Integration of P2Y receptor-activated signal transduction pathways in G protein-dependent signalling networks

The role of nucleotides in intracellular energy provision and nucleic acid synthesis has been known for a long time. In the past decade, evidence has been presented that, in addition to these functions, nucleotides are also autocrine and paracrine messenger molecules that initiate and regulate a large number of biological processes. The actions of extracellular nucleotides are mediated by ionotropic P2X and metabotropic P2Y receptors, while hydrolysis by ecto-enzymes modulates the initial signal. An increasing number of studies have been performed to obtain information on the signal transduction pathways activated by nucleotide receptors. The development of specific and stable purinergic receptor agonists and antagonists with therapeutical potential largely contributed to the identification of receptors responsible for nucleotide-activated pathways. This article reviews the signal transduction pathways activated by P2Y receptors, the involved second messenger systems, GTPases and protein kinases, as well as recent findings concerning P2Y receptor signalling in C6 glioma cells. Besides vertical signal transduction, lateral cross-talks with pathways activated by other G protein-coupled receptors and growth factor receptors are discussed

Characterisation of carbonic anhydrase in the symbiotic dinoflagellate Symbiodinium

Photosynthesis by Symbiodinium plays a central role in the coral-algal symbiosis as the majority (around 95%) of the hosts' metabolic demand is derived from photosynthetically fixed carbon. Photosynthesis in Symbiodinium is augmented by the use of a carbon-concentrating mechansism (CCM), of which the enzyme carbonic anhydrase (CA) plays a significant role in the accumulation, transportation and interconversion of inorganic carbon (Ci) forms to ultimately provide CO₂ for the carbon-fixing enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). Environmental changes associated with climate change, such as ocean acidification and warming, represent key threats to coral reef ecosystems and are the major causes of the decline and deterioration of coral reefs worldwide and have prompted a major research focus on how climate related stressors affect coral-algal symbioses. Given the hosts' dependency on the symbionts ability to perform photosynthesis, how climate change will affect Symbiodinium photosynthesis is therefore an area that needs to be investigated. Current understanding of eukaryotic CCM expression is predominately derived from the green alga Chlamydomonas reinhardtii. While it has been demonstrated that Symbiodinium possess a CCM, the signals that trigger the expression of the CCM and subsequent genes involved have not been precisely defined in Symbiodinium. Therefore, the aims of this research were to use sequence tag data for Symbiodinium sp. Clade C3 to characterise the genes encoding CAs involved in the Symbiodinium CCM and to determine if Symbiodinium CAs were modified by external CO₂ concentrations as in other photosynthetic algae; to determine what the combined effects of elevated CO₂ and temperature were on Symbiodinium photosynthesis and CA expression; to examine varying light intensities on the regulation of CA; and to investigate possible long-term effects of CO₂ enrichment on the Symbiodinium transcriptome. To achieve these aims a sequencing project was performed. Bioinformatic analyses including analysis of conserved amino acid residues of CA sequences and homology of translated CA protein sequences with other known CA genes from algae and higher plants was performed. Phylogenetic comparison of the translated protein sequences of CAs with CA sequences from a variety of organisms was also undertaken. Quantitative PCR was used for RNA transcript analysis of the identified CAs, Rubisco and phosphoglycolate phosphatase (PGPase) under the various environmental stressors and Illumina RNA-seq was used to investigate long-term CO₂ effects on the Symbiodinium transcriptome. Two distinct β-CAs and one δ-CA protein were identified and characterised in this study. Both β-CAs were encoded as polyproteins and, were presumably localised to the cytosol while the δ-CA protein is likely localised to the plasma membrane. Phylogenetic analysis revealed that the dinoflagellate β-CAs form a novel group within this gene family, illustrating the diversity that exists within the β-CA class. Expression analysis of CAs in Symbiodinium sp. clade C1 under elevated CO₂ concentrations revealed that CAs are down-regulated by elevated CO₂ conditions as seen in other algae however, expression patterns differ between different phylotypes of Symbiodinium. Exposure to combined elevated CO₂ and temperature illustrated that thermal stress was the main driver of changes in both transcript levels and physiological parameters of Symbiodinium sp. clade F, while CO₂ concentrations relevant to current through to projected future levels of CO₂ had little significant effect overall. Transcript abundance of Symbiodinium CAs under varied light intensities was also examined. High-light environments caused both a decrease in Symbiodinium CA transcripts and photosynthetic efficiency. Lastly, the response of Symbiodinium clade F to long-term elevated CO₂ concentrations highlighted the transcriptome wide changes with elevated CO₂ significantly enhancing processes such as photosynthesis, energy and ATP metabolism and CA transcript abundance while processes such as transmembrane transport and protein phosphorylation were significantly downregulated. The information resulting from this research therefore provides a basis for future investigations into the role and functioning of CAs in Symbiodinium; a means to compare the expression of CAs between stress tolerant and susceptible Symbiodinium species; and a platform to understand how Symbiodinium photosynthesis and therefore the coral-algal symbiosis may be affected by future climate change conditions

Cholesterol Modulates the Rate and Mechanism of Acetylcholine Receptor Internalization

Stability of the nicotinic acetylcholine receptor (AChR) at the cell surface is key to the correct functioning of the cholinergic synapse. Cholesterol (Chol) is necessary for homeostasis of AChR levels at the plasmalemma and for ion translocation. Here we characterize the endocytic pathway followed by muscle-type AChR in Chol-depleted cells (Chol(−)). Under such conditions, the AChR is internalized by a ligand-, clathrin-, and dynamin-independent mechanism. Expression of a dominant negative form of the small GTPase Rac1, Rac1N17, abolishes receptor endocytosis. Unlike the endocytic pathway in control CHO cells (1), accelerated AChR internalization proceeds even upon disruption of the actin cytoskeleton. Under Chol(−) conditions, AChR internalization is furthermore found to require the activity of Arf6 and its effectors Rac1 and phospholipase D. The Arf6-dependent mechanism may constitute the default endocytic pathway followed by the AChR in the absence of external ligands, membrane Chol levels acting as a key homeostatic regulator of cell surface receptor levels