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.

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.

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.

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.

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.

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.