Assistant Professor, Biology
Honors & Awards
NIH Director's New Innovator Award, National Institutes of Health (2015)
Young Investigator Award, Air Force Office of Scientific Research (2010)
Graduate Course Teaching Award, University of California, San Francisco (2006)
NSF Graduate Research Fellowship, National Science Foundation (2005)
Ph.D., Univ. California, San Francisco, Molecular Biology and Genetics (2009)
B.S., Mass. Institute of Technology, Aerospace Engineering (2004)
Current Research and Scholarly Interests
Photosynthesis provides energy for nearly all life on Earth. As humans increasingly change this planet, it is essential that we understand this process and the organisms that perform it. Our lab aims to dramatically accelerate our understanding of photosynthetic organisms by developing and applying novel functional genomics strategies in the green alga Chlamydomonas reinhardtii. In the long run, we dream of engineering photosynthetic organisms to address the challenges that our civilization faces in agriculture, health and energy.
Our lab is focused around three synergistic areas:
I. Systems biology of photosynthetic organisms
Many fundamental systems-level questions about photosynthetic organisms remain unanswered. What is the full set of genes required for photosynthesis? Which parts work together? What do all the uncharacterized parts do?
The green alga Chlamydomonas reinhardtii is a powerful model photosynthetic organism. The green plant photosynthetic apparatus is highly conserved and thus can be studied in Chlamydomonas. Chlamydomonas can grow as a haploid and in the absence of a functional photosynthetic apparatus, allowing rapid isolation of mutants of interest. Its unicellular nature and short doubling time enable higher throughput experiments than alternative systems.
We are developing transformative tools to enable high-throughput studies of gene function in Chlamydomonas. We have developed a new tool, which increases the pace at which mutated genes in Chlamydomonas can be identified by >1,000-fold. We are presently using this tool to develop a genome-wide collection of Chlamydomonas insertion mutants as a powerful resource for the research community.
II. Molecular mechanisms of efficient photosynthesis
Photosynthetic organisms growing in nearly all environments must cope with rapid fluctuations in light intensity. The sunlight intensity in most environments can change dramatically in a fraction of a second due to e.g. clouds or leaves moving in the wind. Yet, almost nothing is known about the molecular mechanisms that enable efficient photosynthesis under fluctuating light. We recently discovered that plants have evolved a mechanism that enhances photosynthetic efficiency in changing light environments. We found that this mechanism works by accelerating fluxes of ions across the photosynthetic (thylakoid) membrane.
The Chlamydomonas Carbon Concentrating Mechanism (CCM) allows it to use CO2 much more efficiently than C3 crop plants. If we understood how this CCM works, we could engineer it into crop plants to increase their growth rates and reduce their need for water and fertilizer. We are working with our collaborators in the NSF project Combining Algal and Plant Photosynthesis to identify and transfer CCM components into the model C3 plant Arabidopsis, as a first step towards ultimately enhancing CO2 uptake in wheat and rice.
III. Lipid metabolism in photosynthetic eukaryotes
We are discovering and characterizing new genes with roles in algal lipid metabolism and its regulation. Photosynthetic organisms have the potential to play an important role in the production of renewable fuels and high-value lipids. Yet, many key aspects of lipid metabolism remain poorly characterized. For example, fatty acids are made in the chloroplast, but we don't understand how they get out of the chloroplast and to the rest of the cell. We have developed a new method that is allowing us to identify large numbers of new genes with roles in algal lipid metabolism. We are now using this method to systematically identify novel genes with roles in algal lipid metabolism.
A fluorescence-activated cell sorting-based strategy for rapid isolation of high-lipid Chlamydomonas mutants
2015; 81 (1): 147-159
There is significant interest in farming algae for the direct production of biofuels and valuable lipids. Chlamydomonas reinhardtii is the leading model system for studying lipid metabolism in green algae, but current methods for isolating mutants with perturbed lipid content in this organism are slow and tedious. Here, we present the Chlamydomonas High Lipid Sorting (CHiLiS) strategy, which enables enrichment of high-lipid mutants by fluorescence-activated cell sorting (FACS) of pooled mutants stained with the lipid-sensitive dye Nile Red. This method only takes five weeks from mutagenesis to mutant isolation. We developed a staining protocol that allows quantitation of lipid content while preserving cell viability. We improved separation of high-lipid mutants from wild-type by using each cell's chlorophyll fluorescence as an internal control. We initially demonstrated 20-fold enrichment of the known high-lipid mutant sta1 from a mixture of sta1 and wild-type cells. We then applied CHiLiS to sort thousands of high-lipid cells from a pool of ~60,000 mutants. Flow cytometry analysis of 24 individual mutants isolated by this approach revealed that ~50% showed a reproducible high lipid phenotype. We further characterized 9 of the mutants with highest lipid content by flame ionization detection and mass spectrometry lipidomics. All mutants analyzed had higher triacylglycerol content and perturbed whole-cell fatty-acid composition. One arbitrarily chosen mutant was evaluated by microscopy, revealing larger lipid droplets than wild-type. The unprecedented throughput of CHiLiS opens the door to a systems-level understanding of green algal lipid biology by enabling genome-saturating isolation of mutants in key genes. This article is protected by copyright. All rights reserved.
View details for DOI 10.1111/tpj.12682
View details for Web of Science ID 000346918400012
View details for PubMedID 25267488
Ion antiport accelerates photosynthetic acclimation in fluctuating light environments
Many photosynthetic organisms globally, including crops, forests and algae, must grow in environments where the availability of light energy fluctuates dramatically. How photosynthesis maintains high efficiency despite such fluctuations in its energy source remains poorly understood. Here we show that Arabidopsis thaliana K(+) efflux antiporter (KEA3) is critical for high photosynthetic efficiency under fluctuating light. On a shift from dark to low light, or high to low light, kea3 mutants show prolonged dissipation of absorbed light energy as heat. KEA3 localizes to the thylakoid membrane, and allows proton efflux from the thylakoid lumen by proton/potassium antiport. KEA3's activity accelerates the downregulation of pH-dependent energy dissipation after transitions to low light, leading to faster recovery of high photosystem II quantum efficiency and increased CO2 assimilation. Our results reveal a mechanism that increases the efficiency of photosynthesis under fluctuating light.
View details for DOI 10.1038/ncomms6439
View details for Web of Science ID 000345625000019
View details for PubMedID 25451040
- High-Throughput Genotyping of Green Algal Mutants Reveals Random Distribution of Mutagenic Insertion Sites and Endonucleolytic Cleavage of Transforming DNA PLANT CELL 2014; 26 (4): 1398-1409
Comprehensive Characterization of Genes Required for Protein Folding in the Endoplasmic Reticulum
2009; 323 (5922): 1693-1697
Protein folding in the endoplasmic reticulum is a complex process whose malfunction is implicated in disease and aging. By using the cell's endogenous sensor (the unfolded protein response), we identified several hundred yeast genes with roles in endoplasmic reticulum folding and systematically characterized their functional interdependencies by measuring unfolded protein response levels in double mutants. This strategy revealed multiple conserved factors critical for endoplasmic reticulum folding, including an intimate dependence on the later secretory pathway, a previously uncharacterized six-protein transmembrane complex, and a co-chaperone complex that delivers tail-anchored proteins to their membrane insertion machinery. The use of a quantitative reporter in a comprehensive screen followed by systematic analysis of genetic dependencies should be broadly applicable to functional dissection of complex cellular processes from yeast to human.
View details for DOI 10.1126/science.1167983
View details for Web of Science ID 000264559800030
View details for PubMedID 19325107
Molecular techniques to interrogate and edit the Chlamydomonas nuclear genome.
2015; 82 (3): 393-412
The success of the green alga Chlamydomonas reinhardtii as a model organism is to a large extent due to the wide range of molecular techniques that are available for its characterization. Here, we review some of the techniques currently used to modify and interrogate the C. reinhardtii nuclear genome and explore several technologies under development. Nuclear mutants can be generated with ultraviolet (UV) light and chemical mutagens, or by insertional mutagenesis. Nuclear transformation methods include biolistic delivery, agitation with glass beads, and electroporation. Transforming DNA integrates into the genome at random sites, and multiple strategies exist for mapping insertion sites. A limited number of studies have demonstrated targeted modification of the nuclear genome by approaches such as zinc-finger nucleases and homologous recombination. RNA interference is widely used to knock down expression levels of nuclear genes. A wide assortment of transgenes has been successfully expressed in the Chlamydomonas nuclear genome, including transformation markers, fluorescent proteins, reporter genes, epitope tagged proteins, and even therapeutic proteins. Optimized expression constructs and strains help transgene expression. Emerging technologies such as the CRISPR/Cas9 system, high-throughput mutant identification, and a whole-genome knockout library are being developed for this organism. We discuss how these advances will propel future investigations.
View details for DOI 10.1111/tpj.12801
View details for PubMedID 25704665
- Waking sleeping algal cells. Proceedings of the National Academy of Sciences of the United States of America 2014; 111 (44): 15610-15611
Alternative Acetate Production Pathways in Chlamydomonas reinhardtii during Dark Anoxia and the Dominant Role of Chloroplasts in Fermentative Acetate Production
2014; 26 (11): 4499-4518
Chlamydomonas reinhardtii insertion mutants disrupted for genes encoding acetate kinases (EC 18.104.22.168) (ACK1 and ACK2) and a phosphate acetyltransferase (EC 22.214.171.124) (PAT2, but not PAT1) were isolated to characterize fermentative acetate production. ACK1 and PAT2 were localized to chloroplasts, while ACK2 and PAT1 were shown to be in mitochondria. Characterization of the mutants showed that PAT2 and ACK1 activity in chloroplasts plays a dominant role (relative to ACK2 and PAT1 in mitochondria) in producing acetate under dark, anoxic conditions and, surprisingly, also suggested that Chlamydomonas has other pathways that generate acetate in the absence of ACK activity. We identified a number of proteins associated with alternative pathways for acetate production that are encoded on the Chlamydomonas genome. Furthermore, we observed that only modest alterations in the accumulation of fermentative products occurred in the ack1, ack2, and ack1 ack2 mutants, which contrasts with the substantial metabolite alterations described in strains devoid of other key fermentation enzymes.
View details for DOI 10.1105/tpc.114.129965
View details for Web of Science ID 000348645200017
View details for PubMedID 25381350
Actin is required for IFT regulation in Chlamydomonas reinhardtii.
2014; 24 (17): 2025-2032
Assembly of cilia and flagella requires intraflagellar transport (IFT), a highly regulated kinesin-based transport system that moves cargo from the basal body to the tip of flagella . The recruitment of IFT components to basal bodies is a function of flagellar length, with increased recruitment in rapidly growing short flagella . The molecular pathways regulating IFT are largely a mystery. Because actin network disruption leads to changes in ciliary length and number, actin has been proposed to have a role in ciliary assembly. However, the mechanisms involved are unknown. In Chlamydomonas reinhardtii, conventional actin is found in both the cell body and the inner dynein arm complexes within flagella [3, 4]. Previous work showed that treating Chlamydomonas cells with the actin-depolymerizing compound cytochalasin D resulted in reversible flagellar shortening , but how actin is related to flagellar length or assembly remains unknown. Here we utilize small-molecule inhibitors and genetic mutants to analyze the role of actin dynamics in flagellar assembly in Chlamydomonas reinhardtii. We demonstrate that actin plays a role in IFT recruitment to basal bodies during flagellar elongation and that when actin is perturbed, the normal dependence of IFT recruitment on flagellar length is lost. We also find that actin is required for sufficient entry of IFT material into flagella during assembly. These same effects are recapitulated with a myosin inhibitor, suggesting that actin may act via myosin in a pathway by which flagellar assembly is regulated by flagellar length.
View details for DOI 10.1016/j.cub.2014.07.038
View details for PubMedID 25155506
J Domain Co-chaperone Specificity Defines the Role of BiP during Protein Translocation
JOURNAL OF BIOLOGICAL CHEMISTRY
2010; 285 (29): 22484-22494
Hsp70 chaperones can potentially interact with one of several J domain-containing Hsp40 co-chaperones to regulate distinct cellular processes. However, features within Hsp70s that determine Hsp40 specificity are undefined. To investigate this question, we introduced mutations into the ER-lumenal Hsp70, BiP/Kar2p, and found that an R217A substitution in the J domain-interacting surface of BiP compromised the physical and functional interaction with Sec63p, an Hsp40 required for ER translocation. In contrast, interaction with Jem1p, an Hsp40 required for ER-associated degradation, was unaffected. Moreover, yeast expressing R217A BiP exhibited defects in translocation but not in ER-associated degradation. Finally, the genetic interactions of the R217A BiP mutant were found to correlate with those of known translocation mutants. Together, our results indicate that residues within the Hsp70 J domain-interacting surface help confer Hsp40 specificity, in turn influencing distinct chaperone-mediated cellular activities.
View details for DOI 10.1074/jbc.M110.102186
View details for Web of Science ID 000279702200060
View details for PubMedID 20430885
Automated identification of pathways from quantitative genetic interaction data
MOLECULAR SYSTEMS BIOLOGY
High-throughput quantitative genetic interaction (GI) measurements provide detailed information regarding the structure of the underlying biological pathways by reporting on functional dependencies between genes. However, the analytical tools for fully exploiting such information lag behind the ability to collect these data. We present a novel Bayesian learning method that uses quantitative phenotypes of double knockout organisms to automatically reconstruct detailed pathway structures. We applied our method to a recent data set that measures GIs for endoplasmic reticulum (ER) genes, using the unfolded protein response as a quantitative phenotype. The results provided reconstructions of known functional pathways including N-linked glycosylation and ER-associated protein degradation. It also contained novel relationships, such as the placement of SGT2 in the tail-anchored biogenesis pathway, a finding that we experimentally validated. Our approach should be readily applicable to the next generation of quantitative GI data sets, as assays become available for additional phenotypes and eventually higher-level organisms.
View details for DOI 10.1038/msb.2010.27
View details for Web of Science ID 000279636000003
View details for PubMedID 20531408
Identification of yeast proteins necessary for cell-surface function of a potassium channel
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2007; 104 (46): 18079-18084
Inwardly rectifying potassium (Kir) channels form gates in the cell membrane that regulate the flow of K(+) ions into and out of the cell, thereby influencing the membrane potential and electrical signaling of many cell types, including neurons and cardiomyocytes. Kir-channel function depends on other cellular proteins that aid in the folding of channel subunits, assembly into tetrameric complexes, trafficking of quality-controlled channels to the plasma membrane, and regulation of channel activity at the cell surface. We used the yeast Saccharomyces cerevisiae as a model system to identify proteins necessary for the functional expression of a mammalian Kir channel at the cell surface. A screen of 376 yeast strains, each lacking one nonessential protein localized to the early secretory pathway, identified seven deletion strains in which functional expression of the Kir channel at the plasma membrane was impaired. Six deletions were of genes with known functions in trafficking and lipid biosynthesis (sur4Delta, csg2Delta, erv14Delta, emp24Delta, erv25Delta, and bst1Delta), and one deletion was of an uncharacterized gene (yil039wDelta). We provide genetic and functional evidence that Yil039wp, a conserved, phosphoesterase domain-containing protein, which we named "trafficking of Emp24p/Erv25p-dependent cargo disrupted 1" (Ted1p), acts together with Emp24p/Erv25p in cargo exit from the endoplasmic reticulum (ER). The seven yeast proteins identified in our screen likely impact Kir-channel functional expression at the level of vesicle budding from the ER and/or the local lipid environment at the plasma membrane.
View details for DOI 10.1073/pnas.0708765104
View details for Web of Science ID 000251077000034
View details for PubMedID 17989219