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.     

Daily changes in the phosphoproteome of the dinoflagellate Lingulodinium

The dinoflagellate Lingulodinium has a large number of daily rhythms, many of which have no biochemical correlates. We examined the possibility that changes in protein phosphorylation may mediate some of the rhythmic changes by comparing proteins prepared from midday (LD6) and midnight (LD18) cultures. We used two different methods, one a 2D gel protocol in which phosphoproteins were identified after staining with ProQ Diamond, and the other an LC-MS/MS identification of tryptic phosphopeptides that had been purified by TiO(2) chromatography. Two differentially phosphorylated proteins, a light harvesting complex protein and Rad24, were identified using the 2D gel protocol. Six differentially phosphorylated proteins, a polyketide synthase, an uncharacterized transporter, a LIM (actin binding) domain and three RNA binding domain proteins, were identified using the phosphopeptide enrichment protocol. We conclude that changes in protein phosphorylation may underlie some of the rhythmic behavior of Lingulodinium.     

Human histone genes are interspersed with members of the Alu family and with other transcribed sequences

We have isolated a series of recombinant λCh4A phages containing human histone genes. Histone H2A, H2B, H3 and H4 genes have been found to be clustered, but are not present in any simple repeat pattern. Hybridization of a blot containing phage DNA with S phase polysomal cDNA indicates the presence of additional sequences complementary to HeLa polysomal RNA sequences. Northern blot analysis using these clones as probes has also shown the presence of sequences complementary to non-histone-coding RNAs, some of which accumulate differentially in different stages of the cell cycle. We have also found, by hybridization with appropriate probes, that histone genes are interspersed with several copies of the Alu DNA family; however, not all of the histone genes are associated with an Alu DNA sequence.     

Circadian photosynthetic reductant flow in the dinoflagellate Lingulodinium is limited by carbon availability

Circadian rhythms are the observed outputs of endogenous daily clocks and are thought to provide a selective advantage to cells adapted to daily light/dark cycles. However, the biochemical links between the clock and the overt rhythms in cell physiology are generally not known. Here, we examine the circadian rhythm in O₂ evolution by cultures of the dinoflagellate Lingulodinium, a rhythm previously ascribed to rhythmic electron flow through photosystem II. We find that O₂ evolution rates increase when CO₂ concentrations are increased, either following addition of DIC or a rapid decrease in culture pH. In medium containing only nitrate as an electron acceptor, O₂ evolution rates mirror the circadian rhythm of nitrate reductase activity in the cells. Furthermore, competition between photosynthetic electron flow to carbon and to nitrate varies in its relative efficiency through the day–night cycle. We also find, using simultaneous and continuous monitoring of pH and O₂ evolution rates over several days, that while culture pH is normally rhythmic, circadian changes in rates of O₂ evolution depend not on the external pH but on levels of internal electron acceptors. We propose that the photosynthetic electron transport rhythm in Lingulodinium is driven by the availability of a reductant sink.     

Hydrogen peroxide and nitric oxide trigger redox-related cyst formation in cultures of the dinoflagellate Lingulodinium polyedrum

The dinoflagellate Lingulodinium polyedrum is a toxin producer that shows the ability of turning to resting cysts as a survival strategy when exposed to environmental unfavorable conditions, such as nitrogen and phosphorus depletion, abrupt changes in temperature or light, and chemical or mechanical stress. Algal adaptation to all these conditions involves hydrogen peroxide (H₂O₂) and nitric oxide (NO) as key redox signals for housekeeping cellular processes. Thus, we aim here to shed light on the role of H₂O₂ and NO (from aqueous decomposition of sodium nitroprusside, SNP) as prooxidant agents and putative redox signals for encystment of the dinoflagellate L. polyedrum. Harsh oxidative stress imposed by 500 μM H₂O₂ treatment forced L. polyedrum cells to rapidly encyst, in less than 30 min, whereas slower cyst formation was observed upon lower H₂O₂ doses. L. polyedrum encystment was marked by a significant increase in the antioxidant carotenoid peridinin, although other photosynthetic pigments (chlorophyll a and β-carotene) and light-harvesting complexes (peridinin complex protein, PCP) were all diminished in cyst forms. Although SOD activity (a frontline antioxidant enzyme) was severely inhibited by increasing doses of H₂O₂, a theoretical compensatory effect was provided by the dose-dependent increase of ascorbate peroxidase activity (APX), which resulted in significant lower levels of lipid peroxidation during cyst formation. Although SNP data cannot be fully compared to those found with H₂O₂ treatments, changes in APX activity and in biomarkers of lipid and protein oxidation matched the dose–responses found in H₂O₂ experiments, revealing similar biochemical and morphological responses against increasing oxidative conditions during cyst formation. Our data significantly contribute to a better understanding of the relationship between encystment, photosynthesis, and antioxidant responses triggered by H₂O₂ and NO in L. polyedrum, a harmful diarrhetic shellfish poisoning toxin (DSPs) producer.     

Actively transcribed GAL genes can be physically linked to the nuclear pore by the SAGA chromatin modifying complex

Recent work has demonstrated that some actively transcribed genes closely associate with nuclear pore complexes (NPC) at the nuclear periphery. The Saccharomyces cerevisiae Mlp1 and Mlp2 proteins are components of the inner nuclear basket of the nuclear pore that mediate interactions with these active genes. To investigate the physical link between the NPC and active loci, we identified proteins that interact with the carboxyl-terminal globular domain of Mlp1 by tandem affinity purification coupled with mass spectrometry. This analysis led to the identification of several components of the Spt-Ada-Gcn5-acetyltransferase (SAGA) histone acetyltransferase complex, Gcn5, Ada2, and Spt7. We utilized co-immunoprecipitation and in vitro binding assays to confirm the interaction between the Mlp proteins and SAGA components. Chromatin immunoprecipitation experiments revealed that Mlp1 and SAGA components associate with the same region of the GAL promoters. Critically, this Mlp-promoter interaction depends on the integrity of the SAGA complex. These results identify a physical association between SAGA and the NPC, and support previous results that relied upon visualization of GAL loci at the nuclear periphery by microscopy (Cabal, G. G. Genovesio, A., Rodriguez-Navarro, S., Zimmer, C., Gadal, O., Lesne, A., Buc, H., Feuerbach-Fournier, F., Olivo-Marin, J.-C., Hurt, E. C., and Nehrbass, U. (2006) Nature 441, 770-773). We propose that a physical interaction between nuclear pore components and the SAGA complex can link the actively transcribed GAL genes to the nuclear pore.