Functional responses between PMP3 small membrane proteins and membrane potential.

The Plasma Membrane Proteolipid 3 (PMP3, UPF0057 family in Uniprot) family consists of abundant small hydrophobic polypeptides with two predicted transmembrane helices. Plant homologues were upregulated in response to drought/salt-stresses and yeast deletion mutants exhibited conditional growth defects. We report here abundant expression of Group I PMP3 homologues (PMP3(i)hs) during normal vegetative growth in both prokaryotic and eukaryotic cells, at a level comparable to housekeeping genes, implicating the regular cellular functions. Expression of eukaryotic PMP3(i)hs was dramatically upregulated in response to membrane potential (Vm) variability (Vmvar ), whereas PMP3(i)hs deletion-knockdown led to Vm changes with conditional growth defects. Bacterial PMP3(i)h yqaE deletion led to a shift of salt sensitivity; Vmvar alternations with exogenous K+ addition downregulated prokaryotic PMP3(i)hs, suggesting [K+ ]-Vmvar axis being a significant feedback element in prokaryotic ionic homeostasis. Remarkably, the eukaryotic homologues functionally suppressed the conditional growth defects in bacterial deletion mutant, demonstrating the conserved cross-kingdom membrane functions by PMP3(i)hs. These data demonstrated a direct reciprocal relationship between PMP3(i)hs expression and Vm differentials in both prokaryotic and eukaryotic cells. Cumulative with PMP3(i)hs ubiquitous abundance, their lipid-binding selectivity and membrane protein colocalization, we propose [PMP3(i)hs]-Vmvar axis as a key element in membrane homeostasis.

Counterion-mediated decompaction of liquid crystalline chromosomes.

Liquid crystalline phases of DNA and nucleosome core particles can be formed in vitro, indicating the crucial roles of these phases in the maintenance and compaction of genomes in vivo. In the present study, sequential levels of liquid crystalline decompaction were identified in highly purified nuclei of Karenia papilionacea in response to the gradual chelation of divalent counterions by ethylenediaminetetraacetic acid (EDTA); the decompaction was observed using polarizing light microscopy, confocal laser scanning microscopy, and transmission electron microscopy and further confirmed utilizing microcalorimetry. Nested fibrous coils in 150 nm arc-like bands of chromatin were observed in the early stages of chromosomal decompaction. The microcalorimetry spectra of isolated nuclei revealed that the dynamic processes of nuclear decompaction occurred in a nonlinear manner; in addition, an EDTA-sensitive thermal transition between 60°C-70°C, corresponding to a liquid-crystalline-phase transition of chromosomes, was found. The results suggested that nested coils of fibrous chromatin filaments are responsible for the establishment and stabilization of the liquid crystalline and birefringence features of the chromosomes of dinoflagellates. The results also indicated that positively charged divalent counterions play significant roles in modulating liquid crystalline phases to compact the chromosomes of dinoflagellates.

Atomistic simulations of electroporation in water preembedded membranes.

Atomistic simulations of electroporation were conducted on water/membrane/water systems, in which the membranes initially contained randomly distributed water molecules that might be introduced by acoustic treatment. The simulation results indicate that the critical strength of an applied electric field to induce electroporation is greatly reduced due to the initially embedded water molecules in the membranes. A lower applied electric field will significantly enhance the viability of cells in electroporation.

Effects of deformability and thermal motion of lipid membrane on electroporation: by molecular dynamics simulations.

Effects of mechanical properties and thermal motion of POPE lipid membrane on electroporation were studied by molecular dynamics simulations. Among simulations in which specific atoms of lipids were artificially constrained at their equilibrium positions using a spring with force constant of 2.0 kcal/(molŲ) in the external electric field of 1.4 kcal/(molŠe), only constraint on lateral motions of lipid tails prohibited electroporation while non-tail parts had little effects. When force constant decreased to 0.2 kcal/(molŲ) in the position constraints on lipid tails in the external electric field of 2.0 kcal/(molŠe), water molecules began to enter the membrane. Position constraints of lipid tails allow water to penetrate from both sides of membrane. Thermal motion of lipids can induce initial defects in the hydrophobic core of membrane, which are favorable nucleation sites for electroporation. Simulations at different temperatures revealed that as the temperature increases, the time taken to the initial pore formation will decrease.

The activity of a wall-bound cellulase is required for and is coupled to cell cycle progression in the dinoflagellate Crypthecodinium cohnii.

Cellulose synthesis, but not its degradation, is generally thought to be required for plant cell growth. In this work, we cloned a dinoflagellate cellulase gene, dCel1, whose activities increased significantly in G(2)/M phase, in agreement with the significant drop of cellulose content reported previously. Cellulase inhibitors not only caused a delay in cell cycle progression at both the G(1) and G(2)/M phases in the dinoflagellate Crypthecodinium cohnii, but also induced a higher level of dCel1p expression. Immunostaining results revealed that dCel1p was mainly localized at the cell wall. Accordingly, the possible role of cellulase activity in cell cycle progression was tested by treating synchronized cells with exogenous dCelp and purified antibody, in experiments analogous to overexpression and knockdown analyses, respectively. Cell cycle advancement was observed in cells treated with exogenous dCel1p, whereas the addition of purified antibody resulted in a cell cycle delay. Furthermore, delaying the G(2)/M phase independently with antimicrotubule inhibitors caused an abrupt and reversible drop in cellulase protein level. Our results provide a conceptual framework for the coordination of cell wall degradation and reconstruction with cell cycle progression in organisms with cell walls. Since cellulase activity has a direct bearing on the cell size, the coupling between cellulase expression and cell cycle progression can also be considered as a feedback mechanism that regulates cell size.

Birefringence and DNA condensation of liquid crystalline chromosomes.

DNA can self-assemble in vitro into several liquid crystalline phases at high concentrations. The largest known genomes are encoded by the cholesteric liquid crystalline chromosomes (LCCs) of the dinoflagellates, a diverse group of protists related to the malarial parasites. Very little is known about how the liquid crystalline packaging strategy is employed to organize these genomes, the largest among living eukaryotes-up to 80 times the size of the human genome. Comparative measurements using a semiautomatic polarizing microscope demonstrated that there is a large variation in the birefringence, an optical property of anisotropic materials, of the chromosomes from different dinoflagellate species, despite their apparently similar ultrastructural patterns of bands and arches. There is a large variation in the chromosomal arrangements in the nuclei and individual karyotypes. Our data suggest that both macroscopic and ultrastructural arrangements affect the apparent birefringence of the liquid crystalline chromosomes. Positive correlations are demonstrated for the first time between the level of absolute retardance and both the DNA content and the observed helical pitch measured from transmission electron microscopy (TEM) photomicrographs. Experiments that induced disassembly of the chromosomes revealed multiple orders of organization in the dinoflagellate chromosomes. With the low protein-to-DNA ratio, we propose that a highly regulated use of entropy-driven force must be involved in the assembly of these LCCs. Knowledge of the mechanism of packaging and arranging these largest known DNAs into different shapes and different formats in the nuclei would be of great value in the use of DNA as nanostructural material.

Telomere maintenance in liquid crystalline chromosomes of dinoflagellates.

The organisation of dinoflagellate chromosomes is exceptional among eukaryotes. Their genomes are the largest in the Eukarya domain, chromosomes lack histones and may exist in liquid crystalline state. Therefore, the study of the structural and functional properties of dinoflagellate chromosomes is of high interest. In this work, we have analysed the telomeres and telomerase in two Dinoflagellata species, Karenia papilionacea and Crypthecodinium cohnii. Active telomerase, synthesising exclusively Arabidopsis-type telomere sequences, was detected in cell extracts. The terminal position of TTTAGGG repeats was determined by in situ hybridisation and BAL31 digestion methods and provides evidence for the linear characteristic of dinoflagellate chromosomes. The length of telomeric tracts, 25-80 kb, is the largest among unicellular eukaryotic organisms to date. Both the presence of long arrays of perfect telomeric repeats at the ends of dinoflagellate chromosomes and the existence of active telomerase as the primary tool for their high-fidelity maintenance demonstrate the general importance of these structures throughout eukaryotes. We conclude that whilst chromosomes of dinoflagellates are unique in many aspects of their structure and composition, their telomere maintenance follows the most common scenario.

Cyclic ADP-ribose links metabolism to multiple fission in the dinoflagellate Crypthecodinium cohnii.

Cellular metabolism is required for cell proliferation. However, the way in which metabolic signals are conveyed to cell cycle decisions is unclear. Cyclic ADP-ribose (cADPR), the NAD(+) metabolite, mobilizes calcium from calcium stores in many cells. We found that dinoflagellate cells with higher metabolic rate underwent multiple fission (MF), a division mode in which cells can exceed twice their sizes at G1. A temperature shift-down experiment suggested that MF involves a commitment point at late G1. In fast-growing cells, cADPR level peaked in G(1) and increased with increasing concentrations of glucose in the medium. Addition of glycolytic poison iodoacetate inhibited cell growth, reduced cADPR levels as well as the commitment of cell cycles in fast-growing cells. Commitment of MF cell cycles was induced by a cell permeant cADPR agonist, but blocked by a specific antagonist of cADPR-induced Ca(2+) release. Our results establish cADPR as a link between cellular metabolism and cell cycle control.

The replication of plastid minicircles involves rolling circle intermediates.

Plastid genomes of peridinin-containing dinoflagellates are unique in that its genes are found on multiple circular DNA molecules known as 'minicircles' of approximately 2-3 kb in size, carrying from one to three genes. The non-coding regions (NCRs) of these minicircles share a conserved core region (250-500 bp) that are AT-rich and have several inverted or direct repeats. Southern blot analysis using an NCR probe, after resolving a dinoflagellate whole DNA extract in pulsed-field gel electrophoresis (PFGE), revealed additional positive bands (APBs) of 6-8 kb in size. APBs preferentially diminished from cells treated with the DNA-replication inhibitor aphidicolin, when compared with 2-3 kb minicircles, implicating they are not large minicircles. The APBs are also exonuclease III-sensitive, implicating the presence of linear DNA. These properties and the migration pattern of the APBs in a 2D-gel electrophoresis were in agreement with a rolling circle type of replication, rather than the bubble-forming type. Atomic force microscopy of 6-8 kb DNA separated by PFGE revealed DNA intermediates with rolling circle shapes. Accumulating data thus supports the involvement of rolling circle intermediates in the replication of the minicircles.

An unusual S-adenosylmethionine synthetase gene from dinoflagellate is methylated.

BACKGROUND: S-Adenosylmethionine synthetase (AdoMetS) catalyzes the formation of S-Adenosylmethionine (AdoMet), the major methyl group donor in cells. AdoMet-mediated methylation of DNA is known to have regulatory effects on DNA transcription and chromosome structure. Transcription of environmental-responsive genes was demonstrated to be mediated via DNA methylation in dinoflagellates. RESULTS: A full-length cDNA encoding AdoMetS was cloned from the dinoflagellate Crypthecodinium cohnii. Phylogenetic analysis suggests that the CcAdoMetS gene, is associated with the clade of higher plant orthrologues, and not to the clade of the animal orthrologues. Surprisingly, three extra stretches of residues (8 to 19 amino acids) were found on CcAdoMetS, when compared to other members of this usually conserved protein family. Modeled on the bacterial AdeMetS, two of the extra loops are located close to the methionine binding site. Despite this, the CcAdoMetS was able to rescue the corresponding mutant of budding yeast. Southern analysis, coupled with methylation-sensitive and insensitive enzyme digestion of C. cohnii genomic DNA, demonstrated that the AdoMetS gene is itself methylated. The increase in digestibility of methylation-sensitive enzymes on AdoMet synthetase gene observed following the addition of DNA methylation inhibitors L-ethionine and 5-azacytidine suggests the presence of cytosine methylation sites within CcAdoMetS gene. During the cell cycle, both the transcript and protein levels of CcAdoMetS peaked at the G1 phase. L-ethionine was able to delay the cell cycle at the entry of S phase. A cell cycle delay at the exit of G2/M phase was induced by 5-azacytidine. CONCLUSION: The present study demonstrates a major role of AdoMet-mediated DNA methylation in the regulation of cell proliferation and that the CcAdoMetS gene is itself methylated.

Mechanical characterization of cellulosic thecal plates in dinoflagellates by nanoindentation.

Dinoflagellates constitute an important group of microorganisms. Symbiotic dinoflagellates are responsible for the primary production of coral reef ecosystems and the phenomenon of their demise is known as "coral bleaching." Blooming of the planktonic dinoflagellates is the major cause of "red tides." Many dinoflagellates have prominent membrane-bound thecal plates at their cell cortices. These thecal plates have high cellulose content and are biologically fabricated into various shapes. However, the mechanical properties of theca have not previously been characterized; understanding these properties, including hardness and elastic modulus, will give insights into the ecological significance and biotechnological potential of bio-fabricated structures. A series of nanoindentation tests were performed on various locations of cellulosic thecal plates isolated from the dinoflagellates Alexandrium catenella and Lingulodinium polyedrum. Despite having transparent properties, thecal plates possess mechanical properties comparable to softwood cell walls, implicating their role as a protective cell covering. Consistent measurements were obtained when indentation was performed at various locations, which contrasts with the high variability of cellulose microfibers from plant sources. The present study demonstrated the novel properties of this potential new source of cellulose.

Concentration-dependent organization of DNA by the dinoflagellate histone-like protein HCc3.

The liquid crystalline chromosomes of dinoflagellates are the alternative to the nucleosome-based organization of chromosomes in the eukaryotes. These nucleosome-less chromosomes have to devise novel ways to maintain active parts of the genome. The dinoflagellate histone-like protein HCc3 has significant sequence identity with the bacterial DNA-binding protein HU. HCc3 also has a secondary structure resembling HU in silico. We have examined HCc3 in its recombinant form. Experiments on DNA-cellulose revealed its DNA-binding activity is on the C-terminal domain. The N-terminal domain is responsible for intermolecular oligomerization as demonstrated by cross-linking studies. However, HCc3 could not complement Escherichia coli HU-deficient mutants, suggesting functional differences. In ligation assays, HCc3-induced DNA concatenation but not ring closure as the DNA-bending HU does. The basic HCc3 was an efficient DNA condensing agent, but it did not behave like an ordinary polycationic compound. HCc3 also induced specific structures with DNA in a concentration-dependent manner, as demonstrated by atomic force microscopy (AFM). At moderate concentration of HCc3, DNA bridging and bundling were observed; at high concentrations, the complexes were even more condensed. These results are consistent with a biophysical role for HCc3 in maintaining extended DNA loops at the periphery of liquid crystalline chromosomes.

Novel method for preparing spheroplasts from cells with an internal cellulosic cell wall.

Protoplast and spheroplast preparations allow the transfer of macromolecules into cells and provide the basis for the generation of engineered organisms. Crypthecodinium cohnii cells harvested from polyethylene glycol-containing agar plates possessed significantly lower levels of cellulose in their cortical layers, which facilitated the delivery of fluorescence-labeled oligonucleotides into these cells.

Alveolata histone-like proteins have different evolutionary origins.

Prokaryotic histone-like proteins (Hlps) are abundant proteins found in bacterial and plastid nucleoids. Hlps are also found in the eukaryotic dinoflagellates and the apicomplexans, two major lineages of the Alveolata. It may be expected that Hlps of both groups were derived from the same ancestral Alveolates. However, our phylogenetic analyses suggest different origins for the dinoflagellate and the apicomplexan Hlps. The apicomplexan Hlps are affiliated with the cyanobacteria and probably originated from Hlps of the plastid genome. The dinoflagellate Hlps and the proteobacterial long Hlps form a clade that branch off from the node with the proteobacterial short Hlps.

Involvement of calcium mobilization from caffeine-sensitive stores in mechanically induced cell cycle arrest in the dinoflagellate Crypthecodinium cohnii.

Mechanical loads can profoundly alter cell growth and cell proliferation. The dinoflagellates are especially sensitive to mechanical stimulation. Many species will be arrested in cell cycle in response to turbulence or shear stress. We demonstrate here that mechanical shaking and caffeine, the ryanodine-receptor agonist, induced an elevation of cytosolic calcium in the dinoflagellate Crypthecodinium cohnii. Dantrolene, a ryanodine-receptor antagonist, dose-dependently inhibited both shaking-induced and caffeine-induced calcium release. Similar to the effect of mechanical shaking, caffeine alone dose-dependently and reversibly induced cell cycle arrest in dinoflagellates. Prolonged shaking substantially abolished the magnitude of caffeine-induced calcium release and vice-versa, suggesting that both agents released calcium from similar stores through ryanodine receptors. Fluorescence-conjugated ryanodine gave positive labeling, which could be blocked by ryanodine, in the cortice of C. cohnii cells. In addition, caffeine or shaking mobilized intracellular chlortetracycline (CTC)-positive membrane-bound calcium, which could be similarly depleted by t-BuBHQ, a SERCA pump inhibitor. Prior treatment with shaking or caffeine also inhibited the ability of the other agent in mobilizing CTC-positive calcium. CTC-positive microsomal fractions could also be induced to release calcium by caffeine and cADPR, the ryanodinee receptor modulator. t-BuBHQ, but not calcium ionophores, induced cell cycle arrest, and the calcium chelator BAPTA-AM was unable to rescue caffeine-induced cell cycle arrest. These data culminate to suggest that mobilization or depletion of caffeine-sensitive calcium stores, but not calcium elevation per se, is involved in the induction of cell cycle arrest by mechanical stimulation. The present study establishes the role of caffeine-sensitive calcium stores in the regulation of cell cycle progression.

Type II topoisomerase activities in both the G1 and G2/M phases of the dinoflagellate cell cycle.

Dinoflagellate genomes are large (up to 200 pg) and are encoded in histoneless chromosomes that are quasi-permanently condensed. This unique combination of chromosomal characteristics presents additional topological and cell cycle control problems for a eukaryotic cell, potentially exhibiting novel regulatory requirements of topoisomerase II. The heterotrophic dinoflagellate Crypthecodinium cohnii was used in this study. The topoisomerase II activities throughout its cell cycle were investigated by DNA flow cytometry following enzyme deactivation. Fluorescence microscopy was also used for studying the chromosome morphology of the treated cells. Two classes of topoisomerase II inhibitors were applied in our study, both of which caused G1 delay as well as G2/M arrest in the C. cohnii cell cycle. At high doses, the topoisomerase poisons amsacrine and ellipticine induced DNA fragmentation in C. cohnii cells. Topoisomerase II activities, as measured by the ability to decatenate kinetoplastid DNA (kDNA), are normally detected throughout the cell cycle in C. cohnii. Our results suggest that the requirement of type II topoisomerase activities during the G1 phase of the cell cycle may relate to the unwinding of quasi-permanently condensed chromosomes for the purpose of transcription. This was also the first time that topoisomerase II activity in dinoflagellate cells was detected.

Lipid biosynthesis and its coordination with cell cycle progression.

The activation of cell cycle regulators at the G1/S boundary has been linked to the cellular protein synthesis rate. It is conceivable that regulatory mechanisms are required to allow cells to coordinate the synthesis of other macromolecules with cell cycle progression. The availability of highly synchronized cells and flow cytometric methods facilitates investigation of the dynamics of lipid synthesis in the entire cell cycle of the heterotrophic dinoflagellate Crypthecodinium cohnii. Flow cytograms of Nile red-stained cells revealed a stepwise increase in the polar lipid content and a continuous increase in neutral lipid content in the dinoflagellate cell cycle. A cell cycle delay at early G1, but not G2/M, was observed upon inhibition of lipid synthesis. However, lipid synthesis continued during cell cycle arrest at the G1/S transition. A cell cycle delay was not observed when inhibitors of cellulose synthesis and fatty acid synthesis were added after the late G1 phase of the cell cycle. This implicates a commitment point that monitors the synthesis of fatty acids at the late G1 phase of the dinoflagellate cell cycle. Reduction of the glucose concentration in the medium down-regulated the G1 cell size with a concomitant forward shift of the commitment point. Inhibition of lipid synthesis up-regulated cellulose synthesis and resulted in an increase in cellulosic contents, while an inhibition of cellulose synthesis had no effects on lipid synthesis. Fatty acid synthesis and cellulose synthesis are apparently coupled to the cell cycle via independent pathways.

Proliferation of dinoflagellates: blooming or bleaching.

The dinoflagellates, a diverse sister group of the malaria parasites, are the major agents causing harmful algal blooms and are also the symbiotic algae of corals. Dinoflagellate nuclei differ significantly from other eukaryotic nuclei by having extranuclear spindles, no nucleosomes and enormous genomes in liquid crystal states. These cytological characteristics were related to the acquisition of prokaryotic genes during evolution (hence Mesokaryotes), which may also account for the biochemical diversity and the relatively slow growth rates of dinoflagellates. The fact that the proliferation of many dinoflagellates is sensitive to turbulence may be due to the physiological requirements of the genome's liquid crystal states. Mechanical stress and anti-microtubule drugs induce cell cycle arrest mainly in G1, implicating a role for the permanent cortical microtubular cytoskeleton in mechanotransduction. The cell cycles of photosynthetic dinoflagellates are also gated by the circadian rhythm, with cell division occurring mainly at the end of the dark phase. Cell growth and the biosynthesis of many toxins occur during the light phase, corresponding to G1 in the cell cycle. The dinoflagellates also embody several options for coupling cell cycle progression to cell growth, enabling them to make the best use of available resources and possibly preparing them for a symbiotic existence.

Monitoring cytosolic calcium in the dinoflagellate Crypthecodinium cohnii with calcium orange-AM.

Calcium plays several important roles in the signal transduction pathways of dinoflagellates. We describe here the development of calcium orange-AM as an intracellular calcium reporter for the heterotrophic dinoflagellate Crypthecodinium cohnii. We demonstrated with confocal microscopy that by restricting the incubation period to 30-45 min, no compartmentalization of the dye occurs in the mitochondria or endoplasmic reticulum. The dye fluorescence responded well to the effects of calcium ionophores and calcium chelators. By calibrating the dye with known calcium concentrations, we determined the intracellular calcium concentration of C. cohnii to be 158 +/- 56 nM, which rose to about 550 nM upon mechanical stimulation.

Cellulose synthesis is coupled to cell cycle progression at G1 in the dinoflagellate Crypthecodinium cohnii.

Cellulosic deposition in alveolar vesicles forms the "internal cell wall" in thecated dinoflagellates. The availability of synchronized single cells, the lack of secondary deposition, and the absence of cellulosic cell plates at division facilitate investigation of the possible roles of cellulose synthesis (CS) in the entire cell cycle. Flow cytograms of cellulosic contents revealed a stepwise process of CS in the dinoflagellate cell cycle, with the highest rate occurring at G(1). A cell cycle delay in G(1), but not G(2)/M, was observed after inhibition of CS. A cell cycle inhibitor of G(1)/S, but not G(2)/M, was able to delay cell cycle progression with a corresponding reduction of CS. The increase of cellulose content in the cell cycle corresponded well to the expected increase of surface area. No differences were observed in the cellulose to surface area ratio between normal and fast-growing G(1) cells, implicating the significance of surface area in linking CS to the coupling of cell growth with cell cycle progression. The coupling of CS to G(1) implicates a novel link between CS and cell cycle control, and we postulate that the coupling mechanism might integrate cell wall integrity to the cell size checkpoint.