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summary Changes in the ultrastructure of the Golgi apparatus during the cell cycle were investigated using a highly synchronized cell division system in a Catharanthus roseus (L.) G. Don suspension culture. During the G2 phase and cytokinesis, diameters of the dictyosomes and the number of cisternae significantly increased. Electron micrographs of cells in cytokinesis suggested that dictyosomes were dividing into two. Numerous secretory vesicles were associated with the dictyosomes in cells of the second G1 phase, the time when cell wall synthesis is most active. Structural changes in the Golgi apparatus are discussed in relation to the cell cycle and changes in cell wall synthesis.

Plasmodium falciparum is the most virulent and widespread of the human malaria parasite species. This parasite has a complex life cycle that involves sexual replication in a mosquito vector and asexual replication in a human host. During the 48-hour intraerythrocytic developmental cycle (IDC), parasites develop and multiply through the morphologically distinct ring, trophozoite and schizont stages. Stage-specific transcriptomic approaches have shown gene expression profiles continually change throughout the IDC. Cultures of tightly synchronized parasites are required to capture the transcriptome specific to a developmental stage. However, the most commonly used synchronization methods require lysis of late stages, potentially perturbing transcription, and often do not result in tightly synchronized cultures. To produce complete transcriptome profiles of the IDC a synchronous culture requires frequent sampling over a 48-hour period, this is both time consuming and labor intensive. Here we develop a method to sample the IDC densely by isolating parasites from an asynchronous culture with fluorescence activated cell sorting (FACS). We sort parasites in tight windows of IDC progression based on their DNA/RNA abundance. We confirmed the tight synchronization and stage specificity by light microscopy and RNAseq profiling. We optimized our protocol for low numbers of sorted cells allowing us to rapidly capture transcriptome profiles across the entire IDC from a single culture flask. This methodology will allow malaria stage-specific studies to perform experiments directly from asynchronous cultures with high accuracy and without the need for labor-intensive time-course experiments.

Photosynthetic performance was examined in Skeletonema costatum (Greville) Cleve. under 12 : 12-h light : dark (LD) cycle at ambient CO2 (350 μL L–1) and elevated CO2 (1000 μL L–1). At ambient CO2, the cellular chlorophyll a content, the light-saturated photosynthetic rate (Pm), the initial slope of the light saturation curves (α), the photochemical efficiency of PSII (Fv / Fm), the apparent carboxylating efficiency (ACE) and the photosynthetic affinity for CO2 [1 / Km(CO2)] all showed rhythmical changes with different amplitudes during the light period. The Pm had similar changing pattern in the light period with the ACE and 1 / Km(CO2) rather than with the α and Fv / Fm, indicating that rhythmical changes of photosynthetic capacity may be mainly controlled by the activity of C-reduction associated with CO2 uptake during the light period. The CO2 enrichment reduced the ACE and the affinity to CO2, and increased the α, cellular chlorophyll a content and Pm based on cell number. By contrast, the changing patterns of all photosynthetic parameters examined here during the light period had almost the same for cells grown at ambient CO2 and elevated CO2, suggesting that the photosynthetic rhythms of S. costatum are not affected by CO2 enrichment.

The non-steroidal anti-inflammatory drug diclofenac (DCF) is one of the commonly used and frequently detected drugs in water bodies, and several studies indicate its toxic effect on plants and algae. Studies performed with asynchronous Chlamydomonas reinhardtii cultures indicated that DCF inhibit the growth of population of the algae. Here, a synchronous population of C. reinhardtii, in which all cells are in the same developmental phase, is used. Following changes in cells size, photosynthetic activity and gene expression, we could compare, at the level of single cell, DCF-mediated effects with the effects caused by atrazine, a triazine herbicide that inhibits photosynthesis and triggers oxidative stress. Application of DCF and atrazine at the beginning of the cell cycle allowed us to follow the changes occurring in the cells in the subsequent stages of their development. Synchronized Chlamydomonas reinhardtii cultures (strain CC-1690, wild type) were exposed to diclofenac sodium salt (135 mg/L) or atrazine (77.6 µg/L). The cell suspension was sampled hourly (0–10 h) in the light period of the cell cycle to determine cell number and volume, photosynthetic pigment content, chlorophyll a fluorescence (OJIP test) in vivo, and selected gene expression (real-time qPCR), namely psbA, psaA, FSD1, MSD3 and APX1. The two toxicants differently influenced C. reinhardtii cells. Both substances decreased photosynthetic “vitality” (PI - performance index) of the cells, albeit for different reasons. While atrazine significantly disrupted the photosynthetic electron transport, resulting in excessive production of reactive oxygen species (ROS) and limited cell growth, DCF caused silencing of photosystem II (PSII) reaction centers, transforming them into “heat sinks”, thus preventing significant ROS overproduction. Oxidative stress caused by atrazine was the probable reason for the rapid appearance of phytotoxic action soon after entering the cells, while the effects of DCF could only be seen several hours after treatment. A comparison of DCF-caused effects with the effects caused by atrazine led us to conclude that, although DCF cannot be regarded as typical photosynthetic herbicide, it exhibits an algicidal activity and can be potentially dangerous for aquatic plants and algae.

Algal cells are highly complex optical systems that can dynamically change their structure. Consequently, absorption and scattering properties of algae change, while the cells are acclimating to different light conditions or during growth and division in a cell cycle. This may be particularly important in algal species that can grow rapidly under very high-light such as Chlorella vulgaris IPPAS C-1 that is studied here. From cell transmittance measured conventionally and using integrating sphere, we evaluated absorption and scattering coefficients and cross sections per cell dry weight and chlorophyll content. This was done for asynchronous cell culture grown in low-light (LL; 220 μmol (photons) m−2 s−1) or high-light (HL; 1760 μmol (photons) m−2 s−1) light, as well as during cell cycle of synchronous culture grown in HL. During the cell cycle, we also determined cell ultrastructural organization by transmission electron microscopy, and correlated its parameters with absorption and scattering cross sections per cell dry weight. We found that the IPPAS C-1 cells of asynchronous culture scatter light more than other cells, however, internal organization of the cells that is decisive for scattering is less sensitive to HL and LL treatment than the cell pigment content that controls absorption. The light scattering and absorption were dynamically changed during cell cycle of synchronous cells grown in the HL. Changes in ratio of chloroplast to protoplast area, reflecting amount of scattering chloroplast membrane (outer, inner) interfaces, best correlated with changes in light scattering. We suggest that the increased light scattering by the HL-acclimated IPPAS C-1 cells might be responsible for increased HL resilience reported in the literature. Biotechnological aspect of this study is that the scattering and absorption properties of phytoplankton cells ought to be calibrated for each particular growth phase or irradiance to which the cells are acclimated.

The cell cycle has fundamental effects on cell cultures and their products. Tools to synchronize cultured cells allow the study of cellular physiology and metabolism at particular cell cycle phases. However, cells are most often arrested by methods that alter their homeostasis and are then cultivated in poorly controlled environments. Cell behavior could then be affected by the synchronization method and culture conditions used, and not just by the particular cell cycle phase under study. Moreover, only a few viable cells are recovered. Here, we designed an integrated system where a large number of cells from a controlled bioreactor culture is separated by centrifugal elutriation at high viabilities. In contrast to current elutriation methods, cells are injected directly from a bioreactor into an injection loop, allowing the introduction of a large number of cells into the separation chamber without stressful centrifugation. A low pulsation peristaltic pump increases the stability of the elutriation chamber. Using this approach, a large number of healthy cells at each cell cycle phase were obtained, allowing their direct inoculation into fully instrumented bioreactors. Hybridoma cells synchronized and cultured in this system behaved as expected for a synchronous culture.

This protocol describes how to visualize the transient DNA compaction in cyanobacteria. DNA compaction is a dramatic cytoplasmic event recently found to occur in some cyanobacteria before cell division. However, due to the large cell size and the transient character, it is difficult to investigate the structure in detail. To overcome the difficulties, first, DNA compaction is reproducibly produced in the cyanobacterium Synechococcus elongatus PCC 7942 by synchronous culture using 12 h each light/dark cycle. Second, DNA compaction is monitored by fluorescence microscopy and captured by rapid freezing. Third, the detailed structure of DNA compacted cells is visualized in three dimensions (3D) by high voltage cryo-electron tomography. This set of methods is widely applicable to investigate transient structures in bacteria, e.g. cell division, chromosome segregation, phage infection etc., which are monitored by fluorescence microscopy and directly visualized by cryo-electron tomography at appropriate time points.

Cultivation of the erythrocytic stages of Plasmodium parasites, specifically the most important and deadly for humans, Plasmodium falciparum, has required a lot of effort and time in order to develop a continuous in vitro culture. Moreover, the development of methods to synchronize P. falciparum parasites (which grow asynchronously in vitro) has become an essential tool in research to study different immulogical, biochemical or physiological aspects of the parasite. We have compared two different synchronization methods, one based on differential permeability of the membrane of parasitized erythrocytes, and the other on the sedimentation behavior in gelatin solution. An optimized method has been established which allows for maintaining a healthy, highly synchronous culture for longer periods of time. Asexual erythrocytic stages of a complete P. falciparum cycle have been obtained, which is the starting point of the stage-specific assays of the activity of new antimalarial drugs.

Cilia and flagella are highly conserved antenna-like organelles that found in nearly all mammalian cell types. They perform sensory and motile functions contributing to numerous physiological and developmental processes. Defects in their assembly and function are implicated in a wide range of human diseases ranging from retinal degeneration to cancer. Chlamydomonas reinhardtii is an algal model system for studying mammalian cilium formation and function. Here, we report a simple synchronization method that allows detection of small changes in ciliary length by minimizing variability in the population. We find that this method alters the key relationship between cell size and the amount of protein accumulated for flagellar growth. This provides a rapid alternative to traditional methods of cell synchronization for uncovering novel regulators of cilia., The unicellular green alga Chlamydomonas reinhardtii is an ideal model organism for studies of ciliary function and assembly. In assays for biological and biochemical effects of various factors on flagellar structure and function, synchronous culture is advantageous for minimizing variability. Here, we have characterized a method in which 100% synchronization is achieved with respect to flagellar length but not with respect to the cell cycle. The method requires inducing flagellar regeneration by amputation of the entire cell population and limiting regeneration time. This results in a maximally homogeneous distribution of flagellar lengths at 3 h postamputation. We found that time-limiting new protein synthesis during flagellar synchronization limits variability in the unassembled pool of limiting flagellar protein and variability in flagellar length without affecting the range of cell volumes. We also found that long- and short-flagella mutants that regenerate normally require longer and shorter synchronization times, respectively. By minimizing flagellar length variability using a simple method requiring only hours and no changes in media, flagellar synchronization facilitates the detection of small changes in flagellar length resulting from both chemical and genetic perturbations in Chlamydomonas. This method increases our ability to probe the basic biology of ciliary size regulation and related disease etiologies. IMPORTANCE Cilia and flagella are highly conserved antenna-like organelles that found in nearly all mammalian cell types. They perform sensory and motile functions contributing to numerous physiological and developmental processes. Defects in their assembly and function are implicated in a wide range of human diseases ranging from retinal degeneration to cancer. Chlamydomonas reinhardtii is an algal model system for studying mammalian cilium formation and function. Here, we report a simple synchronization method that allows detection of small changes in ciliary length by minimizing variability in the population. We find that this method alters the key relationship between cell size and the amount of protein accumulated for flagellar growth. This provides a rapid alternative to traditional methods of cell synchronization for uncovering novel regulators of cilia.

In budding yeast, a mother cell can produce a finite number of daughter cells over its life. The accumulation of a variety of types of damaged components has an impact on the aging process. Asymmetrical inheritance during cell division causes these aberrant intracellular constituents to be retained in mother cells and prevents them from segregating to daughter cells. However, the understanding of asymmetrical inheritance of individual proteins that are damaged or old age, and their relevance to the aging process, has been limited. The aim of this study is to propose a proteomics strategy for asymmetrical inheritance of preexisting proteins between mother and daughter cells. During synchronous culture for one generation, newly synthesized proteins were labeled with stable isotope amino acids to discriminate preexisting proteins originally expressed in mother cells, followed by separation of mother and daughter cells using a conventional method based on biotin labeling. Isotope incorporation ratios for individual proteins were quantified using mass spectrometry. We successfully identified 21 proteins whose preexisting versions were asymmetrically inherited in mother cells, including plasma membrane transporter involved in the aging process and organelle-anchoring proteins related to the stress response to misfolded proteins. Thus, our approach would be useful for making catalog of asymmetrically inherited proteins.

Cyanidioschyzon merolae 10D was originally isolated from a mixture of hot spring water sampled in Naples, Italy. Currently, this strain is available in the Microbial Culture Collection at the National Institute of Environmental Studies in Japan. The strain has been cultured in 2× Allen’s medium or its derivatives. The optimal growth conditions for this strain are as follows: pH 2.5, 42 °C, and ~100 μmol photons m−2 s−1, allowing the cell density to reach ~5 × 108 cells mL−1. C. merolae can also grow slowly at >20 °C. We generally store stock cultures at room temperature or at 30 °C under a low light condition (~20 μmol photons m−2 s−1) or as frozen stock in liquid nitrogen. Cell cycle progression can be synchronized by subjecting the culture to a 12-h light/12-h dark cycle. In addition, cells can be arrested at the S or M phases by adding relevant inhibitors. The shapes of cells and chloroplasts are clearly observed by phase-contrast or differential interference contrast microscopy. Because C. merolae lacks a cell wall, cellular contents (e.g., DNA, RNA, and proteins) are easily extracted.

Cell division in several lineages of eukaryotic algae occurs predominantly during the night. Cell cycle progression is shown to be regulated by circadian rhythms. However, the advantages and their underlying mechanisms conferred by this restriction of cell division to night are poorly understood. By using the unicellular red alga Cyanidioschyzon merolae, we recently showed that the retinoblastoma (RB)-E2F-DP pathway inhibits G1/S transition during the daytime. In C. merolae, E2F is phosphorylated in a time-dependent manner, peaking during the evening, which in turn permits the phosphorylation of RB only when the cell has grown to a certain size threshold. In addition, it is suggested that temporal segregation of photosynthesis during the daytime, which produces reactive oxygen species (ROS), and DNA replication and mitosis during the night is important for eukaryotic algae. Because the temporal segregation of respiratory activity and cell cycle progression has been observed in yeasts and mammalian cells, the temporal restriction of cell cycle progression is probably important for facilitating the safe multiplication of eukaryotic cells.

A cell replicates by duplicating cellular contents and then dividing into two daughter cells. This is known as the cell cycle, which is characterized by the dynamics of the cell nucleus. However, eukaryotic cells contain organelles other than the nucleus, such as mitochondria, chloroplasts, peroxisomes, endoplasmic reticulum (ER), Golgi bodies, and vacuoles. To understand reproduction of cells, the division and inheritance of organelles as well as the mitotic cycle should be investigated. However, model animal cells and plant cells contain multiples of these organelles which exhibit complex shapes. Therefore, it has been difficult to elucidate their division and inheritance. In contrast, the red alga Cyanidioschyzon merolae possesses a minimum set of organelles. Because of its simple cellular architecture, the dynamics and mechanisms underlying its cell division and inheritance have been investigated. In addition, cell cycle stages of C. merolae have been determined using an S phase-specific marker proliferating cell nuclear antigen (PCNA) and an M phase-specific marker H3S10ph. In this chapter, I introduce the timing of division and inheritance of organelles during the cell cycle in C. merolae and application of the synchronous culture to transcriptome analysis for identifying novel genes related to the division and inheritance of organelles.

The cells of Cyanidioschyzon merolae have advantageous features for omics analyses. The genomes of the cell nucleus, mitochondrion, and chloroplast have been completely sequenced in C. merolae and are thus ready for omics analyses utilizing complete genome information. Phase-specific microarray analysis of a synchronous culture of C. merolae revealed the gene expression profiles nearing the organelle division cycle and mitotic cycle. In ammonium-depleted cultures of C. merolae, the gene expression patterns of cells were analyzed, and the nitrate assimilation pathway was revealed. Microarray analysis for chloroplast-encoded and mitochondrion-encoded genes of C. merolae was also conducted. The results revealed that organelle gene expressions correlate to the light period or the progression of the cell cycle. Proteomic analyses of organelles and sub-organelles of C. merolae have been promoted using MALDI TOF-MS. Comparative proteomic analysis of chloroplasts between interphase and metaphase revealed the dynamic changes in protein composition. Proteins composing the dividing machineries of chloroplasts and peroxisomes were identified using isolation techniques for sub-organellar structure. Isolated vacuoles and storage glucans were also analyzed, and proteins associated with the functions of these organelles were found.