Aqueous enzymatic protein and lipid release from the microalgae Chlamydomonas reinhardtii

Sierra, Laura Soto; Wilken, Lisa R.; Dixon, Chelsea

doi:10.1186/s40643-020-00328-4

Cited by 20 publications

(6 citation statements)

References 48 publications

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“…It has been established that nitrogen deprivation induces accumulation of TAGs and starch. TAGs, act as a storage of carbon rich compounds, a resource for future recovery (26). Concurrent to earlier research it has been observed that Chlamydomonas spp.…”

Section: Stress In the Algal Cultures Has Led To An Increase In Accumulation Of Lipidssupporting

confidence: 56%

Evaluation of growth pattern and biochemical components of Chlamydomonas reinhardtii Dangeard

Sharma

2022

Plant Sci. Today

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Chlamydomonas reinhardtii Dangeard is the simplest motile, unicellular fresh water alga of class Chlorophyceae. It also functions as the most efficient model system for converting solar energy to chemical energy in the form of various metabolites. The objective of the present work is to deal with the growth conditions/growth kinetics of Chlamydomonas reinhardtii Dangeard, required for optimal biomass production. The parameter used to study growth of algae was, photosynthetic measurement, biomass, proteins and lipid measurement, which vary with the change in the cultural conditions. Present investigations reveal that change in protein content is positively correlated with the increase in biomass, revealing that the algae can grow rapidly in laboratory/cultural conditions. Lipid content shows a negative correlation with proteins and biomass. Lipids are known to have a role as structural components, in hydration and also in signaling events. Lipids, mainly the triacyl glycerides (TAGs) act as storage compounds enabling the microalgae to survive in adverse environmental conditions. Lipidic content increases in Chlamydomonas reinhardtii increases with optimal light and nutrient system. The increase is in the form of triacyl glycerides which serve as precursors for the production of biodiesel and bioethanol. Conclusion- Further research is required to investigate the interactions of biomolecules and growth of algae.

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Section: Stress In the Algal Cultures Has Led To An Increase In Accumulation Of Lipidssupporting

confidence: 56%

Evaluation of growth pattern and biochemical components of Chlamydomonas reinhardtii Dangeard

Sharma

2022

Plant Sci. Today

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“…Each plate of cells was resuspended in 10 ml of M-N/5 (minimal minus nitrogen / 5) [ 109 ] and rocked in light at room temperature for 3 hrs. Cells were spun at 2,000 x g for 5 min, then resuspended in 10 ml of autolysin [ 114 ] for 1 hr. Next, cells were spun at 2,000 x g for 5 min and resuspended in 750 μl of cell lysis buffer (20 mM Tris-HCl pH 7.5, 5 mM MgCl 2 (Sigma, 232–094, lot #088J00292), 300 mM NaCl (Fisher Scientific, BP358-212, lot #976181), 5 mM Dithiothreitol (RPI Research Products, D11000-5.0, lot #12936482), 0.1% Triton-X100 (EM Science, TX1566-1, lot #31050) [ 64 ], with 20 μM phenylmethylsulfonyl fluoride (Sigma, P-7626, lot #050M1688) and the Protease Inhibitor Cocktail for plants (Sigma, p9599, lot #086K4075).…”

Section: Methodsmentioning

confidence: 99%

Gene dosage of independent dynein arm motor preassembly factors influences cilia assembly in Chlamydomonas reinhardtii

Penny,

Dutcher

2024

PLoS Genet

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Motile cilia assembly utilizes over 800 structural and cytoplasmic proteins. Variants in approximately 58 genes cause primary ciliary dyskinesia (PCD) in humans, including the dynein arm (pre)assembly factor (DNAAF) gene DNAAF4. In humans, outer dynein arms (ODAs) and inner dynein arms (IDAs) fail to assemble motile cilia when DNAAF4 function is disrupted. In Chlamydomonas reinhardtii, a ciliated unicellular alga, the DNAAF4 ortholog is called PF23. The pf23-1 mutant assembles short cilia and lacks IDAs, but partially retains ODAs. The cilia of a new null allele (pf23-4) completely lack ODAs and IDAs and are even shorter than cilia from pf23-1. In addition, PF23 plays a role in the cytoplasmic modification of IC138, a protein of the two-headed IDA (I1/f). As most PCD variants in humans are recessive, we sought to test if heterozygosity at two genes affects ciliary function using a second-site non-complementation (SSNC) screening approach. We asked if phenotypes were observed in diploids with pairwise heterozygous combinations of 21 well-characterized ciliary mutant Chlamydomonas strains. Vegetative cultures of single and double heterozygous diploid cells did not show SSNC for motility phenotypes. When protein synthesis is inhibited, wild-type Chlamydomonas cells utilize the pool of cytoplasmic proteins to assemble half-length cilia. In this sensitized assay, 8 double heterozygous diploids with pf23 and other DNAAF mutations show SSNC; they assemble shorter cilia than wild-type. In contrast, double heterozygosity of the other 203 strains showed no effect on ciliary assembly. Immunoblots of diploids heterozygous for pf23 and wdr92 or oda8 show that PF23 is reduced by half in these strains, and that PF23 dosage affects phenotype severity. Reductions in PF23 and another DNAAF in diploids affect the ability to assemble ODAs and IDAs and impedes ciliary assembly. Thus, dosage of multiple DNAAFs is an important factor in cilia assembly and regeneration.

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“…Each plate was resuspended in 10 ml of M-N/5 and rocked in light at room temperature for 3 hr. Cells were spun at 2,000 x g for 5 min, resuspended in 10 ml of autolysin [144] for 1 hr. Next cells were spun at 2,000 x g for 5 min and resuspended in 600-750 μl of cell lysis buffer (20 mM Tris–HCl pH 7.5, 5 mM MgCl 2 , 300 mM NaCl, 5 mM Dithiothreitol, 0.1% Triton-X100 [93], with 20 μM phenylmethylsulfonyl fluoride (Sigma, Lot # 050M1688) and the Protease Inhibitor Cocktail for plants (Sigma, p9599, lot #086K4075).…”

Section: Methodsmentioning

confidence: 99%

Reduced dosage of multiple dynein arm preassembly factors limits cilia regeneration inChlamydomonas reinhardtii

Penny,

Dutcher

2023

Preprint

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Motile cilia are complex organelles comprised of >800 structural proteins. Variants in 58 of these genes cause primary ciliary dyskinesia (PCD) in humans. We used a second-site non-complementation approach in diploidChlamydomonas reinhardtiistrains to investigate whether reduced dosage of different combinations of motile cilia proteins affects cilia growth / regeneration. Temperature-sensitive mutants in intraflagellar transport genes fail to complement in heterozygous diploids at the restrictive temperature, which may be due to poisonous interactions or a reduction in protein levels. Diploid strains heterozygous for 211 double mutant combinations in 21 genes are phenotypically recessive when unperturbed and do not show SSNC. Consequently, we developed a sensitized screen. When protein synthesis is inhibited, cells utilize the existing cytoplasmic pool of proteins, which is insufficient to build full-length cilia. Six double heterozygous strains in dynein arm preassembly factors (DNAAFs)pf13, pf23, wdr92,andoda8regrow cilia that are shorter than wild-type diploids, which suggests that double heterozygosity limits the pool of assembled dynein arms further. We isolated a new null allele (pf23-4)that shows a more severe loss of ODAs and IDAs in cilia than the previously studiedpf23-1hypomorphic allele. We also identified a new role for PF23 in cytoplasmic modification of IC138, protein of the I1/finner dynein arm. In our SSNC assay, thepf23-4allele also exhibits a more severe phenotype thanpf23-1. Whole cell extract immunoblots show a reduction of PF23 protein to one-half of wild-type levels in both single and double heterozygous genotypes. Thepf13mutant shows SSNC with mutants in two outer dynein arm structural proteins, ODA6, andODA9. We suggest that reduction of multiple dynein preassembly factors limits the pool of assembled dynein arms needed for cilia assembly and regeneration. Our data support PF23 as a scaffolding hub protein involved in dynein arm assembly.Author SummaryMotile cilia are essential for movement of cells and fluids. In humans, motile cilia defects cause primary ciliary dyskinesia (PCD), a rare disease characterized by recurring respiratory infections, left-right asymmetry defects, ear infections, and infertility. Many genes are involved in motile cilia function and assembly. We useChlamydomonas reinhardtiito study the effects of changes in protein levels in different combinations of proteins needed for motile cilia. Our results show that the proteins that help fold and assemble the dynein motors in the cytoplasm are sensitive to changes in the dosage of these preassembly factors. Reductions in proteins involved in multiple steps of the pathway prevents efficient dynein preassembly and cilia regeneration under stressful cellular conditions.

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Aqueous enzymatic protein and lipid release from the microalgae Chlamydomonas reinhardtii

Cited by 20 publications

References 48 publications

Evaluation of growth pattern and biochemical components of Chlamydomonas reinhardtii Dangeard

Evaluation of growth pattern and biochemical components of Chlamydomonas reinhardtii Dangeard

Gene dosage of independent dynein arm motor preassembly factors influences cilia assembly in Chlamydomonas reinhardtii

Reduced dosage of multiple dynein arm preassembly factors limits cilia regeneration inChlamydomonas reinhardtii

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