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Using single cell cultivation system for on-chip monitoring of the interdivision timer in Chlamydomonas reinhardtii cell cycle
Kazunori Matsumura, Toshiki Yagi, Akihiro Hattori, Mikhail Soloviev, Kenji Yasuda
Journal of Nanobiotechnology , 2010, DOI: 10.1186/1477-3155-8-23
Abstract: Proliferating eukaryotic cells maintain a relatively constant size by coordinating their growth with the progression of the cell cycle [1], and their responses to changing environmental conditions, which are mainly evident in the G1 phase [2-4]. When sufficient nutrients are not available, cells delay their progress through the G1 phase or enter a specialized resting state known as G0 [5]. If sufficient nutrients are available, cells in early G1 or G0 phase pass through a control point that in the yeast cell cycle is referred to as the 'start' [6,7] and in the mammalian cell cycle is referred to as the 'restriction point' [5,8]. After passing through this control point, cells are committed to initiating DNA replication and proceed to the S phase even if sufficient nutrients are no longer available [5,9]. Both size-dependent and time-dependent controllers have been proposed to determine the length of the G1 phase [7]: the 'sizer' determines whether the cell has reached the threshold size needed to progress to the next phase, and the 'timer' determines whether the cells have been in the G1 phase long enough. The exact molecular mechanisms behind these controllers remain unknown because experimentalists have not been able to control environmental conditions, such as nutrient conditions, cell-cell interactions and cell cycle phase synchronization, well enough for their effects to be analyzed quantitatively.Several groups used microfluidic-type devices for studying the mechanisms of cell cycle regulation and division control under the controlled conditions [10-18]. We have earlier developed an on-chip cultivation system for use with the unicellular green algae Chlamydomonas reinhardtii. The photosynthetic algae Chlamydomonas uses light as the source of energy. This property allows one to easily manipulate and vary the amount of energy supplied to the cells by varying the light, whilst maintaining other environmental conditions (such as the carbon dioxide concentration in
A Forward Genetic Approach in Chlamydomonas reinhardtii as a Strategy for Exploring Starch Catabolism  [PDF]
Hande Tun?ay, Justin Findinier, Thierry Duchêne, Virginie Cogez, Charlotte Cousin, Gilles Peltier, Steven G. Ball, David Dauvillée
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0074763
Abstract: A screen was recently developed to study the mobilization of starch in the unicellular green alga Chlamydomonas reinhardtii. This screen relies on starch synthesis accumulation during nitrogen starvation followed by the supply of nitrogen and the switch to darkness. Hence multiple regulatory networks including those of nutrient starvation, cell cycle control and light to dark transitions are likely to impact the recovery of mutant candidates. In this paper we monitor the specificity of this mutant screen by characterizing the nature of the genes disrupted in the selected mutants. We show that one third of the mutants consisted of strains mutated in genes previously reported to be of paramount importance in starch catabolism such as those encoding β-amylases, the maltose export protein, and branching enzyme I. The other mutants were defective for previously uncharacterized functions some of which are likely to define novel proteins affecting starch mobilization in green algae.
Cellular oxido-reductive proteins of Chlamydomonas reinhardtii control the biosynthesis of silver nanoparticles
Indu Barwal, Peeyush Ranjan, Suneel Kateriya, Subhash Yadav
Journal of Nanobiotechnology , 2011, DOI: 10.1186/1477-3155-9-56
Abstract: The C. reinhardtii cell free extract (in vitro) and in vivo cells mediated synthesis of silver nanoparticles reveals SNPs of size range 5 ± 1 to 15 ± 2 nm and 5 ± 1 to 35 ± 5 nm respectively. In vivo biosynthesized SNPs were localized in the peripheral cytoplasm and at one side of flagella root, the site of pathway of ATP transport and its synthesis related enzymes. This provides an evidence for the involvement of oxidoreductive proteins in biosynthesis and stabilization of SNPs. Alteration in size distribution and decrease of synthesis rate of SNPs in protein-depleted fractions confirmed the involvement of cellular proteins in SNPs biosynthesis. Spectroscopic and SDS-PAGE analysis indicate the association of various proteins on C. reinhardtii mediated in vivo and in vitro biosynthesized SNPs. We have identified various cellular proteins associated with biosynthesized (in vivo and in vitro) SNPs by using MALDI-MS-MS, like ATP synthase, superoxide dismutase, carbonic anhydrase, ferredoxin-NADP+ reductase, histone etc. However, these proteins were not associated on the incubation of pre-synthesized silver nanoparticles in vitro.Present study provides the indication of involvement of molecular machinery and various cellular proteins in the biosynthesis of silver nanoparticles. In this report, the study is mainly focused towards understanding the role of diverse cellular protein in the synthesis and capping of silver nanoparticles using C. reinhardtii as a model system.Silver nanoparticles (SNPs) have extensive applications in civil, therapeutic and industrial areas as catalyst, cryogenic superconductor, biosensor, microelectronic and bacteriostatic materials [1-3], etc. These SNPs have been synthesized by various physical, chemical and biological methods. Among the various known synthesis methods, biosynthesis of silver nanoparticles is preferred as it is environmentally safe, low cost and less toxic [4]. These biologically synthesized silver nanoparticles (SNPs) could
Circadian rhythms lit up in Chlamydomonas
Ghislain Breton, Steve A Kay
Genome Biology , 2006, DOI: 10.1186/gb-2006-7-4-215
Abstract: Chlamydomonas reinhardtii is often referred to as 'green yeast' to convey the usefulness of this eukaryotic unicellular green alga as a model organism for plant research [1]. Like a plant cell, Chlamydomonas possesses a cell wall and a chloroplast, but like animal sperm cells, it has a flagellum. This structure enables it to carry out phototaxis, moving towards or away from light and so maximizing light perception for photosynthesis and minimizing photodamage. When given a light source, C. reinhardtii can be grown in large quantities in a simple medium containing only inorganic salts. In the dark, it can also grow non-photosynthetically with acetate as its sole carbon source. As most of its life cycle is in the haploid phase, C. reinhardtii is amenable to genetic screens, which have led to major discoveries in the fields of chloroplast biogenesis and the structure of eukaryotic flagella and the basal bodies from which they derive [2].In the past decade, the C. reinhardtii community has embraced the genomic revolution [3]. In addition to the mitochondrial and chloroplast genomes, the 110 Mb nuclear genome has been sequenced and is currently being annotated using the information generated by two large-scale expressed sequence tag (EST) projects. More than 15,000 genes have now been identified and this information is accessible through the C. reinhardtii genome portal [4]. Genome-scale analyses of protein sequences have described the repertoire of cell-cycle regulatory proteins, tyrosine kinases and flagella and basal body genes [5-8]. The EST sequencing projects have also been instrumental in designing cDNA macroarrays and oligonucleotide arrays [3,9,10]. In addition, the generation of a sequence database has facilitated the data-mining processes needed to identify large protein complexes by mass spectrometry [11,12]. Finally, progress made toward facilitating map-based cloning, and insertional mutagenesis has turned this little green eukaryotic cell into a key model
Predicting the Physiological Role of Circadian Metabolic Regulation in the Green Alga Chlamydomonas reinhardtii  [PDF]
Sascha Sch?uble, Ines Heiland, Olga Voytsekh, Maria Mittag, Stefan Schuster
PLOS ONE , 2011, DOI: 10.1371/journal.pone.0023026
Abstract: Although the number of reconstructed metabolic networks is steadily growing, experimental data integration into these networks is still challenging. Based on elementary flux mode analysis, we combine sequence information with metabolic pathway analysis and include, as a novel aspect, circadian regulation. While minimizing the need of assumptions, we are able to predict changes in the metabolic state and can hypothesise on the physiological role of circadian control in nitrogen metabolism of the green alga Chlamydomonas reinhardtii.
Selenoprotein-Transgenic Chlamydomonas reinhardtii  [PDF]
Qintang Hou,Shi Qiu,Qiong Liu,Jing Tian,Zhangli Hu,Jiazuan Ni
Nutrients , 2013, DOI: 10.3390/nu5030624
Abstract: Selenium (Se) deficiency is associated with the occurrence of many diseases. However, excessive Se supplementation, especially with inorganic Se, can result in toxicity. Selenoproteins are the major forms of Se in vivo to exert its biological function. Expression of those selenoproteins, especially with the application of a newly developed system, is thus very important for studying the mechanism of Se in nutrition. The use of Chlamydomonas reinhardtii ( C. reinhardtii) as a biological vector to express an heterogeneous protein is still at the initial stages of development. In order to investigate the possibility of using this system to express selenoproteins, human 15-KDa selenoprotein (Sep15), a small but widely distributed selenoprotein in mammals, was chosen for the expression platform test. Apart from the wild-type human Sep15 gene fragment, two Sep15 recombinants were constructed containing Sep15 open reading frame (ORF) and the selenocysteine insertion sequence (SECIS) element from either human Sep15 or C. reinhardtii selenoprotein W1, a highly expressed selenoprotein in this alga. Those Sep15-containing plasmids were transformed into C. reinhardtii CC-849 cells. Results showed that Sep15 fragments were successfully inserted into the nuclear genome and expressed Sep15 protein in the cells. The transgenic and wild-type algae demonstrated similar growth curves in low Se culture medium. To our knowledge, this is the first report on expressing human selenoprotein in green alga.
Rapid Induction of Lipid Droplets in Chlamydomonas reinhardtii and Chlorella vulgaris by Brefeldin A  [PDF]
Sangwoo Kim, Hanul Kim, Donghwi Ko, Yasuyo Yamaoka, Masumi Otsuru, Maki Kawai-Yamada, Toshiki Ishikawa, Hee-Mock Oh, Ikuo Nishida, Yonghua Li-Beisson, Youngsook Lee
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0081978
Abstract: Algal lipids are the focus of intensive research because they are potential sources of biodiesel. However, most algae produce neutral lipids only under stress conditions. Here, we report that treatment with Brefeldin A (BFA), a chemical inducer of ER stress, rapidly triggers lipid droplet (LD) formation in two different microalgal species, Chlamydomonas reinhardtii and Chlorella vulgaris. LD staining using Nile red revealed that BFA-treated algal cells exhibited many more fluorescent bodies than control cells. Lipid analyses based on thin layer chromatography and gas chromatography revealed that the additional lipids formed upon BFA treatment were mainly triacylglycerols (TAGs). The increase in TAG accumulation was accompanied by a decrease in the betaine lipid diacylglyceryl N,N,N-trimethylhomoserine (DGTS), a major component of the extraplastidic membrane lipids in Chlamydomonas, suggesting that at least some of the TAGs were assembled from the degradation products of membrane lipids. Interestingly, BFA induced TAG accumulation in the Chlamydomonas cells regardless of the presence or absence of an acetate or nitrogen source in the medium. This effect of BFA in Chlamydomonas cells seems to be due to BFA-induced ER stress, as supported by the induction of three homologs of ER stress marker genes by the drug. Together, these results suggest that ER stress rapidly triggers TAG accumulation in two green microalgae, C. reinhardtii and C. vulgaris. A further investigation of the link between ER stress and TAG synthesis may yield an efficient means of producing biofuel from algae.
A Novel Negative Fe-Deficiency-Responsive Element and a TGGCA-Type-Like FeRE Control the Expression of FTR1 in Chlamydomonas reinhardtii
Xiaowen Fei,Mats Eriksson,Yajun Li,Xiaodong Deng
Journal of Biomedicine and Biotechnology , 2010, DOI: 10.1155/2010/790247
Abstract: We have reported three Fe-deficiency-responsive elements (FEREs), FOX1, ATX1, and FEA1, all of which are positive regulatory elements in response to iron deficiency in Chlamydomonas reinhardtii. Here we describe FTR1, another iron regulated gene and mutational analysis of its promoter. Our results reveal that the FeREs of FTR1 distinguish itself from other iron response elements by containing both negative and positive regulatory regions. In FTR1, the ?291/?236 region from the transcriptional start site is necessary and sufficient for Fe-deficiency-inducible expression. This region contains two positive FeREs with a TGGCA-like core sequence: the FtrFeRE1 (ATGCAGGCT) at ?287/?279 and the FtrFeRE2 (AAGCGATTGCCAGAGCGC) at ?253/?236. Furthermore, we identified a novel FERE, FtrFeRE3 (AGTAACTGTTAAGCC) localized at ?319/?292, which negatively influences the expression of FTR1.
Expression of human soluble TRAIL in Chlamydomonas reinhardtii chloroplast
Zongqi Yang,yinü Li,Feng Chen,Dong Li,Zhifang Zhang,Yanxin Liu,Dexian Zheng,Yong Wang,Guifang Shen
Chinese Science Bulletin , 2006, DOI: 10.1007/s11434-006-2041-0
Abstract: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces selectively apoptosis in various tumor cells and virus-infected cells, but rarely in normal cells. A chloroplast expression vector, p64TRAIL, containing the cDNA coding for the soluble TRAIL (sTRAIL), was constructed with clpP-trnL-petB-chlL-rpl23-rpl2 as Chlamydomonas reinhardtii plastid homologous recombinant fragments and spectinomycin-resistant aadA gene as a select marker. The plasmid p64TRAIL was transferred into the chloroplast genome of C. reinhardtii by the biolistic method. Three independently transformed lines were obtained by 100 mg/L spectinomycin selection. PCR amplification, Southern blot analysis of the sTRAIL coding region DNA and cultivation cells in the dark all showed that the exogenous DNA had been integrated into chloroplast genome of C. reinhardtii. Western blot analysis showed that human soluble TRAIL was expressed in C. reinhardtii chloroplast. The densitometric analysis of Western blot indicated that the expressed human sTRAIL protein in the chloroplasts of C. reinhardtii accounted for about 0.43%–0.67% of the total soluble proteins. These experimental results demonstrated the possibility of using transgenic chloroplasts of green alga as bioreactors for production of biopharmaceuticals.
Measurement of Lipid Droplet Accumulation Kinetics in Chlamydomonas reinhardtii Using Seoul-Fluor  [PDF]
Jae Woo Park,Sang Cheol Na,Youngjun Lee,Sanghee Lee,Seung Bum Park,Noo Li Jeon
Energies , 2013, DOI: 10.3390/en6115703
Abstract: Alternative energy resources have become an important issue due to the limited stocks of petroleum-based fuel. Microalgae, a source of renewable biodiesel, use solar light to convert CO 2 into lipid droplets (LDs). Quantification of LDs in microalgae is required for developing and optimizing algal bioprocess engineering. However, conventional quantification methods are both time and labor-intensive and difficult to apply in high-throughput screening systems. LDs in plant and mammalian cells can be visualized by staining with various fluorescence probes such as the Nile Red, BODIPY, and Seoul-Fluor (SF) series. This report describes the optimization of LD staining in Chlamydomonas reinhardtii with SF probes via systematic variations of dye concentration, staining time, temperature, and pH. A protocol for quantitative measurement of accumulation kinetics of LDs in C. reinhardtii was developed using a spectrofluorimeter and the accuracy of LD size measurement was confirmed by transmission electron microscopy (TEM). Our results indicate that our spectrofluorimeter-based measurement approach can monitor kinetics of intracellular LDs (in control and nitrogen-source-starved Chlamydomonas reinhardtii) accumulation that has not been possible in the case of conventional imaging-based methods. Our results presented here confirmed that an SF44 can be a powerful tool for in situ monitoring and tracking of intracellular LDs formation.
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