S6 protein kinase activates Juvenile Hormone and vitellogenin production in the cockroach Blattella germanica

Nutritional status regulates different processes, such as growth and development, through TOR (target of rapamycin) and insulin receptor signalling pathways. The ribosomal S6 protein kinase (S6K) is a downstream element of both pathways. Using cDNA from the German cockroach Blattella germanica (L.), two S6K isoform sequences (BgS6K) are cloned. The long isoform differs from the short one by the insertion of a 22‐amino acid duplication, involving the key phosphorylation position Thr³⁹⁰. As a result of this, the long isoform presents a new, potentially regulatory phosphorylation site. RNA interference knockdown of both BgS6K isoforms induces an increase in the length of the last nymphal instar, together with a reduction in the mRNA levels of a number of enzymes of the Juvenile Hormone biosynthetic pathway in the corpora allata, vitellogenin mRNA levels in the fat body and basal oocyte length. Thus, BgS6K is important for nymphal development and is necessary for the full induction of Juvenile Hormone synthesis and vitellogenin production in adult females.     

Akt and RhoA inhibition promotes anoikis of aggregated B16F10 melanoma cells

In the highly metastatic B16F10 melanoma cell line, activation of the signalling molecules that promote cell proliferation and survival on conventional adhesive culture dishes may also be responsible for the growth and resistance to anoikis of aggregates on a non-adhesive substratum. We have examined the influence of bacterial ADP-ribosyltransferases C3-like exoenzymes, which selectively modify RhoA, B and C proteins and inhibit signal pathways controlled by them. RNA interference [siRNA (small interfering RNA) Akt (also known as protein kinase B)] and a PI3K (phosphoinositide 3-kinase) inhibitor were used to analyse the changes caused by inhibiting the PI3K/Akt pathway. Inhibiting the activation of RhoA, B, C and Akt expression resulted in a decrease of the number of cells cultured in aggregates, and caspase 3 activation. RhoA activation and RhoB and RhoC expression were controlled by Akt, but not RhoA expression. Inhibiting Akt and RhoA reduced the expression of α5 integrin, and inactivated FAK (focal adhesion kinase) in B16F10 cells cultured as aggregates. Thus, inhibiting Rho subfamily proteins and Akt expression inactivates the FAK pathway and induces anoikis in anoikis-resistant cells. The activation of RhoA in melanoma cells can depend on PI3K/Akt activation, suggesting that PI3K/Akt is a suitable target for new therapeutic approaches.     

Dissecting the roles of osmolyte accumulation during stress

Many plants accumulate organic osmolytes in response to the imposition of environmental stresses that cause cellular dehydration. Although an adaptive role for these compounds in mediating osmotic adjustment and protecting subcellular structure has become a central dogma in stress physiology, the evidence in favour of this hypothesis is largely correlative. Transgenic plants engineered to accumulate proline, mannitol, fructans, trehalose, glycine betaine or ononitol exhibit marginal improvements in salt and/or drought tolerance. While these studies do not dismiss causative relationships between osmolyte levels and stress tolerance, the absolute osmolyte concentrations in these plants are unlikely to mediate osmotic adjustment. Metabolic benefits of osmolyte accumulation may augment the classically accepted roles of these compounds. In re-assessing the functional significance of compatible solute accumulation, it is suggested that proline and glycine betaine synthesis may buffer cellular redox potential. Disturbances in hexose sensing in transgenic plants engineered to produce trehalose, fructans or mannitol may be an important contributory factor to the stress-tolerant phenotypes observed. Associated effects on photoassimilate allocation between root and shoot tissues may also be involved. Whether or not osmolyte transport between subcellular compartments or different organs represents a bottleneck that limits stress tolerance at the whole-plant level is presently unclear. None the less, if osmolyte metabolism impinges on hexose or redox signalling, then it may be important in long-range signal transmission throughout the plant.     

NaCl-induced changes in cytosolic free Ca2+ in Arabidopsis thaliana are heterogeneous and modified by external ionic composition

Increases in cytosolic free Ca²⁺ ([Ca²⁺]_cyt) are common to many stress-activated signalling pathways, including the response to saline environments. We have investigated the nature of NaCl-induced [Ca²⁺]_cyt signals in whole Arabidopsis thaliana seedlings using aequorin. We found that NaCl-induced increases in [Ca²⁺]_cyt are heterogeneous and mainly restricted to the root. Both the concentration of NaCl and the composition of the solution bathing the root have profound effects on the magnitude and dynamics of NaCl-induced increases in [Ca²⁺]_cyt. Alteration of external K⁺ concentration caused changes in the temporal and spatial pattern of [Ca²⁺]_cyt increase, providing evidence for Na⁺-induced Ca²⁺ influx across the plasma membrane. The effects of various pharmacological agents on NaCl-induced increases in [Ca²⁺]_cyt indicate that NaCl may induce influx of Ca²⁺ through both plasma membrane and intracellular Ca²⁺-permeable channels. Analysis of spatiotemporal [Ca²⁺]_cyt dynamics using photon-counting imaging revealed additional levels of complexity in the [Ca²⁺]_cyt signal that may reflect the oscillatory nature of NaCl-induced changes in single cells.     

Calcium signalling in early embryos

The onset of development in most species studied is triggered by one of the largest and longest calcium transients known to us. It is the most studied and best understood aspect of the calcium signals that accompany and control development. Its properties and mechanisms demonstrate what embryos are capable of and thus how the less-understood calcium signals later in development may be generated. The downstream targets of the fertilization calcium signal have also been identified, providing some pointers to the probable targets of calcium signals further on in the process of development.In one species or another, the fertilization calcium signal involves all the known calcium-releasing second messengers and many of the known calcium-signalling mechanisms. These calcium signals also usually take the form of a propagating calcium wave or waves. Fertilization causes the cell cycle to resume, and therefore fertilization signals are cell-cycle signals. In some early embryonic cell cycles, calcium signals also control the progress through each cell cycle, controlling mitosis.Studies of these early embryonic calcium-signalling mechanisms provide a background to the calcium-signalling events discussed in the articles in this issue.     

The interaction between urokinase receptor and vitronectin in cell adhesion and signalling

The extracellular matrix (ECM) is a complex structural entity surrounding and supporting cells present in all tissue and organs. Cell-matrix interactions play fundamental roles during embryonic development, morphogenesis, tissue homoeostasis, wound healing, and tumourigenesis. Cell-matrix communication is kept in balance by physical contact and by transmembrane integrin receptors providing the dynamic link between the extracellular and intracellular environments through bi-directional signalling. The urokinase-type plasminogen activator receptor (uPAR) is a plasma membrane receptor overexpressed during inflammation and in almost all human cancers. One of its functions is to endorse ECM remodelling through the activation of plasminogen and downstream proteases, including matrix-metalloproteases (MMPs). Beside its role in ECM degradation, uPAR modulates cell-matrix contact through a direct engagement with the ECM component, vitronectin (Vn), and by regulating the activity state of integrins thus promoting or inhibiting integrin signalling and integrin-mediated cell adhesion to other ECM components, like fibronectin and collagen. In this review we have centred our attention on the non-proteolytic function of uPAR as a mediator of cell adhesion and downstream signalling.     

Positive feedback in Cav‐1‐ROS signalling in PSCs mediates metabolic coupling between PSCs and tumour cells

Caveolin‐1 (Cav‐1) is the principal structural component of caveolae, and its dysregulation occurs in cancer. However, the role of Cav‐1 in pancreatic cancer (PDAC) tumorigenesis and metabolism is largely unknown. In this study, we aimed to investigate the effect of pancreatic stellate cell (PSC) Cav‐1 on PDAC metabolism and aggression. We found that Cav‐1 is expressed at low levels in PDAC stroma and that the loss of stromal Cav‐1 is associated with poor survival. In PSCs, knockdown of Cav‐1 promoted the production of reactive oxygen species (ROS), while ROS production further reduced the expression of Cav‐1. Positive feedback occurs in Cav‐1‐ROS signalling in PSCs, which promotes PDAC growth and induces stroma‐tumour metabolic coupling in PDAC. In PSCs, positive feedback in Cav‐1‐ROS signalling induced a shift in energy metabolism to glycolysis, with up‐regulated expression of glycolytic enzymes (hexokinase 2 (HK‐2), 6‐phosphofructokinase (PFKP) and pyruvate kinase isozyme type M2 (PKM2)) and transporter (Glut1) expression and down‐regulated expression of oxidative phosphorylation (OXPHOS) enzymes (translocase of outer mitochondrial membrane 20 (TOMM20) and NAD(P)H dehydrogenase [quinone] 1 (NQO1)). These events resulted in high levels of glycolysis products such as lactate, which was secreted by up‐regulated monocarboxylate transporter 4 (MCT4) in PSCs. Simultaneously, PDAC cells took up these glycolysis products (lactate) through up‐regulated MCT1 to undergo OXPHOS, with down‐regulated expression of glycolytic enzymes (HK‐2, PFKP and PKM2) and up‐regulated expression of OXPHOS enzymes (TOMM20 and NQO1). Interrupting the metabolic coupling between the stroma and tumour cells may be an effective method for tumour therapy.     

Tomato ethylene receptor-CTR interactions: visualization of NEVER-RIPE interactions with multiple CTRs at the endoplasmic reticulum

In the model plant Arabidopsis, members of a family of two-componentsystem His kinase-like ethylene receptors have direct protein–proteininteractions with a single downstream Ser/Thr kinase CTR1. Thesecomponents of the ethylene signalling network found in Arabidopsisare conserved in the climacteric fruit tomato, but both theethylene receptors and CTR1-like proteins (LeCTRs) in tomatoare encoded by multigene families. Here, using a yeast two-hybridinteraction assay, it is shown that the tomato receptors LeETR1,LeETR2, and NEVER-RIPE (NR) can interact with multiple LeCTRs.In vivo protein localization studies with fluorescent taggedproteins revealed that the ethylene receptor NR was targetedto the endoplasmic reticulum (ER) when transiently expressedin onion epidermal cells, whereas the four LeCTR proteins werefound in the cytoplasm and nucleus. When co-expressed with NR,three LeCTRs (1, 3, and 4), but not LeCTR2, also adopted thesame ER localization pattern in an NR receptor-dependent mannerbut not in the absence of NR. The receptor–CTR interactionswere confirmed by biomolecular fluorescence complementation(BiFC) showing that NR could form a protein complex with LeCTR1,3, and 4. This suggested that ethylene receptors recruit theseLeCTR proteins to the ER membrane through direct protein–proteininteraction. The receptor–CTR interactions and localizationobserved in the study reinforce the idea that ethylene receptorstransmit the signal to the downstream CTRs and show that a singlereceptor can interact with multiple CTR proteins. It remainsunclear whether the different LeCTRs are functionally redundantor have unique roles in ethylene signalling. Key words: BiFC, endoplasmic reticulum, Ser/Thr kinase, tomato ethylene signalling, two-component system His kinase     

A fluorescence-immunohistochemical study on phosphorylation of ERK1/2, p38, and STAT3 in rat dorsal root ganglia following noxious stimulation of hind paw sensory neurons

A fluorescence-immunohistochemical investigation was performed in lumbar dorsal root ganglia (DRGs) neurons of the rat with regard to ERK1/2-, p38- and STAT3-phosphorylation in response to nociceptor activation in the rat. The stimuli applied were perineural capsaicin treatment of the sciatic nerve, mustard oil application to the hind paw and heat or cold stimulation of the hind paw. The time points of investigations were 15 min/30 min after perineural capsaicin, 30 min/2 h/4 h for mustard oil, 10 min/4 h for cold and 30 min/2 h/8 h for the heat stimulus. All four stimuli lead to a time-dependent, significant 2-3 fold increase in the number of small and medium size DRG cells displaying cytoplasmic staining for p-ERK1/2, but to no activation of satellite cells. Phosphorylated p38 immunoreactivity was increased in the cytoplasma of DRG cells at 2 h after the mustard oil treatment of the hind paw and 30 min after the perineural capsaicin application to the sciatic nerve axons, but not following heat or cold stimuli to the hind paws. Phospho-STAT3 staining was characteristically observed as nuclear and cytoplasmic staining. It was found increased after the perineural capsaicin application to the sciatic nerve axons, however, no marked increase was found with the other 3 noxious stimuli. The present results show that sensory neurons respond with a selective long-lasting increase in p-ERK1/2 in small and medium-size DRG cells, when their axons or axon terminals are stimulated by capsaicin, mustard oil, noxious heat or noxious cold.     

Gamete signalling underlies the evolution of mating types and their number

The gametes of unicellular eukaryotes are morphologically identical, but are nonetheless divided into distinct mating types. The number of mating types varies enormously and can reach several thousand, yet most species have only two. Why do morphologically identical gametes need to be differentiated into self-incompatible mating types, and why is two the most common number of mating types? In this work, we explore a neglected hypothesis that there is a need for asymmetric signalling interactions between mating partners. Our review shows that isogamous gametes always interact asymmetrically throughout sex and argue that this asymmetry is favoured because it enhances the efficiency of the mating process. We further develop a simple mathematical model that allows us to study the evolution of the number of mating types based on the strength of signalling interactions between gametes. Novel mating types have an advantage as they are compatible with all others and rarely meet their own type. But if existing mating types coevolve to have strong mutual interactions, this restricts the spread of novel types. Similarly, coevolution is likely to drive out less attractive mating types. These countervailing forces specify the number of mating types that are evolutionarily stable.This article is part of the themed issue ‘Weird sex: the underappreciated diversity of sexual reproduction’.