A post-genomic approach to understanding sphingolipid metabolism in Arabidopsis thaliana

AIMS: To highlight the importance of sphingolipids and their metabolites in plant biology. SCOPE: The completion of the arabidopsis genome provides a platform for the identification and functional characterization of genes involved in sphingolipid biosynthesis. Using the yeast Saccharomyces cerevisiae as an experimental model, this review annotates arabidopsis open reading frames likely to be involved in sphingolipid metabolism. A number of these open reading frames have already been subject to functional characterization, though the majority still awaits investigation. Plant-specific aspects of sphingolipid biology (such as enhanced long chain base heterogeneity) are considered in the context of the emerging roles for these lipids in plant form and function. CONCLUSIONS: Arabidopsis provides an excellent genetic and post-genomic model for the characterization of the roles of sphingolipids in higher plants.     

Ca2+ signaling, intracellular pH and cell volume in cell proliferation

Mitogens control progression through the cell cycle in non-transformed cells by complex cascades of intracellular messengers, such as Ca²⁺ and protons, and by cell volume changes. Intracellular Ca²⁺ and proton concentrations are critical for linking external stimuli to proliferation, motility, apoptosis and differentiation. This review summarizes the role in cell proliferation of calcium release from intracellular stores and the Ca²⁺ entry through plasma membrane Ca²⁺ channels. In addition, the impact of intracellular pH and cell volume on cell proliferation is discussed.     

Purified Wnt-5a increases differentiation of midbrain dopaminergic cells and dishevelled phosphorylation

The Wnt family of lipoproteins regulates several aspects of the development of the nervous system. Recently, we reported that Wnt-3a enhances the proliferation of midbrain dopaminergic precursors and that Wnt-5a promotes their differentiation into dopaminergic neurones. Here we report the purification of hemagglutinin-tagged Wnt-5a using a three-step purification method similar to that previously described for Wnt-3a. Haemagglutinin-tagged Wnt-5a was biologically active and induced the differentiation of immature primary midbrain precursors into tyrosine hydroxylase-positive dopaminergic neurones. Using a substantia nigra-derived dopaminergic cell line (SN4741), we found that Wnt-5a, unlike Wnt-3a, did not promote beta-catenin phosphorylation or stabilization. However, both Wnt-5a and Wnt-3a activated dishevelled, as assessed by a phosphorylation-dependent mobility shift. Moreover, the activity of Wnt-5a on dishevelled was blocked by pre-treatment with acyl protein thioesterase-1, indicating that palmitoylation of Wnt-5a is necessary for its function. Thus, our results suggest that Wnt-3a and Wnt-5a, respectively, activate canonical and non-canonical Wnt signalling pathways in ventral midbrain dopaminergic cells. Furthermore, we identify dishevelled as a key player in transducing both Wnt canonical and non-canonical signals in dopaminergic cells.     

ATP release via anion channels

ATP serves not only as an energy source for all cell types but as an ‘extracellular messenger’ for autocrine and paracrine signalling. It is released from the cell via several different purinergic signal efflux pathways. ATP and its Mg²⁺ and/or H⁺ salts exist in anionic forms at physiological pH and may exit cells via some anion channel if the pore physically permits this. In this review we survey experimental data providing evidence for and against the release of ATP through anion channels. CFTR has long been considered a probable pathway for ATP release in airway epithelium and other types of cells expressing this protein, although non-CFTR ATP currents have also been observed. Volume-sensitive outwardly rectifying (VSOR) chloride channels are found in virtually all cell types and can physically accommodate or even permeate ATP⁴⁻ in certain experimental conditions. However, pharmacological studies are controversial and argue against the actual involvement of the VSOR channel in significant release of ATP. A large-conductance anion channel whose open probability exhibits a bell-shaped voltage dependence is also ubiquitously expressed and represents a putative pathway for ATP release. This channel, called a maxi-anion channel, has a wide nanoscopic pore suitable for nucleotide transport and possesses an ATP-binding site in the middle of the pore lumen to facilitate the passage of the nucleotide. The maxi-anion channel conducts ATP and displays a pharmacological profile similar to that of ATP release in response to osmotic, ischemic, hypoxic and salt stresses. The relation of some other channels and transporters to the regulated release of ATP is also discussed.     

Expression of scaffolding, signalling and contractile-filament proteins in human myometria: effects of pregnancy and labour

Successful parturition requires the co-ordination of numerous myometrial signalling events to allow for timely and efficient uterine contractions. Late pregnancy and labour onset in humans may be associated with changes in the expression of myometrial proteins implicated in such uterine contractile signal integration. Accordingly, in myometria from non-pregnant women and pregnant women, not in labour or in labour, we examined the content of putative plasmalemmal scaffolding proteins (caveolin-1 and -2) and compared these to the proportions of signal transducing rho-associated kinases (ROKalpha and beta) and contractile filament-associated proteins alpha-actin, myosin regulatory light chain (MLC(20)) and h-caldesmon. There was no effect of pregnancy or labour on the proportion of caveolin, ROK betaor alpha-actin. However, pregnancy was associated with a decrease in ROKalpha and MLC(20) such that ROK alpha: alpha-actin and MLC(20): alpha-actin ratios were reduced compared to myometria of non-pregnant women. In contrast, h-caldesmon was up-regulated in pregnancy resulting in an elevated h-caldesmon: alpha-actin ratio. There were, however, no further significant changes in ROK alpha, MLC(20) or h-caldesmon expression with spontaneous or oxytocin-induced labour. These data suggest that the mechanism(s) integrating myometrial signalling events with the onset of human labour does not involve differential alterations of the cellular expressions of caveolins, ROK, alpha-actin, MLC(20) or h-caldesmon.     

Negative feedback via RSK modulates Erk‐dependent progression from naïve pluripotency

Mitogen‐activated protein kinase (MAPK)/extracellular signal‐regulated kinase (ERK) signalling is implicated in initiation of embryonic stem (ES) cell differentiation. The pathway is subject to complex feedback regulation. Here, we examined the ERK‐responsive phosphoproteome in ES cells and identified the negative regulator RSK1 as a prominent target. We used CRISPR/Cas9 to create combinatorial mutations in RSK family genes. Genotypes that included homozygous null mutations in Rps6ka1, encoding RSK1, resulted in elevated ERK phosphorylation. These RSK‐depleted ES cells exhibit altered kinetics of transition into differentiation, with accelerated downregulation of naïve pluripotency factors, precocious expression of transitional epiblast markers and early onset of lineage specification. We further show that chemical inhibition of RSK increases ERK phosphorylation and expedites ES cell transition without compromising multilineage potential. These findings demonstrate that the ERK activation profile influences the dynamics of pluripotency progression and highlight the role of signalling feedback in temporal control of cell state transitions.