Spatial Distribution of Flagellated Microalgae Chlamydomonas reinhardtii in a Quasi-Two-Dimensional Space
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PROVIDER: S-EPMC10146014 | biostudies-literature | 2023 Apr
REPOSITORIES: biostudies-literature
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Project description:Correct folding and post-translational modifications are vital for therapeutic proteins to elicit their biological functions. Osteopontin (OPN), a bone regenerative protein present in a range of mammalian cells, is an acidic phosphoprotein with multiple potential phosphorylation sites. In this study, the ability of unicellular microalgae, Chlamydomonas reinhardtii, to produce phosphorylated recombinant OPN in its chloroplast is investigated. This study further explores the impact of phosphorylation and expression from a "plant-like" algae on separation of OPN. Chromatography resins ceramic hydroxyapatite (CHT) and Gallium-immobilized metal affinity chromatography (Ga-IMAC) were assessed for their binding specificity to phosphoproteins. Non-phosphorylated recombinant OPN expressed in E. coli was used to compare the specificity of interaction of the resins to phosphorylated OPN. We observed that CHT binds OPN by multimodal interactions and was better able to distinguish phosphorylated proteins in the presence of 250 mM NaCl. Ga-IMAC interaction with OPN was not selective to phosphorylation, irrespective of salt, as the resin bound OPN from both algal and bacterial sources. Anion exchange chromatography proved an efficient capture method to partially separate major phosphorylated host cell protein impurities such as Rubisco from OPN.
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Project description:Despite the fact that microplastics (MPs) facilitate the adsorption of environmental organic pollutants and influence their toxicity for organisms, more study is needed on the combination of MPs and antibiotics pollutant effects. In this study, polystyrene MPs (1 and 5 μm) and sulfadiazine (SDZ) were examined separately and in combination on freshwater microalga, Chlamydomonas reinhardtii. The results suggest that both the MPs and SDZ alone and in combination inhibited the growth of microalgae with an increasing concentration of MPs and SDZ (5-200 mg l-1); however, the inhibition rate was reduced by combination. Upon exposure for 7 days, both the MPs and SDZ inhibited algal growth, reduced chlorophyll content, and enhanced superoxide dismutase (SOD) activities, whereas glutathione peroxidase (GSH-Px) activity was elevated only with the exposure of 1 μm MPs. Fluorescence microscopy and scanning electron microscopy also indicated that particle size contributed to the combined toxicity by aggregating MPs with periphery pollutants. Further, the amount of extracellular secretory protein increased in the presence of MPs and SDZ removal ratio decreased when MPs and SDZ coexisted, suggesting that MPs affected SDZ metabolism by microalgae. The particle size of microplastics affected the toxicity of MPs on microalgae and the combined effect of MPs and SDZ could be mitigated by MPs adsorption. These findings provide insight into microalgae responses to the combination of MPs and antibiotics in water ecosystems.
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Project description:Hydrogen peroxide (H2O2) is recognized as an important signaling molecule in plants. We sought to establish a genetically encoded, fluorescent H2O2 sensor that allows H2O2 monitoring in all major subcompartments of a Chlamydomonas cell. To this end, we used the Chlamydomonas Modular Cloning toolbox to target the hypersensitive H2O2 sensor reduction-oxidation sensitive green fluorescent protein2-Tsa2ΔCR to the cytosol, nucleus, mitochondrial matrix, chloroplast stroma, thylakoid lumen, and endoplasmic reticulum (ER). The sensor was functional in all compartments, except for the ER where it was fully oxidized. Employing our novel sensors, we show that H2O2 produced by photosynthetic linear electron transport (PET) in the stroma leaks into the cytosol but only reaches other subcellular compartments if produced under nonphysiological conditions. Furthermore, in heat-stressed cells, we show that cytosolic H2O2 levels closely mirror temperature up- and downshifts and are independent from PET. Heat stress led to similar up- and downshifts of H2O2 levels in the nucleus and, more mildly, in mitochondria but not in the chloroplast. Our results thus suggest the establishment of steep intracellular H2O2 gradients under normal physiological conditions with limited diffusion into other compartments. We anticipate that these sensors will greatly facilitate future investigations of H2O2 biology in plant cells.
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Project description:A general ejection theory of polymer is developed in a two- and three-dimensional space. A polymer is confined initially in a cavity and ejects spontaneously to the outer space through a nanopore channel without the help of any external stimulus. A reflective wall boundary is set at the pore entrance to prevent the falling of the head monomer of chain into the cavity. Three stages are distinguished in a process: (1) an entering stage, in which the head monomer enters the pore to search for a way to traverse the pore channel, (2) a main ejection stage, in which the chain body is transported from the cavity to the outer space, (3) a leaving stage, in which the tail monomer passes through and leaves the pore channel. Depending on the number of the monomers remaining in the cavity, the main ejection stage can be divided into the confined and the non-confined stages. The non-confined stage can be further split into the thermal escape and the entropic pulling stages. The Onsager's variational principle is applied to derive the kinetics equation of ejection. The escape time is calculated from the corresponding Kramers' escape problem. Extensive molecular dynamics simulations are then performed in a quasi two-dimensional space to verify the theory. The variation of the ejection speed is carefully examined. The decreasing behavior of the number of monomers in the cavity is studied in details. The scaling properties of the spending time at each processing stage are investigated systematically by varying the chain length, the cavity diameter, and the initial volume fraction of chain. The results of simulation support firmly the predictions of the theory, cross-checked in the studies of various topics. In combining with the previous investigations in the three-dimensional space, the generalized theory is very robust, able to explain the two seemly different phenomena, polymer ejection and polymer translocation, together under the same theoretical framework in the two space dimensions.
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Project description:Spontaneous mutations are the source of new genetic variation and are thus central to the evolutionary process. In molecular evolution and quantitative genetics, the nature of genetic variation depends critically on the distribution of effects of mutations on fitness and other quantitative traits. Spontaneous mutation accumulation (MA) experiments have been the principal approach for investigating the overall rate of occurrence and cumulative effect of mutations but have not allowed the phenotypic effects of individual mutations to be studied directly. Here, we crossed MA lines of the green alga Chlamydomonas reinhardtii with its unmutated ancestral strain to create haploid recombinant lines, each carrying an average of 50% of the accumulated mutations in a large number of combinations. With the aid of the genome sequences of the MA lines, we inferred the genotypes of the mutations, assayed their growth rate as a measure of fitness, and inferred the distribution of fitness effects (DFE) using a Bayesian mixture model. We infer that the DFE is highly leptokurtic (L-shaped). Of mutations with absolute fitness effects exceeding 1%, about one-sixth increase fitness in the laboratory environment. The inferred distribution of effects for deleterious mutations is consistent with a strong role for nearly neutral evolution. Specifically, such a distribution predicts that nucleotide variation and genetic variation for quantitative traits will be insensitive to change in the effective population size.
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Project description:Microalgae-based biodiesel production has many advantages over crude oil extraction and refinement, thus attracting more and more concern. Protein ubiquitination is a crucial mechanism in eukaryotes to regulate physiological responses and cell development, which is highly related to algal biodiesel production. Cullins as the molecular base of cullin-RING E3 ubiquitin ligases (CRLs), which are the largest known class of ubiquitin ligases, control the life activities of eukaryotic cells. Here, three cullins (CrCULs) in the green microalgae Chlamydomonas reinhardtii were identified and characterized. To investigate the roles of CrCULs in lipid metabolism, the gene expression profiles of CrCULs under nutrition starvation were examined. Except for down-regulation under nitrogen starvation, the CrCUL3 gene was induced by sulfur and iron starvation. CrCUL2 seemed insensitive to nitrogen and sulfur starvation because it only had changes after treatment for eight days. CrCUL4 exhibited an expression peak after nitrogen starvation for two days but this declined with time. All CrCULs expressions significantly increased under iron deficiency at two and four days but decreased thereafter. The silencing of CrCUL2 and CrCUL4 expression using RNAi (RNA interference) resulted in biomass decline and lipids increase but an increase of 20% and 28% in lipid content after growth for 10 days, respectively. In CrCUL2 and CrCUL4 RNAi lines, the content of fatty acids, especially C16:0 and C18:0, notably increased as well. However, the lipid content and fatty acids of the CrCUL3 RNAi strain slightly changed. Moreover, the subcellular localization of CrCUL4 showed a nuclear distribution pattern. These results suggest CrCUL2 and CrCUL4 are regulators for lipid accumulation in C. reinhardtii. This study may offer an important complement of lipid biosynthesis in microalgae.
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Project description: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.
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Project description:The coupling of Chlamydomonas reinhardtii biomass production for nutrients removal of Escherichia coli anaerobic broth (EAB) is thought to be an economically feasible option for the cultivation of microalgae. The feasibility of growing microalgae in using EAB high in nutrients for the production of more biomass was examined. EAB comprised of nutrient-abundant effluents, which can be used to produce microalgae biomass and remove environment pollutant simultaneously. In this study, C. reinhardtii 21gr (cc1690) was cultivated in different diluted E. coli anaerobic broth supplemented with trace elements under mixotrophic and heterotrophic conditions. The results showed that C. reinhardtii grown in 1×, 1/2×, 1/5× and 1/10×E. coli anaerobic broth under mixotrophic conditions exhibited specific growth rates of 2.71, 2.68, 1.45, and 1.13 day-1, and biomass production of 201.9, 184.2, 175.5, and 163.8 mg L-1, respectively. Under heterotrophic conditions, the specific growth rates were 1.80, 1.86, 1.75, and 1.02 day-1, and biomass production were 45.6, 29.4, 15.8, and 12.1 mg L-1, respectively. The removal efficiency of chemical oxygen demand, total-nitrogen and total-phosphorus from 1×E. coli anaerobic broth was 21.51, 22.41, and 15.53%. Moreover, the dry biomass had relatively high carbohydrate (44.3%) and lipid content (18.7%). Therefore, this study provides an environmentally sustainable as well economical method for biomass production in promising model microalgae and subsequently paves the way for industrial use.
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Project description:Efficient protein secretion is a desirable trait for any recombinant protein expression system, together with simple, low-cost, and defined media, such as the typical media used for photosynthetic cultures of microalgae. However, low titers of secreted heterologous proteins are usually obtained, even with the most extensively studied microalga Chlamydomonas reinhardtii, preventing their industrial application. In this study, we aimed to expand and evaluate secretory signal peptides (SP) for heterologous protein secretion in C. reinhardtii by comparing previously described SP with untested sequences. We compared the SPs from arylsulfatase 1 and carbonic anhydrase 1, with those of untried SPs from binding protein 1, an ice-binding protein, and six sequences identified in silico. We identified over 2000 unique SPs using the SignalP 4.0 software. mCherry fluorescence was used to compare the protein secretion of up to 96 colonies for each construct, non-secretion construct, and parental wild-type cc1690 cells. Supernatant fluorescence varied according to the SP used, with a 10-fold difference observed between the highest and lowest secretors. Moreover, two SPs identified in silico secreted the highest amount of mCherry. Our results demonstrate that the SP should be carefully selected and that efficient sequences can be coded in the C. reinhardtii genome. The SPs described here expand the portfolio available for research on heterologous protein secretion and for biomanufacturing applications.
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Project description:The green microalga Chlamydomonas reinhardtii is emerging as a promising cell biofactory for secreted recombinant protein (RP) production. In recent years, the generation of the broadly used cell wall-deficient mutant strain UVM4 has allowed for a drastic increase in secreted RP yields. However, purification of secreted RPs from the extracellular space of C. reinhardtii strain UVM4 is challenging. Previous studies suggest that secreted RPs are trapped in a matrix of cell wall protein aggregates populating the secretome of strain UVM4, making it difficult to isolate and purify the RPs. To better understand the nature and behaviour of these extracellular protein aggregates, we analysed and compared the extracellular proteome of the strain UVM4 to its cell-walled ancestor, C. reinhardtii strain 137c. When grown under the same conditions, strain UVM4 produced a unique extracellular proteomic profile, including a higher abundance of secreted cell wall glycoproteins. Further characterization of high molecular weight extracellular protein aggregates in strain UVM4 revealed that they are largely comprised of pherophorins, a specific class of cell wall glycoproteins. Our results offer important new insights into the extracellular space of strain UVM4, including strain-specific secreted cell wall proteins and the composition of the aggregates possibly related to impaired RP purification. The discovery of pherophorins as a major component of extracellular protein aggregates will inform future strategies to remove or prevent aggregate formation, enhance purification of secreted RPs, and improve yields of recombinant biopharmaceuticals in this emerging cell biofactory. KEY POINTS: • Extracellular protein aggregates hinder purification of recombinant proteins in C. reinhardtii • Unassembled cell wall pherophorins are major components of extracellular protein aggregates • Known aggregate composition informs future strategies for recombinant protein purification.
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