Publications by authors named "Daniela Strenkert"

22 Publications

  • Page 1 of 1

Single-cell visualization and quantification of trace metals in lysosome-related organelles.

Proc Natl Acad Sci U S A 2021 Apr;118(16)

California Institute for Quantitative Biosciences, University of California, Berkeley, CA 94720;

The acidocalcisome is an acidic organelle in the cytosol of eukaryotes, defined by its low pH and high calcium and polyphosphate content. It is visualized as an electron-dense object by transmission electron microscopy (TEM) or described with mass spectrometry (MS)-based imaging techniques or multimodal X-ray fluorescence microscopy (XFM) based on its unique elemental composition. Compared with MS-based imaging techniques, XFM offers the additional advantage of absolute quantification of trace metal content, since sectioning of the cell is not required and metabolic states can be preserved rapidly by either vitrification or chemical fixation. We employed XFM in to determine single-cell and organelle trace metal quotas within algal cells in situations of trace metal overaccumulation (Fe and Cu). We found up to 70% of the cellular Cu and 80% of Fe sequestered in acidocalcisomes in these conditions and identified two distinct populations of acidocalcisomes, defined by their unique trace elemental makeup. We utilized the mutant, defective in polyphosphate synthesis and failing to accumulate Ca, to show that Fe sequestration is not dependent on either. Finally, quantitation of the Fe and Cu contents of individual cells and compartments via XFM, over a range of cellular metal quotas created by nutritional and genetic perturbations, indicated excellent correlation with bulk data from corresponding cell cultures, establishing a framework to distinguish the nutritional status of single cells.
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http://dx.doi.org/10.1073/pnas.2026811118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8072209PMC
April 2021

Widespread polycistronic gene expression in green algae.

Proc Natl Acad Sci U S A 2021 Feb;118(7)

UCLA DOE Institute for Genomics and Proteomics, University of California, Los Angeles, CA 90095;

Polycistronic gene expression, common in prokaryotes, was thought to be extremely rare in eukaryotes. The development of long-read sequencing of full-length transcript isomers (Iso-Seq) has facilitated a reexamination of that dogma. Using Iso-Seq, we discovered hundreds of examples of polycistronic expression of nuclear genes in two divergent species of green algae: and Here, we employ a range of independent approaches to validate that multiple proteins are translated from a common transcript for hundreds of loci. A chromatin immunoprecipitation analysis using trimethylation of lysine 4 on histone H3 marks confirmed that transcription begins exclusively at the upstream gene. Quantification of polyadenylated [poly(A)] tails and poly(A) signal sequences confirmed that transcription ends exclusively after the downstream gene. Coexpression analysis found nearly perfect correlation for open reading frames (ORFs) within polycistronic loci, consistent with expression in a shared transcript. For many polycistronic loci, terminal peptides from both ORFs were identified from proteomics datasets, consistent with independent translation. Synthetic polycistronic gene pairs were transcribed and translated in vitro to recapitulate the production of two distinct proteins from a common transcript. The relative abundance of these two proteins can be modified by altering the Kozak-like sequence of the upstream gene. Replacement of the ORFs with selectable markers or reporters allows production of such heterologous proteins, speaking to utility in synthetic biology approaches. Conservation of a significant number of polycistronic gene pairs between , , and five other species suggests that this mechanism may be evolutionarily ancient and biologically important in the green algal lineage.
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http://dx.doi.org/10.1073/pnas.2017714118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7896298PMC
February 2021

An epigenetic gene silencing pathway selectively acting on transgenic DNA in the green alga Chlamydomonas.

Nat Commun 2020 12 8;11(1):6269. Epub 2020 Dec 8.

Max-Planck-Institut für Molekulare Pflanzenphysiologie, Am Mühlenberg 1, D-14476, Potsdam-Golm, Germany.

Silencing of exogenous DNA can make transgene expression very inefficient. Genetic screens in the model alga Chlamydomonas have demonstrated that transgene silencing can be overcome by mutations in unknown gene(s), thus producing algal strains that stably express foreign genes to high levels. Here, we show that the silencing mechanism specifically acts on transgenic DNA. Once a permissive chromatin structure has assembled, transgene expression can persist even in the absence of mutations disrupting the silencing pathway. We have identified the gene conferring the silencing and show it to encode a sirtuin-type histone deacetylase. Loss of gene function does not appreciably affect endogenous gene expression. Our data suggest that transgenic DNA is recognized and then quickly inactivated by the assembly of a repressive chromatin structure composed of deacetylated histones. We propose that this mechanism may have evolved to provide protection from potentially harmful types of environmental DNA.
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http://dx.doi.org/10.1038/s41467-020-19983-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7722844PMC
December 2020

Coexpressed subunits of dual genetic origin define a conserved supercomplex mediating essential protein import into chloroplasts.

Proc Natl Acad Sci U S A 2020 12 3;117(51):32739-32749. Epub 2020 Dec 3.

Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94143;

In photosynthetic eukaryotes, thousands of proteins are translated in the cytosol and imported into the chloroplast through the concerted action of two translocons-termed TOC and TIC-located in the outer and inner membranes of the chloroplast envelope, respectively. The degree to which the molecular composition of the TOC and TIC complexes is conserved over phylogenetic distances has remained controversial. Here, we combine transcriptomic, biochemical, and genetic tools in the green alga Chlamydomonas () to demonstrate that, despite a lack of evident sequence conservation for some of its components, the algal TIC complex mirrors the molecular composition of a TIC complex from The Chlamydomonas TIC complex contains three nuclear-encoded subunits, Tic20, Tic56, and Tic100, and one chloroplast-encoded subunit, Tic214, and interacts with the TOC complex, as well as with several uncharacterized proteins to form a stable supercomplex (TIC-TOC), indicating that protein import across both envelope membranes is mechanistically coupled. Expression of the nuclear and chloroplast genes encoding both known and uncharacterized TIC-TOC components is highly coordinated, suggesting that a mechanism for regulating its biogenesis across compartmental boundaries must exist. Conditional repression of Tic214, the only chloroplast-encoded subunit in the TIC-TOC complex, impairs the import of chloroplast proteins with essential roles in chloroplast ribosome biogenesis and protein folding and induces a pleiotropic stress response, including several proteins involved in the chloroplast unfolded protein response. These findings underscore the functional importance of the TIC-TOC supercomplex in maintaining chloroplast proteostasis.
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http://dx.doi.org/10.1073/pnas.2014294117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7768757PMC
December 2020

From economy to luxury: Copper homeostasis in Chlamydomonas and other algae.

Biochim Biophys Acta Mol Cell Res 2020 11 13;1867(11):118822. Epub 2020 Aug 13.

Department of Biology, Brookhaven National Laboratory, Upton, NY, United States of America.

Plastocyanin and cytochrome c, abundant proteins in photosynthesis, are readouts for cellular copper status in Chlamydomonas and other algae. Their accumulation is controlled by a transcription factor copper response regulator (CRR1). The replacement of copper-containing plastocyanin with heme-containing cytochrome c spares copper and permits preferential copper (re)-allocation to cytochrome oxidase. Under copper-replete situations, the quota depends on abundance of various cuproproteins and is tightly regulated, except under zinc-deficiency where acidocalcisomes over-accumulate Cu(I). CRR1 has a transcriptional activation domain, a Zn-dependent DNA binding SBP-domain with a nuclear localization signal, and a C-terminal Cys-rich region that represses the zinc regulon. CRR1 activates >60 genes in Chlamydomonas through GTAC-containing CuREs; transcriptome differences are recapitulated in the proteome. The differentially-expressed genes encode assimilatory copper transporters of the CTR/SLC31 family including a novel soluble molecule, redox enzymes in the tetrapyrrole pathway that promote chlorophyll biosynthesis and photosystem 1 accumulation, and other oxygen-dependent enzymes, which may influence thylakoid membrane lipids, specifically polyunsaturated galactolipids and γ-tocopherol. CRR1 also down-regulates 2 proteins in Chlamydomonas: for plastocyanin, by activation of proteolysis, while for the di‑iron subunit of the cyclase in chlorophyll biosynthesis, through activation of an upstream promoter that generates a poorly-translated 5' extended transcript containing multiple short ORFs that inhibit translation. The functions of many CRR1-target genes are unknown, and the copper protein inventory in Chlamydomonas includes several whose functions are unexplored. The comprehensive picture of cuproproteins and copper homeostasis in this system is well-suited for reverse genetic analyses of these under-investigated components in copper biology.
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http://dx.doi.org/10.1016/j.bbamcr.2020.118822DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7484467PMC
November 2020

Manganese co-localizes with calcium and phosphorus in acidocalcisomes and is mobilized in manganese-deficient conditions.

J Biol Chem 2019 11 16;294(46):17626-17641. Epub 2019 Sep 16.

Department of Chemistry and Biochemistry, UCLA, Los Angeles, California 90095

Exposing cells to excess metal concentrations well beyond the cellular quota is a powerful tool for understanding the molecular mechanisms of metal homeostasis. Such improved understanding may enable bioengineering of organisms with improved nutrition and bioremediation capacity. We report here that can accumulate manganese (Mn) in proportion to extracellular supply, up to 30-fold greater than its typical quota and with remarkable tolerance. As visualized by X-ray fluorescence microscopy and nanoscale secondary ion MS (nanoSIMS), Mn largely co-localizes with phosphorus (P) and calcium (Ca), consistent with the Mn-accumulating site being an acidic vacuole, known as the acidocalcisome. Vacuolar Mn stores are accessible reserves that can be mobilized in Mn-deficient conditions to support algal growth. We noted that Mn accumulation depends on cellular polyphosphate (polyP) content, indicated by 1) a consistent failure of mutant strains, which are deficient in polyphosphate synthesis, to accumulate Mn and 2) a drastic reduction of the Mn storage capacity in P-deficient cells. Rather surprisingly, X-ray absorption spectroscopy, EPR, and electron nuclear double resonance revealed that only little Mn is stably complexed with polyP, indicating that polyP is not the final Mn ligand. We propose that polyPs are a critical component of Mn accumulation in Chlamydomonas by driving Mn relocation from the cytosol to acidocalcisomes. Within these structures, polyP may, in turn, escort vacuolar Mn to a number of storage ligands, including phosphate and phytate, and other, yet unidentified, compounds.
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http://dx.doi.org/10.1074/jbc.RA119.009130DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6873200PMC
November 2019

Multiomics resolution of molecular events during a day in the life of Chlamydomonas.

Proc Natl Acad Sci U S A 2019 02 18;116(6):2374-2383. Epub 2019 Jan 18.

Institute for Genomics and Proteomics, University of California, Los Angeles, CA 90095;

The unicellular green alga displays metabolic flexibility in response to a changing environment. We analyzed expression patterns of its three genomes in cells grown under light-dark cycles. Nearly 85% of transcribed genes show differential expression, with different sets of transcripts being up-regulated over the course of the day to coordinate cellular growth before undergoing cell division. Parallel measurements of select metabolites and pigments, physiological parameters, and a subset of proteins allow us to infer metabolic events and to evaluate the impact of the transcriptome on the proteome. Among the findings are the observations that exhibits lower respiratory activity at night compared with the day; multiple fermentation pathways, some oxygen-sensitive, are expressed at night in aerated cultures; we propose that the ferredoxin, FDX9, is potentially the electron donor to hydrogenases. The light stress-responsive genes , , and show an acute response to lights-on at dawn under abrupt dark-to-light transitions, while genes also exhibit a later, second burst in expression in the middle of the day dependent on light intensity. Each response to light (acute and sustained) can be selectively activated under specific conditions. Our expression dataset, complemented with coexpression networks and metabolite profiling, should constitute an excellent resource for the algal and plant communities.
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http://dx.doi.org/10.1073/pnas.1815238116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6369806PMC
February 2019

In situ architecture of the algal nuclear pore complex.

Nat Commun 2018 06 18;9(1):2361. Epub 2018 Jun 18.

Structural and Computational Biology Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117, Heidelberg, Germany.

Nuclear pore complexes (NPCs) span the nuclear envelope and mediate nucleocytoplasmic exchange. They are a hallmark of eukaryotes and deeply rooted in the evolutionary origin of cellular compartmentalization. NPCs have an elaborate architecture that has been well studied in vertebrates. Whether this architecture is unique or varies significantly in other eukaryotic kingdoms remains unknown, predominantly due to missing in situ structural data. Here, we report the architecture of the algal NPC from the early branching eukaryote Chlamydomonas reinhardtii and compare it to the human NPC. We find that the inner ring of the Chlamydomonas NPC has an unexpectedly large diameter, and the outer rings exhibit an asymmetric oligomeric state that has not been observed or predicted previously. Our study provides evidence that the NPC is subject to substantial structural variation between species. The divergent and conserved features of NPC architecture provide insights into the evolution of the nucleocytoplasmic transport machinery.
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http://dx.doi.org/10.1038/s41467-018-04739-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6006428PMC
June 2018

High-throughput sequencing of the chloroplast and mitochondrion of Chlamydomonas reinhardtii to generate improved de novo assemblies, analyze expression patterns and transcript speciation, and evaluate diversity among laboratory strains and wild isolates.

Plant J 2018 02 7;93(3):545-565. Epub 2018 Jan 7.

Department of Chemistry and Biochemistry, University of California, Los Angeles, CA, 90095, USA.

Chlamydomonas reinhardtii is a unicellular chlorophyte alga that is widely studied as a reference organism for understanding photosynthesis, sensory and motile cilia, and for development of an algal-based platform for producing biofuels and bio-products. Its highly repetitive, ~205-kbp circular chloroplast genome and ~15.8-kbp linear mitochondrial genome were sequenced prior to the advent of high-throughput sequencing technologies. Here, high coverage shotgun sequencing was used to assemble both organellar genomes de novo. These new genomes correct dozens of errors in the prior genome sequences and annotations. Genome sequencing coverage indicates that each cell contains on average 83 copies of the chloroplast genome and 130 copies of the mitochondrial genome. Using protocols and analyses optimized for organellar transcripts, RNA-Seq was used to quantify their relative abundances across 12 different growth conditions. Forty-six percent of total cellular mRNA is attributable to high expression from a few dozen chloroplast genes. RNA-Seq data were used to guide gene annotation, to demonstrate polycistronic gene expression, and to quantify splicing of psaA and psbA introns. In contrast to a conclusion from a recent study, we found that chloroplast transcripts are not edited. Unexpectedly, cytosine-rich polynucleotide tails were observed at the 3'-end of all mitochondrial transcripts. A comparative genomics analysis of eight laboratory strains and 11 wild isolates of C. reinhardtii identified 2658 variants in the organellar genomes, which is 1/10th as much genetic diversity as is found in the nucleus.
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http://dx.doi.org/10.1111/tpj.13788DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5775909PMC
February 2018

A bioactive peptide amidating enzyme is required for ciliogenesis.

Elife 2017 05 17;6. Epub 2017 May 17.

Department of Molecular Biology and Biophysics, University of Connecticut Health Center, Farmington, United States.

The pathways controlling cilium biogenesis in different cell types have not been fully elucidated. We recently identified peptidylglycine α-amidating monooxygenase (PAM), an enzyme required for generating amidated bioactive signaling peptides, in and mammalian cilia. Here, we show that PAM is required for the normal assembly of motile and primary cilia in , planaria and mice. PAM knockdown lines failed to assemble cilia beyond the transition zone, had abnormal Golgi architecture and altered levels of cilia assembly components. Decreased PAM gene expression reduced motile ciliary density on the ventral surface of planaria and resulted in the appearance of cytosolic axonemes lacking a ciliary membrane. The architecture of primary cilia on neuroepithelial cells in mouse embryos was also aberrant. Our data suggest that PAM activity and alterations in post-Golgi trafficking contribute to the observed ciliogenesis defects and provide an unanticipated, highly conserved link between PAM, amidation and ciliary assembly.
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http://dx.doi.org/10.7554/eLife.25728DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5461114PMC
May 2017

Genetically Programmed Changes in Photosynthetic Cofactor Metabolism in Copper-deficient Chlamydomonas.

J Biol Chem 2016 09 20;291(36):19118-31. Epub 2016 Jul 20.

From the Institute for Genomics and Proteomics, University of California, Los Angeles, California 90095, the Department of Chemistry and Biochemistry, UCLA, Los Angeles, California 90095,

Genetic and genomic studies indicate that copper deficiency triggers changes in the expression of genes encoding key enzymes in various chloroplast-localized lipid/pigment biosynthetic pathways. Among these are CGL78 involved in chlorophyll biosynthesis and HPPD1, encoding 4-hydroxyphenylpyruvate dioxygenase catalyzing the committed step of plastoquinone and tocopherol biosyntheses. Copper deficiency in wild-type cells does not change the chlorophyll content, but a survey of chlorophyll protein accumulation in this situation revealed increased accumulation of LHCSR3, which is blocked at the level of mRNA accumulation when either CGL78 expression is reduced or in the crd1 mutant, which has a copper-nutrition conditional defect at the same step in chlorophyll biosynthesis. Again, like copper-deficient crd1 strains, cgl78 knock-down lines also have reduced chlorophyll content concomitant with loss of PSI-LHCI super-complexes and reduced abundance of a chlorophyll binding subunit of PSI, PSAK, which connects LHCI to PSI. For HPPD1, increased mRNA results in increased abundance of the corresponding protein in copper-deficient cells concomitant with CRR1-dependent increased accumulation of γ-tocopherols, but not plastoquinone-9 nor total tocopherols. In crr1 mutants, where increased HPPD1 expression is blocked, plastochromanol-8, derived from plastoquinone-9 and purported to also have an antioxidant function, is found instead. Although not previously found in algae, this metabolite may occur only in stress conditions.
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http://dx.doi.org/10.1074/jbc.M116.717413DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5009281PMC
September 2016

Dissecting the contributions of GC content and codon usage to gene expression in the model alga Chlamydomonas reinhardtii.

Plant J 2015 Nov 16;84(4):704-17. Epub 2015 Oct 16.

Max-Planck-Institut für Molekulare Pflanzenphysiologie (MPI-MP), Am Mühlenberg 1, D-14476, Potsdam-Golm, Germany.

The efficiency of gene expression in all organisms depends on the nucleotide composition of the coding region. GC content and codon usage are the two key sequence features known to influence gene expression, but the underlying molecular mechanisms are not entirely clear. Here we have determined the relative contributions of GC content and codon usage to the efficiency of nuclear gene expression in the unicellular green alga Chlamydomonas reinhardtii. By comparing gene variants that encode an identical amino acid sequence but differ in their GC content and/or codon usage, we show that codon usage is the key factor determining translational efficiency and, surprisingly, also mRNA stability. By contrast, unfavorable GC content affects gene expression at the level of the chromatin structure by triggering heterochromatinization. We further show that mutant algal strains that permit high-level transgene expression are less susceptible to epigenetic transgene suppression and do not establish a repressive chromatin structure at the transgenic locus. Our data disentangle the relationship between GC content and codon usage, and suggest simple strategies to overcome the transgene expression problem in Chlamydomonas.
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http://dx.doi.org/10.1111/tpj.13033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4715772PMC
November 2015

The thylakoid membrane protein CGL160 supports CF1CF0 ATP synthase accumulation in Arabidopsis thaliana.

PLoS One 2015 2;10(4):e0121658. Epub 2015 Apr 2.

Department of Chemistry and Biochemistry, UCLA, Los Angeles, California, United States of America; Institute for Genomics and Proteomics UCLA, Los Angeles, California, United States of America.

The biogenesis of the major thylakoid protein complexes of the photosynthetic apparatus requires auxiliary proteins supporting individual assembly steps. Here, we identify a plant lineage specific gene, CGL160, whose homolog, atp1, co-occurs with ATP synthase subunits in an operon-like arrangement in many cyanobacteria. Arabidopsis thaliana T-DNA insertion mutants, which no longer accumulate the nucleus-encoded CGL160 protein, accumulate less than 25% of wild-type levels of the chloroplast ATP synthase. Severe cosmetic or growth phenotypes result under either short day or fluctuating light growth conditions, respectively, but this is ameliorated under long day constant light growth conditions where the growth, ATP synthase activity and photosynthetic electron transport of the mutants are less affected. Accumulation of other photosynthetic complexes is largely unaffected in cgl160 mutants, suggesting that CGL160 is a specific assembly or stability factor for the CF1CF0 complex. CGL160 is not found in the mature assembled complex but it does interact specifically with subunits of ATP synthase, predominantly those in the extrinsic CF1 sub-complex. We suggest therefore that it may facilitate the assembly of CF1 into the holocomplex.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0121658PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4383579PMC
April 2016

Copper economy in Chlamydomonas: prioritized allocation and reallocation of copper to respiration vs. photosynthesis.

Proc Natl Acad Sci U S A 2015 Mar 2;112(9):2644-51. Epub 2015 Feb 2.

Department of Chemistry and Biochemistry, and Institute for Genomics and Proteomics, University of California, Los Angeles, CA 90095; and

Inorganic elements, although required only in trace amounts, permit life and primary productivity because of their functions in catalysis. Every organism has a minimal requirement of each metal based on the intracellular abundance of proteins that use inorganic cofactors, but elemental sparing mechanisms can reduce this quota. A well-studied copper-sparing mechanism that operates in microalgae faced with copper deficiency is the replacement of the abundant copper protein plastocyanin with a heme-containing substitute, cytochrome (Cyt) c6. This switch, which is dependent on a copper-sensing transcription factor, copper response regulator 1 (CRR1), dramatically reduces the copper quota. We show here that in a situation of marginal copper availability, copper is preferentially allocated from plastocyanin, whose function is dispensable, to other more critical copper-dependent enzymes like Cyt oxidase and a ferroxidase. In the absence of an extracellular source, copper allocation to Cyt oxidase includes CRR1-dependent proteolysis of plastocyanin and quantitative recycling of the copper cofactor from plastocyanin to Cyt oxidase. Transcriptome profiling identifies a gene encoding a Zn-metalloprotease, as a candidate effecting copper recycling. One reason for the retention of genes encoding both plastocyanin and Cyt c6 in algal and cyanobacterial genomes might be because plastocyanin provides a competitive advantage in copper-depleted environments as a ready source of copper.
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http://dx.doi.org/10.1073/pnas.1422492112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4352834PMC
March 2015

Nitrogen-Sparing Mechanisms in Chlamydomonas Affect the Transcriptome, the Proteome, and Photosynthetic Metabolism.

Plant Cell 2014 Apr 18;26(4):1410-1435. Epub 2014 Apr 18.

Department of Chemistry and Biochemistry, University of California, Los Angeles, California 90095 Institute of Genomics and Proteomics, University of California, Los Angeles, California 90095

Nitrogen (N) is a key nutrient that limits global primary productivity; hence, N-use efficiency is of compelling interest in agriculture and aquaculture. We used Chlamydomonas reinhardtii as a reference organism for a multicomponent analysis of the N starvation response. In the presence of acetate, respiratory metabolism is prioritized over photosynthesis; consequently, the N-sparing response targets proteins, pigments, and RNAs involved in photosynthesis and chloroplast function over those involved in respiration. Transcripts and proteins of the Calvin-Benson cycle are reduced in N-deficient cells, resulting in the accumulation of cycle metabolic intermediates. Both cytosolic and chloroplast ribosomes are reduced, but via different mechanisms, reflected by rapid changes in abundance of RNAs encoding chloroplast ribosomal proteins but not cytosolic ones. RNAs encoding transporters and enzymes for metabolizing alternative N sources increase in abundance, as is appropriate for the soil environmental niche of C. reinhardtii. Comparison of the N-replete versus N-deplete proteome indicated that abundant proteins with a high N content are reduced in N-starved cells, while the proteins that are increased have lower than average N contents. This sparing mechanism contributes to a lower cellular N/C ratio and suggests an approach for engineering increased N-use efficiency.
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http://dx.doi.org/10.1105/tpc.113.122523DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4036562PMC
April 2014

Dissecting the heat stress response in Chlamydomonas by pharmaceutical and RNAi approaches reveals conserved and novel aspects.

Mol Plant 2013 Nov 27;6(6):1795-813. Epub 2013 May 27.

Molekulare Biotechnologie und Systembiologie, TU Kaiserslautern, Paul-Ehrlich-Str. 23, D-67663 Kaiserslautern, Germany.

To study how conserved fundamental concepts of the heat stress response (HSR) are in photosynthetic eukaryotes, we applied pharmaceutical and antisense/amiRNA approaches to the unicellular green alga Chlamydomonas reinhardtii. The Chlamydomonas HSR appears to be triggered by the accumulation of unfolded proteins, as it was induced at ambient temperatures by feeding cells with the arginine analog canavanine. The protein kinase inhibitor staurosporine strongly retarded the HSR, demonstrating the importance of phosphorylation during activation of the HSR also in Chlamydomonas. While the removal of extracellular calcium by the application of EGTA and BAPTA inhibited the HSR in moss and higher plants, only the addition of BAPTA, but not of EGTA, retarded the HSR and impaired thermotolerance in Chlamydomonas. The addition of cycloheximide, an inhibitor of cytosolic protein synthesis, abolished the attenuation of the HSR, indicating that protein synthesis is necessary to restore proteostasis. HSP90 inhibitors induced a stress response when added at ambient conditions and retarded attenuation of the HSR at elevated temperatures. In addition, we detected a direct physical interaction between cytosolic HSP90A/HSP70A and heat shock factor 1, but surprisingly this interaction persisted after the onset of stress. Finally, the expression of antisense constructs targeting chloroplast HSP70B resulted in a delay of the cell's entire HSR, thus suggesting the existence of a retrograde stress signaling cascade that is desensitized in HSP70B-antisense strains.
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http://dx.doi.org/10.1093/mp/sst086DOI Listing
November 2013

Heat shock factor 1 counteracts epigenetic silencing of nuclear transgenes in Chlamydomonas reinhardtii.

Nucleic Acids Res 2013 May 12;41(10):5273-89. Epub 2013 Apr 12.

Max-Planck-Institut für Molekulare Pflanzenphysiologie, Am Mühlenberg 1, D-14476 Potsdam-Golm, Germany.

We found previously that the Chlamydomonas HSP70A promoter counteracts transcriptional silencing of downstream promoters in a transgene setting. To elucidate the underlying mechanisms, we analyzed chromatin state and transgene expression in transformants containing HSP70A-RBCS2-ble (AR-ble) constructs harboring deletions/mutations in the A promoter. We identified histone modifications at transgenic R promoters indicative for repressive chromatin, i.e. low levels of histone H3/4 acetylation and H3-lysine 4 trimethylation and high levels of H3-lysine 9 monomethylation. Transgenic A promoters also harbor lower levels of active chromatin marks than the native A promoter, but levels were higher than those at transgenic R promoters. Strikingly, in AR promoter fusions, the chromatin state at the A promoter was transferred to R. This effect required intact HSE4, HSE1/2 and TATA-box in the A promoter and was mediated by heat shock factor (HSF1). However, time-course analyses in strains inducibly depleted of HSF1 revealed that a transcriptionally competent chromatin state alone was not sufficient for activating the R promoter, but required constitutive HSF1 occupancy at transgenic A. We propose that HSF1 constitutively forms a scaffold at the transgenic A promoter, presumably containing mediator and TFIID, from which local chromatin remodeling and polymerase II recruitment to downstream promoters is realized.
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http://dx.doi.org/10.1093/nar/gkt224DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3664811PMC
May 2013

A protocol for the identification of protein-protein interactions based on 15N metabolic labeling, immunoprecipitation, quantitative mass spectrometry and affinity modulation.

J Vis Exp 2012 Sep 24(67). Epub 2012 Sep 24.

Max Planck Institute of Molecular Plant Physiology.

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in current research and a plethora of methods for their investigation is available. Protein-protein interactions often are highly dynamic and may depend on subcellular localization, post-translational modifications and the local protein environment. Therefore, they should be investigated in their natural environment, for which co-immunoprecipitation approaches are the method of choice. Co-precipitated interaction partners are identified either by immunoblotting in a targeted approach, or by mass spectrometry (LC-MS/MS) in an untargeted way. The latter strategy often is adversely affected by a large number of false positive discoveries, mainly derived from the high sensitivity of modern mass spectrometers that confidently detect traces of unspecifically precipitating proteins. A recent approach to overcome this problem is based on the idea that reduced amounts of specific interaction partners will co-precipitate with a given target protein whose cellular concentration is reduced by RNAi, while the amounts of unspecifically precipitating proteins should be unaffected. This approach, termed QUICK for QUantitative Immunoprecipitation Combined with Knockdown, employs Stable Isotope Labeling of Amino acids in Cell culture (SILAC) and MS to quantify the amounts of proteins immunoprecipitated from wild-type and knock-down strains. Proteins found in a 1:1 ratio can be considered as contaminants, those enriched in precipitates from the wild type as specific interaction partners of the target protein. Although innovative, QUICK bears some limitations: first, SILAC is cost-intensive and limited to organisms that ideally are auxotrophic for arginine and/or lysine. Moreover, when heavy arginine is fed, arginine-to-proline interconversion results in additional mass shifts for each proline in a peptide and slightly dilutes heavy with light arginine, which makes quantification more tedious and less accurate. Second, QUICK requires that antibodies are titrated such that they do not become saturated with target protein in extracts from knock-down mutants. Here we introduce a modified QUICK protocol which overcomes the abovementioned limitations of QUICK by replacing SILAC for (15)N metabolic labeling and by replacing RNAi-mediated knock-down for affinity modulation of protein-protein interactions. We demonstrate the applicability of this protocol using the unicellular green alga Chlamydomonas reinhardtii as model organism and the chloroplast HSP70B chaperone as target protein (Figure 1). HSP70s are known to interact with specific co-chaperones and substrates only in the ADP state. We exploit this property as a means to verify the specific interaction of HSP70B with its nucleotide exchange factor CGE1.
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http://dx.doi.org/10.3791/4083DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3490270PMC
September 2012

Protocol: methodology for chromatin immunoprecipitation (ChIP) in Chlamydomonas reinhardtii.

Plant Methods 2011 Nov 3;7(1):35. Epub 2011 Nov 3.

Max-Planck-Institut für Molekulare Pflanzenphysiologie, Am Mühlenberg 1, D-14476 Potsdam-Golm, Germany.

We report on a detailed chromatin immunoprecipitation (ChIP) protocol for the unicellular green alga Chlamydomonas reinhardtii. The protocol is suitable for the analysis of nucleosome occupancy, histone modifications and transcription factor binding sites at the level of mononucleosomes for targeted and genome-wide studies. We describe the optimization of conditions for crosslinking, chromatin fragmentation and antibody titer determination and provide recommendations and an example for the normalization of ChIP results as determined by real-time PCR.
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http://dx.doi.org/10.1186/1746-4811-7-35DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3225300PMC
November 2011

Transcription factor-dependent chromatin remodeling at heat shock and copper-responsive promoters in Chlamydomonas reinhardtii.

Plant Cell 2011 Jun 24;23(6):2285-301. Epub 2011 Jun 24.

Max-Planck-Institut für Molekulare Pflanzenphysiologie, D-14476 Potsdam-Golm, Germany.

How transcription factors affect chromatin structure to regulate gene expression in response to changes in environmental conditions is poorly understood in the green lineage. To shed light on this issue, we used chromatin immunoprecipitation and formaldehyde-assisted isolation of regulatory elements to investigate the chromatin structure at target genes of HSF1 and CRR1, key transcriptional regulators of the heat shock and copper starvation responses, respectively, in the unicellular green alga Chlamydomonas reinhardtii. Generally, we detected lower nucleosome occupancy, higher levels of histone H3/4 acetylation, and lower levels of histone H3 Lys 4 (H3K4) monomethylation at promoter regions of active genes compared with inactive promoters and transcribed and intergenic regions. Specifically, we find that activated HSF1 and CRR1 transcription factors mediate the acetylation of histones H3/4, nucleosome eviction, remodeling of the H3K4 mono- and dimethylation marks, and transcription initiation/elongation. By this, HSF1 and CRR1 quite individually remodel and activate target promoters that may be inactive and embedded into closed chromatin (HSP22F/CYC6) or weakly active and embedded into partially opened (CPX1) or completely opened chromatin (HSP70A/CRD1). We also observed HSF1-independent histone H3/4 deacetylation at the RBCS2 promoter after heat shock, suggesting interplay of specific and presumably more generally acting factors to adapt gene expression to the new requirements of a changing environment.
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http://dx.doi.org/10.1105/tpc.111.085266DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3160021PMC
June 2011

An inducible artificial microRNA system for Chlamydomonas reinhardtii confirms a key role for heat shock factor 1 in regulating thermotolerance.

Curr Genet 2010 Aug 7;56(4):383-9. Epub 2010 May 7.

Max-Planck-Institut für Molekulare Pflanzenphysiologie, Potsdam-Golm, Germany.

Several RNA silencing strategies employing antisense or inverted repeat constructs have been applied to Chlamydomonas reinhardtii. Problems inherent to these strategies, like off-target effects by unpredictable generation of siRNAs, were solved previously by constructs allowing for routine expression of specific artificial microRNAs (amiRNAs). Yet missing was a routine tool for inducible amiRNA expression, which to establish was the aim of this work. For this, we equipped a recently developed amiRNA expression vector with the NIT1 promoter, which is repressed by ammonium and activated by nitrate. We tested this conditional amiRNA vector with heat shock factor 1 (HSF1) as target. HSF1 transcripts in transformants were already reduced ~2 h after transfer from ammonium to nitrate-containing medium. In contrast, HSF1 protein levels declined only ~8 h after the shift and were strongly reduced after 24 h, suggesting that HSF1 is a stable protein and diluted out by growth. HSF1 levels recovered partly when transformant cells were shifted back to ammonium for 72 h. Transformants developed thermosensitivity only on nitrate and thermosensitivity correlated with strong reduction in HSF1 levels, hence supporting our earlier conclusion that HSF1 is a key regulator for thermotolerance in Chlamydomonas.
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http://dx.doi.org/10.1007/s00294-010-0304-4DOI Listing
August 2010