments combined with application of the electron transport inhibitor 3-(3',4'-dichlorophenyl)-1,1'-dimethyl urea have been performed to induce defined redox signals in the thylakoid membrane. Using DNA macroarrays we assessed the impact of such redox signals on the expression of nuclear genes for chloroplast proteins. In addition, studies on mutants with lesions in cytosolic photoreceptors or in chloroplast-to-nucleus communication indicate that the defective components in the mutants are not essential for the perception and/or transduction of light-induced redox signals. A stable redox state of glutathione suggest that neither glutathione itself nor reactive oxygen species are involved in the observed regulation events pointing to the thylakoid membrane as the main origin of the regulatory pathways. Our data indicate a distinct role of photosynthetic redox signals in the cellular network regulating plant gene expression. These redox signals appear to act independently and/or above of cytosolic photoreceptor or known chloroplast-to-nucleus communication avenues.

Acclimatization↗

Inositol 1,4,5-trisphosphate may mediate closure of K+ channels by light and darkness in Samanea saman motor cells.

Leaflet movements of Samanea saman (Jacq.) Merr. depend in part upon circadian-rhythmic, light-regulated K+ fluxes across the plasma membranes of extensor and flexor cells in opposing regions of the leaf-moving organ, the pulvinus. We previously showed that blue light appears to close open K+ channels in flexor protoplasts during the dark period (subjective night) (Kim et al., 1992, Plant Physiol 99; 1532-1539). In contrast, transfer to darkness apparently closes open K+ channels in extensor protoplasts during the light period (subjective day) (Kim et al., 1993, Science 260; 960-962). We now report that both these channel-closing stimuli increase inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] levels in the appropriate protoplasts. If extensor cells are given a pulse of red light followed by transfer to darkness, channels still apparently close (Kim et al., 1993) but changes in Ins(1,4,5)P3 levels are complex with an initial decrease under red light followed by accumulation. Neomycin, an inhibitor of polyphosphoinositide hydrolysis, inhibits both blue-light-induced Ins(1,4,5)P3 production and K(+)-channel closure in flexor protoplasts and both dark-induced Ins(1,4,5)P3 production and K+ channel closure in extensor protoplasts. The G-protein activator, mastoparan, mimics blue light and darkness in that it both increases Ins(1,4,5)P3 levels and closes K+ channels in the appropriate cell type at the appropriate time. These results indicate that phospholipase C-catalyzed hydrolysis of phosphoinositides, possibly activated by a G protein, is an early step in the signal-transduction pathway by which blue light and darkness close K+ channels in S. saman pulvinar cells.

Darkness↗

Hodgkin's lymphoma and CD30 signal transduction.

Advances in molecular biology have shed light on the biological basis of Hodgkin's lymphoma (HL). Knowledge of the biological basis has enabled us to understand that most Hodgkin and Reed-Sternberg (H-RS) cells are derived from germinal center B-cells and constitutive nuclear factor kappaB (NF-kappaB) activation is a common molecular feature. Molecular mechanisms responsible for constitutive NF-kappaB activation, Epstein Barr virus latent membrane protein 1, and defective IkappaBalpha and IkappaB kinase activation have been clarified in the past several years. A recent study revealed the biological link between 2 characteristic features of H-RS cells: CD30 overexpression and constitutive NF-kappaB activation. Ligand-independent signaling by overexpressed CD3O was shown to be a common mechanism that induced constitutive NF-kappaB activation in these cells. These results suggest the self-growth-promoting potential of H-RS cells and redefine the biology of HL composed of H-RS cells and lymphocytes.

Hodgkin Disease↗

An arrow for wingless to take-off.

The Wnt family of secreted glycoproteins is involved in the regulation of diverse developmental processes. The classical Wnt/beta-catenin pathway has been thoroughly investigated resulting in the identification of a plethora of components involved in the activation of beta-catenin target genes. Moreover, two additional Wnt-triggered pathways have been identified. These various signalling cascades require at least one component that confers signalling specificity. This function is fulfilled at least in part by the Wnt receptor Frizzled. The recent identification of a potential Frizzled co-receptor, an LDL-receptor-related-protein (LRP), sheds more light on Wnt-signal transduction specificity and promises more exciting revelations.

Animals↗

MLC3F transgene expression in iv mutant mice reveals the importance of left-right signalling pathways for the acquisition of left and right atrial but not ventricular compartment identity.

Abstract Transcriptional differences between left and right cardiac chambers are revealed by an nlacZ reporter transgene controlled by regulatory sequences of the MLC3F gene, which is expressed in the left ventricle (LV), atrioventricular canal (AVC), and right atrium (RA). To examine the role of left-right signalling in the acquisition of left and right chamber identity, we have investigated MLC3F transgene expression in iv mutant mice. iv/iv mice exhibit randomised direction of heart looping and an elevated frequency of associated laterality defects, including atrial isomerism. At fetal stages, 3F-nlacZ-2E transgene expression remains confined to the morphological LV, AVC, and RA in L-loop hearts, although these appear on the opposite side of the body. In cases of morphologically distinguishable right atrial appendage isomerism, both atrial appendages show strong transgene expression. Conversely, specimens with morphological left atrial appendage isomerism show only weak expression in both atrial appendages. The earliest left-right atrial differences in the expression of the 3F-nlacZ-2E transgene are observed at E8.5. DiI labelling experiments confirmed that transcriptional regionalisation of the 3F-nlacZ-2E transgene at this stage reflects future atrial chamber identity. In some iv/iv embryos at E8.5, the asymmetry of 3F-nlacZ-2E expression was lost, suggesting atrial isomerism at the transcriptional level prior to chamber formation. These data suggest that molecular specification of left and right atrial but not ventricular chambers is dependent on left-right axial cues.

Animals↗

Characterization of a Neurospora crassa photolyase-deficient mutant generated by repeat induced point mutation of the phr gene.

We produced a photolyase-deficient mutant by repeat induced point mutation using the Neurospora crassa photolyase gene cloned previously. This mutation identified a new gene, phr, which was mapped on the right arm of linkage group I by both RFLP mapping and conventional mapping. To investigate the relationship between photoreactivation and dark repair processes, especially excision repair, double mutants of phr with representative repair-defective mutants of different types were constructed and tested for UV sensitivity and photoreactivation. The results show that the phr mutation has no influence on dark repair. Tests with CPD and TC(6-4) photoproduct-specific antibodies demonstrated that the phr mutant is defective in CPD photolyase and confirmed that there is no TC(6-4) photolyase activity in N. crassa. Furthermore, N. crassa photolyase is not a blue light receptor in the signal transduction that induces carotenoid biosynthesis.

Blotting, Southern↗

Integration of photo- and chemosensory signaling pathways in Chlamydomonas.

The behavior of Chlamydomonas reinhardtii Dangeard is regulated by both light and chemical stimuli. Generation of a transmembrane photoreceptor current is the earliest so far resolved event in phototaxis of green flagellates. Tryptone rapidly inhibits the photoreceptor current in gametes of C. reinhardtii and induces their accumulation. The time-course, concentration dependence and induction during gametogenesis of these two processes coincide. On the other hand, tryptone causes a weak stimulation of the photoreceptor current in the absence of any behavioral responses in vegetative cells. This shows that the tryptone-induced inhibition of the photoreceptor current in C. reinhardtii is due to activation of a gamete-specific chemosensory system, and that integration of the photo- and chemosensory signals already occurs at the initial steps of the signaling pathways.

Animals↗

Accumulation response of chloroplasts induced by mechanical stimulation in bryophyte cells.

Chloroplast movement has been studied in many plants but mainly as a model system for light signaling. However, we recently showed that the avoidance response of chloroplasts is also induced by mechanical stimulation in fern protonemal cells. Here we report the discovery of a mechanically induced accumulation response of chloroplasts in bryophytes. When mechanical stimulation was directly applied with a capillary to a part of a cell, chloroplasts moved towards and accumulated at the pressed site within 30 min after the onset of stimulation in all species tested. The accumulation movement of chloroplasts was inhibited by Cremart but not by cytochalasin B in red-light-grown protonemata of Physcomitrella patens (Hedw.) B., S. & G. To determine the contribution of external Ca(2+) to the response, we examined the effects on the accumulation movement of gadolinium (Ga(3+)), an inhibitor of stretch-activated ion channels, and lanthanum (La(3+)), a potent inhibitor of calcium channels. Mechano-relocation of chloroplasts was abolished by these drugs, but no effects were observed on photo-relocation of chloroplasts, irrespective of light colors and intensity. These results suggest that influx of external Ca(2+) through the plasma membrane is essential for the early steps in signaling of mechano-relocation of chloroplasts whose motility system is dependent on microtubules.

Adiantum↗

Differentially expressed genes associated with dormancy or germination of Arabidopsis thaliana seeds.

Differential display analysis using dormant and non-dormant Arabidopsis thaliana (L.) Heynh seeds resulted in a set of genes that were associated with either dormancy or germination. Expression of the germination-associated genes AtRPL36B and AtRPL27B, encoding two ribosomal proteins, was undetectable in the dry seed, low in dormant seed, and high under conditions that allowed completion of germination. Expression of these genes was also found to be light-regulated and to correlate with germination speed. Expression of the dormancy-associated genes ATS2 and ATS4, encoding a caleosin-like protein and a protein similar to a low-temperature-induced protein respectively, was high in the dry seed and decreased during germination. Expression of ATS2 and ATS4 was high in primary and secondary dormant seed but low in after-ripened or chilled seed. The expression of both genes was also light-regulated, but no relationship with temperature-dependent germination speed was found.

Arabidopsis↗

Sensitivity of Halobacterium salinarium to attractant light stimuli does not change periodically.

Halobacterium salinarium swims alternately in both directions of its cell axis. The average time between two reversals of the swimming direction is modulated by light stimuli. It is a matter of dispute whether the sensitivity to attractant stimuli depends on the time of stimulation during an interval. This question is crucial for model descriptions of the system. I have confirmed constancy of responsiveness with cells adapted to constant conditions and have reconstructed contradicting results. These are shown to be based on inadequate experimental and evaluative methods. The assumption of self-sustained oscillations which modulate sensitivity can not be justified from the attractant response.

Chemotaxis↗

DELLA-mediated cotyledon expansion breaks coat-imposed seed dormancy.

Seed dormancy is a key adaptive trait in plants responsible for the soil seed bank. The long established hormone-balance theory describes the antagonistic roles of the dormancy promoting plant hormone abscisic acid (ABA), and the germination promoting hormone gibberellin (GA) in dormancy control. Light, temperature, and other dormancy-breaking signals function to modulate the synthesis and perception of these hormones in the seed. However, the way in which these hormones control dormancy in the imbibed seed remains unknown. Here, we show that the DELLA protein regulators of the GA response are required for dormancy and describe a model through which hormone signal integration and dormancy regulation is achieved. We demonstrate that cotyledon expansion precedes radicle emergence during Arabidopsis seed germination and that a striking correlation exists between final seedling cotyledon size and seed dormancy in the DELLA mutants. Furthermore, twelve previously characterized seed-dormancy mutants are also defective in the control of cotyledon size in a manner consistent with their effect on germination potential. We propose that DELLA-mediated, light-, temperature-, and hormone-responsive cotyledon expansion prior to radicle emergence overcomes dormancy imposed by the seed coat and underlies seed-dormancy control in Arabidopsis.

Abscisic Acid↗

The S8 serine, C1A cysteine and A1 aspartic protease families in Arabidopsis.

The Arabidopsis thaliana genome has over 550 protease sequences representing all five catalytic types: serine, cysteine, aspartic acid, metallo and threonine (MEROPS peptidase database, http://merops.sanger.ac.uk/), which probably reflect a wide variety of as yet unidentified functions performed by plant proteases. Recent indications that the 26S proteasome, a T1 family-threonine protease, is a regulator of light and hormone responsive signal transduction highlight the potential of proteases to participate in many aspects of plant growth and development. Recent discoveries that proteases are required for stomatal distribution, embryo development and disease resistance point to wider roles for four additional multigene families that include some of the most frequently studied (yet poorly understood) plant proteases: the subtilisin-like, serine proteases (family S8), the papain-like, cysteine proteases (family C1A), the pepsin-like, aspartic proteases (family A1) and the plant matrixin, metalloproteases (family M10A). In this report, 54 subtilisin-like, 30 papain-like and 59 pepsin-like proteases from Arabidopsis, are compared with S8, C1A and A1 proteases known from other plant species at the functional, phylogenetic and gene structure levels. Examples of structural conservation between S8, C1A and A1 genes from rice, barley, tomato and soybean and those from Arabidopsis are noted, indicating that some common, essential plant protease roles were established before the divergence of monocots and eudicots. Numerous examples of tandem duplications of protease genes and evidence for a variety of restricted expression patterns suggest that a high degree of specialization exists among proteases within each family. We propose that comprehensive analysis of the functions of these genes in Arabidopsis will firmly establish serine, cysteine and aspartic proteases as regulators and effectors of a wide range of plant processes.

Amino Acid Sequence↗

Regulation of vertebrate myotome development by the p38 MAP kinase-MEF2 signaling pathway.

Biochemical and cell culture studies have characterized the myocyte enhancer factor 2 (MEF2) transcriptional regulatory proteins as obligatory partners for the myogenic regulatory factors (MRFs) in the differentiation of myogenic cells in culture. However, the role of MEF2 activation in somitic myogenesis has not been fully characterized. Here, we report a critical interaction between the p38 mitogen-activated protein kinase (p38 MAPK) and MEF2 in the developing somite myotome. We document expression of MEF2A and p38 MAPK proteins in the somite of 9.5 dpc mouse embryos concurrent with Myf 5 protein expression. We also observed that abrogation of p38 MAPK signaling blocks MEF2 activation using a MEF2 transgenic 'sensor' mouse. Inhibition of p38 MAPK signaling concurrently inhibited myogenic differentiation in somite cultures and in embryos in vivo using transplacental injection of a p38 inhibitor (SB203580). Finally, we document that commitment to the myogenic lineage is not appreciably affected by p38 MAPK inhibition since the activation of an early marker of myogenic commitment (Myf 5) occurs normally when p38 MAPK signaling is inhibited. Thus, we present novel evidence indicating a crucial role for p38 MAPK signaling to the MEF2 transcriptional regulators during early mammalian somite development and myotome formation.

Animals↗

Signaling networks in the plant circadian system.

Significant advances have been made during the past year in the genetic and molecular dissection of the plant circadian system. Several proteins involved in circadian clock regulation have been identified and the way that their interactions contribute to temporal organization is starting to emerge. In addition, genomic approaches have identified hundreds of genes under clock control, providing a molecular basis to our understanding of how the clock coordinates plant physiology and development with daily and seasonal environmental cycles.

Arabidopsis↗

Putative blue-light photoreceptors from Arabidopsis thaliana and Sinapis alba with a high degree of sequence homology to DNA photolyase contain the two photolyase cofactors but lack DNA repair activity.

The putative blue-light photoreceptor genes of Arabidopsis thaliana and Sinapis alba (mustard) are highly homologous to the DNA repair genes encoding DNA photolyases. The photoreceptors from both organisms were overexpressed in Escherichia coli, purified, and characterized. The photoreceptors contain two chromophores which were identified as flavin adenine dinucleotide and methenyltetrahydrofolate. This chromophore composition suggests that the blue light photoreceptor may initiate signal transduction by a novel pathway which involves electron transfer. Despite the high degree of sequence identity to and identical chromophore composition with photolyases, neither photoreceptor has any photoreactivating activity.

Arabidopsis↗