Insertion sequence elements in Cupriavidus metallidurans CH34: distribution and role in adaptation

Cupriavidus metallidurans CH34 is a β-proteobacterium well equipped to cope with harsh environmental conditions such as heavy metal pollution. The strain carries two megaplasmids specialized in the response to heavy metals and a considerable number of genomic islands, transposons and insertion sequence (IS) elements. The latter were characterized in detail in this study, which revealed nine new IS elements totaling to 21 distinct IS elements from 10 different IS families and reaching a total of 57 intact IS copies in CH34. Analysis of all fully sequenced bacterial genomes revealed that relatives of these IS elements were mostly found in the Burkholderiaceae family (β-proteobacteria) to which C. metallidurans belongs. Three IS elements were 100% conserved in other bacteria suggesting recent interaction and horizontal transfer between these strains. In addition, a number of these IS elements were associated with genomic islands, gene inactivation or rearrangements that alter the autotrophic growth capacities of CH34. The latter rearrangements gave the first molecular evidence for the mutator phenotype that is characteristic for various C. metallidurans strains. Furthermore, differential expression of some IS elements (or adjacent genes in the same strand orientation) was found under heavy metal stress, an environmental stress to which C. metallidurans CH34 is well adapted. These observations indicate that these IS elements play an active role in C. metallidurans CH34 lifestyle, including its metabolic potential and adaptation under selective pressure.     

The phosphatidylinositol transfer protein RdgBβ binds 14-3-3 via its unstructured C-terminus, whereas its lipid-binding domain interacts with the integral membrane protein ATRAP (angiotensin II type I receptor-associated protein)

PITPs [PI (phosphatidylinositol) transfer proteins] bind and transfer PI between intracellular membranes and participate in many cellular processes including signalling, lipid metabolism and membrane traffic. The largely uncharacterized PITP RdgBβ (PITPNC1; retinal degeneration type B β), contains a long C-terminal disordered region following its defining N-terminal PITP domain. In the present study we report that the C-terminus contains two tandem phosphorylated binding sites (Ser(274) and Ser(299)) for 14-3-3. The C-terminus also contains PEST sequences which are shielded by 14-3-3 binding. Like many proteins containing PEST sequences, the levels of RdgBβ are regulated by proteolysis. RdgBβ is degraded with a half-life of 4 h following ubiquitination via the proteasome. A mutant RdgBβ which is unable to bind 14-3-3 is degraded even faster with a half-life of 2 h. In vitro, RdgBβ is 100-fold less active than PITPα for PI transfer, and RdgBβ proteins (wild-type and a mutant that cannot bind 14-3-3) expressed in COS-7 cells or endogenous proteins from heart cytosol do not exhibit transfer activity. When cells are treated with PMA, the PITP domain of RdgBβ interacts with the integral membrane protein ATRAP (angiotensin II type I receptor-associated protein; also known as AGTRAP) causing membrane recruitment. We suggest that RdgBβ executes its function following recruitment to membranes via its PITP domain and the C-terminal end of the protein could regulate entry to the hydrophobic cavity.     

MK-STYX, a catalytically inactive phosphatase regulating mitochondrially dependent apoptosis

Evasion of apoptosis is a significant problem affecting an array of cancers. In order to identify novel regulators of apoptosis, we performed an RNA interference (RNAi) screen against all kinases and phosphatases in the human genome. We identified MK-STYX (STYXL1), a catalytically inactive phosphatase with homology to the mitogen-activated protein kinase (MAPK) phosphatases. Despite this homology, MK-STYX knockdown does not significantly regulate MAPK signaling in response to growth factors or apoptotic stimuli. Rather, RNAi-mediated knockdown of MK-STYX inhibits cells from undergoing apoptosis induced by cellular stressors activating mitochondrion-dependent apoptosis. This MK-STYX phenotype mimics the loss of Bax and Bak, two potent guardians of mitochondrial apoptotic potential. Similar to loss of both Bax and Bak, cells without MK-STYX expression are unable to release cytochrome c. Proapoptotic members of the BCL-2 family (Bax, Bid, and Bim) are unable to trigger cytochrome c release in MK-STYX-depleted cells, placing the apoptotic deficiency at the level of mitochondrial outer membrane permeabilization (MOMP). MK-STYX was found to localize to the mitochondria but is neither released from the mitochondria upon apoptotic stress nor proximal to the machinery currently known to control MOMP, indicating that MK-STYX regulates MOMP using a distinct mechanism.     

Entrainment ranges of forced phase oscillators

In the vertebrate spinal cord, a neural circuit called the central pattern generator produces the basic locomotory rhythm. Short and long distance intersegmental connections serve to maintain coordination along the length of the body. As a way of examining the influence of such connections, we consider a model of a chain of coupled phase oscillators in which one oscillator receives a periodic forcing stimulus. For a certain range of forcing frequencies, the chain will match the stimulus frequency, a phenomenon called entrainment. Motivated by recent experiments in lampreys, we derive analytical expressions for the range of forcing frequencies that entrain the chain, and how that range depends on the forcing location. For short intersegmental connections, in which an oscillator is connected only to its nearest neighbors, we describe two ways in which entrainment is lost: internally, in which oscillators within the chain no longer oscillate at the same frequency; and externally, in which the the chain no longer has the same frequency as the forcing. By analyzing chains in which every oscillator is connected to every other oscillator (i.e., all-to-all connections), we show that the presence of connections with lengths greater than one do not necessarily change the entrainment ranges based on the nearest–neighbor model. We derive a criterion for the ratio of connection strengths under which the connections of length greater than one do not change the entrainment ranges produced in the nearest–neighbor model, provided entrainment is lost externally. However, when this criterion holds, the range of entrained frequencies is a monotonic function of forcing location, unlike experimental results, in which entrainment ranges are larger near the middle of the chain than at the ends. Numerically, we show that similar non-monotonic entrainment ranges are possible if the ratio criterion does not hold, suggesting that in the lamprey central pattern generator, intersegmental connection strengths are not a simple function of the connection length.     

lines and bowl affect the specification of cyst stem cells and niche cells in the Drosophila testis

To function properly, tissue-specific stem cells must reside in a niche. The Drosophila testis niche is one of few niches studied in vivo. Here, a single niche, comprising ten hub cells, maintains both germline stem cells (GSC) and somatic stem cells (CySC). Here, we show that lines is an essential CySC factor. Surprisingly, lines-depleted CySCs adopted several characteristics of hub cells, including the recruitment of new CySCs. This led us to examine the developmental relationship between CySCs and hub cells. In contrast to a previous report, we did not observe significant conversion of steady-state CySC progeny to hub fate. However, we found that these two cell types derive from a common precursor pool during gonadogenesis. Furthermore, lines mutant embryos exhibited gonads containing excess hub cells, indicating that lines represses hub cell fate during gonadogenesis. In many tissues, lines acts antagonistically to bowl, and we found that this is true for hub specification, establishing bowl as a positively acting factor in the development of the testis niche.     

Mutations in the nucleotide binding pocket of MreB can alter cell curvature and polar morphology in Caulobacter

The maintenance of cell shape in Caulobacter crescentus requires the essential gene mreB, which encodes a member of the actin superfamily and the target of the antibiotic, A22. We isolated 35 unique A22-resistant Caulobacter strains with single amino acid substitutions near the nucleotide binding site of MreB. Mutations that alter cell curvature and mislocalize the intermediate filament crescentin cluster on the back surface of MreB's structure. Another subset have variable cell widths, with wide cell bodies and actively growing thin extensions of the cell poles that concentrate fluorescent MreB. We found that the extent to which MreB localization is perturbed is linearly correlated with the development of pointed cell poles and variable cell widths. Further, we find that a mutation to glycine of two conserved aspartic acid residues that are important for nucleotide hydrolysis in other members of the actin superfamily abolishes robust midcell recruitment of MreB but supports a normal rate of growth. These mutant strains provide novel insight into how MreB's protein structure, subcellular localization, and activity contribute to its function in bacterial cell shape.     

Kinematics of swimming in two burrowing anguilliform fishes

Anguilliform or eel-like fishes are typically bottom dwellers, some of which are specialized burrowers. Although specializations for burrowing are predicted to affect the kinematics of swimming, it remains unknown to what extent this is actually the case. Here we examine swimming kinematics and efficiency of two burrowing anguilliform species, Pisodonophis boro and Heteroconger hassi, with different degrees of specialization for burrowing. Our data suggest that differences in the swimming kinematics may indeed be related to the differences in burrowing specialization and style between both species. The resemblance between the swimming kinematics of P. boro and previously published data for Anguilla anguilla and Anguilla rostrata may be linked with the relatively limited burrowing specialization of P. boro and suggests an overall stereotypy in anguilliform forward-swimming patterns. The body of H. hassi, in contrast, is more specialized for tail-first burrowing and backward swimming bears a striking resemblance to the backward burrowing motions observed in this species. These motions differ significantly from backward swimming in Anguilla and in P. boro. The kinematics of forward swimming are, however, comparable across species. Thus, our data suggest that specializations for burrowing may affect swimming kinematics in anguilliform fishes, but also that forward swimming and burrowing are not necessarily incompatible. Future studies comparing the kinematics and mechanics of burrowing in these and other anguilliform fishes are needed to better understand how specializations for burrowing constrain backward swimming in H. hassi.     

Receptors for oreksins: structure, localisation and activation mechanisms

The review concerns morpho-functional characteristics of orexin receptors. The data on their structure, signal transduction pathways, biological effects of orexin receptors 1 or 2 depending on association with different G-proteins are presented. Localisation of orexin receptors in various CNS structures as well as in peripheral organs mediates regulation of different physiological functions by orexins. Low concentration of orexins in peripheral blood and orexin-containing cells in ganglions and internal organs suggests a possibility to activate orexin-sensitive cells distantly, paracrinely or autocrinely. The data on effects of selective or non-selective orexin receptor antagonists is analysed.     

Strigolactones interact with ethylene and auxin in regulating root-hair elongation in Arabidopsis

Strigolactones (SLs) or derivatives thereof have been identified as phytohormones, and shown to act as long-distance shoot-branching inhibitors. In Arabidopsis roots, SLs have been suggested to have a positive effect on root-hair (RH) elongation, mediated via the MAX2 F-box. Two other phytohormones, auxin and ethylene, have been shown to have positive effects on RH elongation. Hence, in the present work, Arabidopsis RH elongation was used as a bioassay to determine epistatic relations between SLs, auxin, and ethylene. Analysis of the effect of hormonal treatments on RH elongation in the wild type and hormone-signalling mutants suggested that SLs and ethylene regulate RH elongation via a common regulatory pathway, in which ethylene is epistatic to SLs, whereas the effect of SLs on RH elongation requires ethylene synthesis. SL signalling was not needed for the auxin response, whereas auxin signalling was not necessary, but enhanced RH response to SLs, suggesting that the SL and auxin hormonal pathways converge for regulation of RH elongation. The ethylene pathway requirement for the RH response to SLs suggests that ethylene forms a cross-talk junction between the SL and auxin pathways.