The P2Y6 receptor signals through Gαq /Ca2+ /PKCα and Gα13 /ROCK pathways to drive the formation of membrane protrusions and dictate cell migration.

Cell migration is a ubiquitous process necessary to maintain and restore tissue functions. However, in cancer, cell migration leads to metastasis development and thus worsens the prognosis. Although the mechanism of cell migration is well understood, the identification of new targets modulating cell migration and deciphering their signaling events could lead to new therapies to restore tissue functions in diseases, such as inflammatory bowel disease, or to block metastatic development in different forms of cancer. Previous research has identified the G-protein-coupled P2Y6 receptor as an innovative target that could dictate cell migration under normal and pathological conditions. Surprisingly, there is little information on the cellular events triggered by activated P2Y6 during cell migration. Here, we demonstrated that P2Y6 activation stimulated A549 human lung cancer cells and Caco-2 colorectal cancer cell migration. Activated P2Y6 increased the number of filopodia and focal adhesions; two migratory structures required for cell migration. The generation of these structures involved Gαq /calcium/protein kinases C (PKC) and Gα13 /RHO-associated protein kinase-dependent pathways that dictate the formation of the migratory structures. These pathways led to the stabilization of the actin cytoskeleton through a PKC-dependent phosphorylation of cofilin. These results support the idea that the P2Y6 receptor represents a target of interest to modulate cell migration and revealed an intricate dialogue between two Gα-protein signaling pathways.

The expression of the P2Y6 receptor is regulated at the transcriptional level by p53.

Inflammatory bowel disease (IBD) is a risk factor for the development of colorectal cancer (CRC) for which mutation to p53 is an early event leading to dysplasia. Interestingly, P2RY6 mRNA increases in both pathologies. In this study, we investigated if p53 and p53R273H mutant, commonly found in CRC and IBD, were involved in the transcriptional regulation of P2RY6. First, the P2RY6 promoter was defined as a region corresponding to -1600 to +273 nucleotides relative to the putative TATA-less transcriptional starting site found at position 73,264,505 of NCBI reference sequence NC_000010.11. We cloned this promoter region along with 5'-deletion constructs in the pGL4.10[luc2] vector for luciferase assays to delineate the minimal promoter region. We observed that p53 wt and p53R273H differentially regulated the transcription of the P2RY6 gene. In fact, increasing quantity of p53R273H enhanced the capacity of p53 wt to stimulate the transactivation of the P2RY6 promoter but this cooperative effect was lost when p53R273H was present in a ratio of 3:1. In accordance with the luciferase assays, ChIP analysis revealed that endogenous p53 wt was significantly associated with the P2RY6 proximal promoter, whereas the interaction of the p53R273H with the P2RY6 promoter was not significant. Although further studies are required to fully elucidate the molecular determinant controlling P2Y6 expression in diseases, we propose, for the first time, a molecular mechanism involving a collaboration between p53 wt and p53R273H to regulate the expression of this receptor.

Assessing Transcriptional Responses to Light by the Dinoflagellate Symbiodinium.

The control of transcription is poorly understood in dinoflagellates, a group of protists whose permanently condensed chromosomes are formed without histones. Furthermore, while transcriptomes contain a number of proteins annotated as transcription factors, the majority of these are cold shock domain proteins which are also known to bind RNA, meaning the number of true transcription factors is unknown. Here we have assessed the transcriptional response to light in the photosynthetic species Symbiodinium kawagutii. We find that three genes previously reported to respond to light using qPCR do not show differential expression using northern blots or RNA-Seq. Interestingly, global transcript profiling by RNA-Seq at LD 0 (dawn) and LD 12 (dusk) found only seven light-regulated genes (FDR = 0.1). qPCR using three randomly selected genes out of the seven was only able to validate differential expression of two. We conclude that there is likely to be less light regulation of gene expression in dinoflagellates than previously thought and suggest that transcriptional responses to other stimuli should also be more thoroughly evaluated in this class of organisms.

miRNAs Do Not Regulate Circadian Protein Synthesis in the Dinoflagellate Lingulodinium polyedrum.

Dinoflagellates have been shown to express miRNA by bioinformatics and RNA blot (Northern) analyses. However, it is not yet known if miRNAs are able to alter gene expression in this class of organisms. We have assessed the possibility that miRNA may mediate circadian regulation of gene expression in the dinoflagellate Lingulodinium polyedrum using the Luciferin Binding Protein (LBP) as a specific example. LBP is a good candidate for regulation by miRNA since mRNA levels are constant over the daily cycle while protein synthesis is restricted by the circadian clock to a period of several hours at the start of the night phase. The transcriptome contains a potential DICER and an ARGONAUTE, suggesting the machinery for generating miRNAs is present. Furthermore, a probe directed against an abundant Symbiodinium miRNA cross reacts on Northern blots. However, L. polyedrum has no small RNAs detectable by ethidium bromide staining, even though higher plant miRNAs run in parallel are readily observed. Illumina sequencing of small RNAs showed that the majority of reads did not have a match in the L. polyedrum transcriptome, and those that did were almost all sense strand mRNA fragments. A direct search for 18-26 nucleotide long RNAs capable of forming duplexes with a 2 base 3' overhang detected 53 different potential miRNAs, none of which was able to target any of the known circadian regulated genes. Lastly, a microscopy-based test to assess synthesis of the naturally fluorescent LBP in single cells showed that neither double-stranded nor antisense lbp RNA introduced into cells by microparticle bombardment prior to the time of LBP synthesis were able to reduce the amount of LBP produced. Taken together, our results indicate that circadian control of protein synthesis in L. polyedrum is not mediated by miRNAs.

The main nitrate transporter of the dinoflagellate Lingulodinium polyedrum is constitutively expressed and not responsible for daily variations in nitrate uptake rates.

Dinoflagellates are unicellular eukaryotes capable of forming spectacular harmful algal blooms (HABs). Eutrophication of coastal waters by fertilizer runoff, nitrate in particular, has contributed to recent increases in the frequency, magnitude and geographic extent of HABs. Although physiological nitrate uptake and assimilation in dinoflagellates have often been measured in the field and in the laboratory, no molecular components involved in nitrate transport have yet been reported. This study reports the first identification and characterization of dinoflagellate nitrate transporters, found in the transcriptome of the bloom-forming Lingulodinium polyedrum. Of the 23 putative transporters found by BLAST searches, only members of the nitrate transporter 2 (NRT2) family contained all key amino acids known to be essential for nitrate transport. The dinoflagellate NRT2 sequences have 12 predicted transmembrane domains, as do the NRT2 sequences of bacteria, plants and fungi. The NRT2 sequences in Lingulodinium appear to have two different evolutionary origins, as determined by phylogenetic analyses. The most expressed transcript of all putative nitrate transporters was determined by RNA-Seq to be LpNRT2.1. An antibody raised against this transporter showed that the same amount of protein was found at different times over the light dark cycle and with different sources of N. Finally, global nitrate uptake was assessed using a 15N tracer, which showed that the process was not under circadian-control as previously suggested, but simply light-regulated.

The Symbiodinium kawagutii genome illuminates dinoflagellate gene expression and coral symbiosis.

Dinoflagellates are important components of marine ecosystems and essential coral symbionts, yet little is known about their genomes. We report here on the analysis of a high-quality assembly from the 1180-megabase genome of Symbiodinium kawagutii. We annotated protein-coding genes and identified Symbiodinium-specific gene families. No whole-genome duplication was observed, but instead we found active (retro)transposition and gene family expansion, especially in processes important for successful symbiosis with corals. We also documented genes potentially governing sexual reproduction and cyst formation, novel promoter elements, and a microRNA system potentially regulating gene expression in both symbiont and coral. We found biochemical complementarity between genomes of S. kawagutii and the anthozoan Acropora, indicative of host-symbiont coevolution, providing a resource for studying the molecular basis and evolution of coral symbiosis.

The Lingulodinium circadian system lacks rhythmic changes in transcript abundance.

BACKGROUND: Almost all cells display circadian rhythms, approximately 24-hour period changes in their biochemistry, physiology or behavior. These rhythms are orchestrated by an endogenous circadian clock whose mechanism is based on transcription-translation feedback loops (TTFL) where the translated products of clock genes act to inhibit their own transcription. RESULTS: We have used RNA-Seq to measure the abundance of all transcripts in an RNA-Seq-derived de novo gene catalog in two different experiments. One compared midday and midnight in a light-dark cycle (ZT6 and ZT18) and under constant light (CT6 and CT18). The second compared four different times (ZT2, ZT6, ZT14 and ZT18) under a light dark cycle. We show here that despite an elaborate repertoire of biological rhythms, the unicellular dinoflagellate Lingulodinium had no detectable daily variation in the abundance of any transcript in an RNA-Seq-derived de novo gene catalog. We also examined the timing of the bioluminescence and photosynthesis rhythms in the presence of the transcription inhibitors actinomycin D and cordycepin. We found that the timing of the two rhythms was unchanged even when transcription rates had decreased to roughly 5% the levels of untreated cells. CONCLUSIONS: The lack of detectable daily variation in transcript levels indicates that the endogenous circadian timer of Lingulodinium does not require rhythmic RNA. If the circadian timer is considered as a limit cycle oscillator, then cellular time in this organism must be defined by variations in state variables that do not include the amount of a clock gene transcript.

The dinoflagellate Lingulodinium polyedrum responds to N depletion by a polarized deposition of starch and lipid bodies.

Dinoflagellates are important contributors to the marine phytoplankton and global carbon fixation, but are also infamous for their ability to form the spectacular harmful algal blooms called red tides. While blooms are often associated with high available nitrogen, there are instances where they are observed in oligotrophic environments. In order to maintain their massive population in conditions of nitrogen limitation, dinoflagellates must have evolved efficient adaptive mechanisms. Here we report the physiological responses to nitrogen deprivation in Lingulodinium polyedrum. We find that this species reacts to nitrogen stress, as do most plants and microalgae, by stopping cell growth and diminishing levels of internal nitrogen, in particular in the form of protein and chlorophyll. Photosynthesis is maintained at high levels for roughly a week following nitrate depletion, resulting in accumulated photosynthetic products in the form of starch. During the second week, photosynthesis rates decrease due to a reduction in the number of chloroplasts and the accumulation of neutral lipid droplets. Surprisingly, the starch granules and lipid droplets are seen to accumulate at opposite poles of the cell. Lastly, we observe that cells acclimated to nitrogen-depleted conditions resume normal growth after addition of inorganic nitrogen, but are able to maintain high cell densities far longer than cells grown continuously in nitrogen-replete conditions.

Putting the N in dinoflagellates.

The cosmopolitan presence of dinoflagellates in aquatic habitats is now believed to be a direct consequence of the different trophic modes they have developed through evolution. While heterotrophs ingest food and photoautotrophs photosynthesize, mixotrophic species are able to use both strategies to harvest energy and nutrients. These different trophic modes are of particular importance when nitrogen nutrition is considered. Nitrogen is required for the synthesis of amino acids, nucleic acids, chlorophylls, and toxins, and thus changes in the concentrations of various nitrogenous compounds can strongly affect both primary and secondary metabolism. For example, high nitrogen concentration is correlated with rampant cell division resulting in the formation of the algal blooms commonly called red tides. Conversely, nitrogen starvation results in cell cycle arrest and induces a series of physiological, behavioral and transcriptomic modifications to ensure survival. This review will combine physiological, biochemical, and transcriptomic data to assess the mechanism and impact of nitrogen metabolism in dinoflagellates and to compare the dinoflagellate responses with those of diatoms.

Dinoflagellate tandem array gene transcripts are highly conserved and not polycistronic.

Dinoflagellates are an important component of the marine biota, but a large genome with high-copy number (up to 5,000) tandem gene arrays has made genomic sequencing problematic. More importantly, little is known about the expression and conservation of these unusual gene arrays. We assembled de novo a gene catalog of 74,655 contigs for the dinoflagellate Lingulodinium polyedrum from RNA-Seq (Illumina) reads. The catalog contains 93% of a Lingulodinium EST dataset deposited in GenBank and 94% of the enzymes in 16 primary metabolic KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways, indicating it is a good representation of the transcriptome. Analysis of the catalog shows a marked underrepresentation of DNA-binding proteins and DNA-binding domains compared with other algae. Despite this, we found no evidence to support the proposal of polycistronic transcription, including a marked underrepresentation of sequences corresponding to the intergenic spacers of two tandem array genes. We also have used RNA-Seq to assess the degree of sequence conservation in tandem array genes and found their transcripts to be highly conserved. Interestingly, some of the sequences in the catalog have only bacterial homologs and are potential candidates for horizontal gene transfer. These presumably were transferred as single-copy genes, and because they are now all GC-rich, any derived from AT-rich contexts must have experienced extensive mutation. Our study not only has provided the most complete dinoflagellate gene catalog known to date, it has also exploited RNA-Seq to address fundamental issues in basic transcription mechanisms and sequence conservation in these algae.

S-phase and M-phase timing are under independent circadian control in the dinoflagellate Lingulodinium.

In many phytoplankton species, cell division (mitosis) usually occurs at defined times of day. This timing is also observed under constant conditions, indicating that it is regulated by a circadian clock rather than by a simple response to the light-dark cycle. For those algae with cell cycles longer than a day, the clock opens a window of opportunity for mitosis at a particular time of day through which cells in an appropriate phase of the cell cycle can pass. Although the timing of mitosis is generally studied due to ease of measurement, for some phytoplankton the timing of S-phase is also circadian. This thus raises the possibility that mitosis is not directly gated by the clock but occurs instead at a defined interval (a constant G2 length) following a circadian controlled S-phase. To determine if the clock exercises independent control over the timing of both S- and M-phase, we measured the timing of both S- and M-phase in cultures of the dinoflagellate Lingulodinium grown under a variety of different photoperiods. We interpret the phase angles of both rhythms, in particular those resulting in a change in the length of G2, as an indication that the clock independently regulates the timing of S-phase and mitosis.