SummaryWe have developed a convenient method to visualize triacylglycerol-filled lipid droplets (LDs) in some species of bacteria, algae and fungi by staining with borondipyrromethene difluoride (BODIPY). When BODIPY was excited by blue light, LDs emitted green fluorescence, which was distinguished easily from the red autofluorescence of chloroplasts. This makes BODIPY staining suitable for the identification of small amounts of LDs, especially in plants. We first ensured that in Chlamydomonas reinhardtii cells growing in nitrogen-replete (+N) and -deficient (-N) media, the spots of BODIPY-stained LDs coincided with those of Nile Red-stained LDs. In addition, it was shown that the LD content per cell in N-starved cells was 200-fold higher than those of the control (+N) using a video-intensified microscope photoncounting system (VIMPCS). BODIPY staining was applied to visualize LD in bacteria, algae and fungi, and included those algae regarded as nonoleaginous. We identified LD spots in unicellular and multicellular bacteria and eukaryotes, namely Cyanidioschyzon merolae, Cyanidium caldarium delta, Chlamydomonas reinhardtii, Klebsormidium nitens and Penicillium sp., but not in Anabaena flos-aquae. We also examined the relationship between the contents of LDs and the genome size in the algae and fungi using VIMPCS but were unable to find a strong relationship between genome size and production of LDs. Finally, the location of LDs was considered in relation to organelles including the endoplasmic reticulum and chloroplasts, which are related to the formation of LDs.Key words Lipid droplets, Genome size, BODIPY, Cyanidioschyzon merolae, Algae, Fungi.There is considerable interest in the development of technologies that harvest lipids from microalgae and fungi and convert them into diesel fuel (Hu et al. 2008, Radakovits et al. 2010, Scott et al. 2010, Wijffels and Barbosa 2010. A widely adopted approach is to collect non-food algal

Yagisawa et al. ESCRT Functions in Algal Cytokinesis perturbed by expressing dominant-negative VPS4, cells with an elongated intercellular bridge accumulated-a phenotype resulting from abscission failure. Our results show that ESCRT mediates cytokinetic abscission in C. merolae. The fact that ESCRT plays a role in cytokinesis in archaea, animals, and early diverged alga C. merolae supports the hypothesis that the function of ESCRT in cytokinesis descended from archaea to a common ancestor of eukaryotes.

The transition from G1 to S phase and subsequent nuclear DNA replication in the cells of many species of eukaryotic algae occur predominantly during the evening and night in the absence of photosynthesis; however, little is known about how day/night changes in energy metabolism and cell cycle progression are coordinated and about the advantage conferred by the restriction of S phase to the night. Using a synchronous culture of the unicellular red alga Cyanidioschyzon merolae, we found that the levels of photosynthetic and respiratory activities peak during the morning and then decrease toward the evening and night, whereas the pathways for anaerobic consumption of pyruvate, produced by glycolysis, are upregulated during the evening and night as reported recently in the green alga Chlamydomonas reinhardtii. Inhibition of photosynthesis by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) largely reduced respiratory activity and the amplitude of the day/night rhythm of respiration, suggesting that the respiratory rhythm depends largely on photosynthetic activity. Even when the timing of G1/S-phase transition was uncoupled from the day/night rhythm by depletion of retinoblastoma-related (RBR) protein, the same patterns of photosynthesis and respiration were observed, suggesting that cell cycle progression and energy metabolism are regulated independently. Progression of the S phase under conditions of photosynthesis elevated the frequency of nuclear DNA double-strand breaks (DSB). These results suggest that the temporal separation of oxygenic energy metabolism, which causes oxidative stress, from nuclear DNA replication reduces the risk of DSB during cell proliferation in C. merolae. IMPORTANCE Eukaryotes acquired chloroplasts through an endosymbiotic event in which a cyanobacterium or a unicellular eukaryotic alga was integrated into a previously nonphotosynthetic eukaryotic cell. Photosynthesis by chloroplasts enabled algae to expand their habitats and led to further evolution of land plants. However, photosynthesis causes greater oxidative stress than mitochondrion-based respiration. In seed plants, cell division is restricted to nonphotosynthetic meristematic tissues and populations of photosynthetic cells expand without cell division. Thus, seemingly, photosynthesis is spatially sequestrated from cell proliferation. In contrast, eukaryotic algae possess photosynthetic chloroplasts throughout their life cycle. Here we show that oxygenic energy conversion (daytime) and nuclear DNA replication (night time) are temporally sequestrated in C. merolae. This sequestration enables “safe” proliferation of cells and allows coexistence of chloroplasts and the eukaryotic host cell, as shown in yeast, where mitochondrial respiration and nuclear DNA replication are temporally sequestrated to reduce the mutation rate.