Clinton A Oakley

Clinton A Oakley
Victoria University of Wellington · School of Biological Sciences

Ph.D.

About

29
Publications
18,695
Reads
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999
Citations
Introduction
I study the physiology and proteomics of symbiosis, primarily the coral/algal mutualism.
Additional affiliations
June 2013 - present
University of Wellington
Position
  • PostDoc Position
Description
  • Proteomics and cellular biology of the cnidarian-dinoflagellate symbiosis.
August 2007 - May 2013
University of Georgia
Position
  • PhD Student
Education
August 2007 - August 2013
University of Georgia
Field of study
  • Plant Biology
August 2002 - June 2006
Washington & Lee University
Field of study
  • Biology

Publications

Publications (29)
Article
Coral bleaching has devastating effects on coral survival and reef ecosystem function, but many of the fundamental cellular effects of thermal stress on cnidarian physiology are unclear. We used label-free liquid chromatography-tandem mass spectrometry to compare the effects of rapidly (33.5 °C, 24 h) and gradually (30 °C and 33.5 °C, 12 d) elevate...
Article
Full-text available
Coral reef ecosystems are metabolically founded on the mutualism between corals and photosynthetic dinoflagellates of the genus Symbiodinium. The glass anemone Aiptasia sp. has become a tractable model for this symbiosis, and recent advances in genetic information have enabled the use of mass spectrometry-based proteomics in this model. We utilized...
Article
Full-text available
The symbiosis between hermatypic corals and their dinoflagellate endosymbionts, genus Symbiodinium, is based on carbon exchange. This symbiosis is disrupted by thermally induced coral bleaching, a stress response in which the coral host expels its algal symbionts as they become physiologically impaired. The disruption of the dissolved inorganic car...
Article
Full-text available
A terminal electron acceptor alternative to mitochondrial cytochrome c oxidase (COX), mitochondrial alternative oxidase (AOX), is ubiquitous in higher plants and represented in nearly every algal taxon but is poorly documented in dinoflagellates. AOX competes for electrons with the conventional COX and has been hypothesized to function as a means o...
Article
Coral reefs of the Florida Keys typically experience seasonal temperatures of 20–31 °C. Deviation outside this range causes physiological impairment of reef-building corals, potentially leading to coral colony death. In January and February 2010, two closely spaced cold fronts, possibly driven by an unusually extreme Arctic Oscillation, caused sudd...
Preprint
Full-text available
Within microeukaryotes, genetic and functional variation sometimes accumulate more quickly than morphological differences. To understand the evolutionary history and ecology of such lineages, it is key to examine diversity at multiple levels of organization. In the dinoflagellate family Symbiodiniaceae, which can form endosymbioses with cnidarians...
Article
Bidirectional nutrient flow between partners is integral to the cnidarian-dinoflagellate endosymbiosis. However, our current knowledge of the transporter proteins that regulate nutrient and metabolite trafficking is nascent. Four transmembrane transporters that likely play an important role in interpartner nitrogen and carbon exchange were investig...
Article
Full-text available
In the cnidarian-dinoflagellate symbiosis, hosts show altered expression of genes involved in growth and proliferation when in the symbiotic state, but little is known about the molecular mechanisms that underlie the host’s altered growth rate. Using tissue-specific transcriptomics, we determined how symbiosis affects expression of cell cycle-assoc...
Article
Full-text available
Endozoicomonas are prevalent, abundant bacterial associates of marine animals, including corals. Their role in holobiont health and functioning, however, remains poorly understood. To identify potential interactions within the coral holobiont, we characterized the novel isolate Endozoicomonas marisrubri sp. nov. 6c and assessed its transcriptomic a...
Article
Full-text available
The Symbiodiniaceae are a taxonomically and functionally diverse family of marine dinoflagellates. Their symbiotic relationship with invertebrates such as scleractinian corals has made them the focus of decades of research to resolve the underlying biology regulating their sensitivity to stressors, particularly thermal stress. Research to-date sugg...
Article
Full-text available
Coral reefs are restricted to warm waters, but are increasingly threatened by coral bleaching induced by sustained high sea surface temperatures. Coral endosymbiont thermal resilience has been proposed to depend, at least in part, on the lipid composition of their thylakoid membranes, which influences photosynthetic performance under sub- and super...
Chapter
Metabolite exchange between coral hosts and their dinoflagellate endosymbionts (family: Symbiodiniaceae) is one of the keys to the ecological success of coral reefs. Due to the physiological variation within Symbiodiniaceae, including amount of organic carbon and stress tolerance provided to the host, symbiont community composition has the potentia...
Article
Tropical lagoon-inhabiting organisms live in highly irradiated ecosystems and are particularly susceptible to thermal stress resulting from climate change. However, despite living close to their thermal maxima, stress response mechanisms found in these organisms are poorly understood. We used a novel physiological-proteomic approach for sponges to...
Article
Full-text available
The algal cell wall is an important cellular component that functions in defense, nutrient utilization, signaling, adhesion, and cell‐cell recognition — processes important in the cnidarian–dinoflagellate symbiosis. The cell wall of symbiodiniacean dinoflagellates is not well characterized. Here, we present a method to isolate cell walls of Symbiod...
Article
The dinoflagellate family Symbiodiniaceae comprises numerous genera and species with large differences in diversity, ecology and geographic distribution. An evolutionarily divergent lineage common in temperate symbiotic cnidarians and designated in the literature by several informal names including ‘temperate–A’, AI, Phylotype A´ (A-prime) and ‘Med...
Article
Full-text available
Some reef corals form stable, dominant or codominant associations with multiple endosymbiotic dinoflagellate species (family Symbiodiniaceae). Given the immense genetic and physiological diversity within this family, Symbiodiniaceae community composition has the potential to impact the nutritional physiology and fitness of the cnidarian host and al...
Article
Full-text available
In oligotrophic waters, cnidarian hosts rely on symbiosis with their photosynthetic dinoflagellate partners (family Symbiodiniaceae) to obtain the nutrients they need to grow, reproduce and survive. For this symbiosis to persist, the host must regulate the growth and proliferation of its symbionts. One of the proposed regulatory mechanisms is arres...
Article
Full-text available
Hosting different symbiont species can affect inter‐partner nutritional fluxes within the cnidarian–dinoflagellate symbiosis. Using nanoscale secondary ion mass spectrometry (NanoSIMS), we measured the spatial incorporation of photosynthetically‐fixed 13C and heterotrophically‐derived 15N into host and symbiont cells of the model symbiotic cnidaria...
Article
Full-text available
The acquisition of thermally tolerant algal symbionts by corals has been proposed as a natural or assisted mechanism of increasing coral reef resilience to anthropogenic climate change, but the cell-level processes determining the performance of new symbiotic associations are poorly understood. We used liquid chromatography–mass spectrometry to inv...
Article
Metabolite exchange is fundamental to the viability of the cnidarian-Symbiodiniaceae symbiosis and survival of coral reefs. Coral holobiont tolerance to environmental change might be achieved through changes in Symbiodiniaceae species composition, but differences in the metabolites supplied by different Symbiodiniaceae species could influence holob...
Chapter
Corals depend on a mutualistic symbiosis with intracellular dinoflagellates of the genus Symbiodinium for their energetic needs. The high productivity of corals in a challenging environment and the necessity of coordinating the metabolism and growth of each partner mean that severe stresses, such as sustained high temperatures, may destabilize the...
Article
Metabolic exchange between cnidarians and their symbiotic dinoflagellates is central to maintaining their mutualistic relationship. Sugars are translocated to the host, while ammonium and nitrate are utilized by the dinoflagellates (Symbiodinium spp.). We investigated membrane protein sequences of each partner to identify potential transporter prot...
Article
Full-text available
The relationship between reef-building corals and phototrophic dinoflagellates of the genus Symbiodinium is fundamental to the functioning of coral reef ecosystems. It has been suggested that reef corals may adapt to climate change by changing their dominant symbiont type to a more thermally tolerant one, although the capacity for such a community...
Article
Full-text available
Experimental manipulation of the symbiosis between cnidarians and photosynthetic dinoflagellates (Symbiodinium spp.) is critical to advance understanding of the cellular mechanisms involved in host-symbiont interactions, and overall coral reef ecology. The anemone Aiptasia sp. is a model for the cnidarian-dinoflagellate symbiosis, and notably it ca...
Article
For cnidarians that can undergo shifts in algal symbiont relative abundance, the underlying algal physiological changes that accompany these shifts are not well known. The sea anemone Anthopleura elegantissima associates with the dinoflagellate Symbiodinium muscatinei and the chlorophyte Elliptochloris marina, symbionts with very different toleranc...
Article
During restoration of bare subsoil, are planted grassland communities with low species richness more susceptible to invasion by non-residents than communities augmented by additional species? What are the mechanisms of invasion resistance in early succession? Lexington, Virginia, USA (37.8°N, −79.4°W). We planted 62 3 × 3 m plots on compacted clay...
Article
Conventional means of assessing net photosynthetic rates of microalgae have largely relied upon the use of oxygen electrodes or carbon isotope radiolabeling. These methods are simple but inadequately resolve simultaneous fluxes of the gaseous substrates and products of photosynthesis, CO2, and O2. Fluorometric methods allow for assessment of the ph...
Article
Full-text available
Members of the marine Roseobacter lineage have been characterized as ecological generalists, suggesting that there will be challenges in assigning well-delineated ecological roles and biogeochemical functions to the taxon. To address this issue, genome sequences of 32 Roseobacter isolates were analyzed for patterns in genome characteristics, gene i...

Questions

Questions (9)
Question
Does anyone have any tips on clearing blocked nanoViper (20 uM ID) tubing in a Dionex 3000 for LC-MS/MS? We're having recurring high pressure errors in our loading pump, and we've isolated it to a specific piece of nanoViper tubing between the loading pump and autosampler. The chemical identity of the clog is unknown, only that it is from a sample and is hydrophobic. Obviously identifying the source of the contaminant is important, we are investigating that as well. The pressure is excessively high but the flow is not completely blocked.
Currently we've connected the tubing directly to the loading pump, with the opposite end open. We have run first water and now 75% ACN through it, in both directions, but with little success in dissolving the block and reducing the pressure so far. Would a stronger/different solvent be useful?
Question
I would like to use STRING for functional analysis of proteome data, as it appears to be quite powerful. Unfortunately I'm working with an organism that isn't in STRING's database, nor is there anything particularly closely related on the species list. STRING requires all proteins to be from a single species on their list. Is there a sound method to use my data in STRING, perhaps using gene orthologs from another species? For example, could I BLAST my differentially expressed protein sequences against the genome of a single model organism (e.g. Arabidopsis) and use the top hits as the gene IDs? I've tried using simple gene names as the input, but typically only about half are present in any single model organism I've tried. I'm aware that there are hazards in using orthologs, but I'm mostly interested in higher-order patterns and GO term enrichment.
Question
We're working on generating proteomics datasets of non-model organisms which show considerable diversity between genera and for which there are several gene model sets available, of varying quality. Most proteome manuscripts use a single model organism and a canonical, well-annotated gene model set (e.g. from Uniprot) for peptide searches using MASCOT, SEQUEST, etc.
Is there any formal analysis available on the effects of using multiple combined gene model sets for peptide/protein matches, particularly in cases where the species of the sample and the species of the gene model set are not identical? In our case, I am considering combining several of the available gene model sets to form one large search database which I can use with samples that are different species (either from the same genus or related genera). Are there pitfalls in this approach, assuming one uses a protein-clustering algorithm to collapse redundant sequences? I would think that it may improve the number of proteins identified due to either a better match of the sample peptide and the gene model (due to a diverse set of models in the database) or by filling in gaps in individual gene model databases.
Question
We are having problems with an ESI LC MS/MS proteomics experiment that are difficult to pin down. Samples were prepared in batches over the course of a week, the first batches worked very well but then performance degraded, resulting in much fewer protein IDs (~1300 proteins reduced to ~300). The problem manifests in the chromatograms as a failure of the sampling peaks to separate (screen captures linked). The 6-second sampling intervals blend together, especially as the hydrophobic solvent (ACN) increases on the gradient. The images illustrate good separation early in the gradient, followed by the peaks beginning to merge, followed by almost a solid signal halfway through the gradient. The .raw file sizes for "good" samples balloon from 400MB to almost 1GB for "bad" samples due to the extra data.
[Raw tissue homogenates are prepared by incubation in a 5% sodium deoxycholate lysis buffer with DTT and 50mm TEAB, incubation at 90C for 15 minutes followed by 60C for 15 minutes, aliquoting of 20ug protein per sample, alkylation with iodoacetamide, 10x dilution in UHPLC-grade water, 18 hour trypsin digestion, acidification with TFA to precipitate detergent, transfer to a new tube, phase transfer with diethyl ether to remove any remaining detergent, and vacuum centrifugation to evaporate any residual ether, followed by desalting with C18 tips. Samples are run on a Dionex 3000 LC and ThermoFisher Orbitrap LTQ using Chromasolv-grade reagents. I've used this protocol and instrumentation with success many times in the past.]
The column has since been changed, the instrument re-calibrated, the spray pattern is good and analysis of cytochrome C standard digests are nearly perfect, but my runs are even worse than before. I attribute the problem to the sample preparation. Most mysteriously, I re-ran a preparation that was very successful a few weeks ago and it now exhibits the same problems (80% loss of protein IDs), and it's been in -80C storage in glass in the interim. What would cause this problem with the chromatograms, given that other users using other sample methods don't seem to be having issues? Can it be attributed to sample contamination from the preparation process, plasticizers, residual detergent/solvents, etc.? In addition to the merged sampling intervals the latest runs have large peaks late in the gradient with m/z of 373.27, 376.26 and 817.58.
Question
We are using a PAM fluorometer to measure the quantum yield of chlorophyll fluorescence in an intact microalga culture. When the culture is dark-adapted for 20-30 minutes, the quantum yield is rather low (0.4), while if it's measured under low light (~50uM quanta) the yield will increase to about 0.6. The alga medium is not agitated and the cells form a biofilm on the bottom of the flask. Could this be a result of a low-oxygen layer forming around the biofilm after being in the dark, inhibiting PQ pool oxidation? Is there another explanation?
Question
We are attempting a shotgun proteomics experiment using LC ESI-MS/MS with an Orbitrap LTQ XL, but are suddenly encountering a problem with excessive background noise. The noise level is fairly constant at ~4E6 intensity for the entire gradient, obscuring many peaks. The peaks that are above the noise are sharp and we still obtain several hundred identifications, but total IDs have dropped by 50-75% and comparison with previous, much more successful, runs shows that the noise in those runs was 0.5-1xE6. The difference between ~5E5 and ~5E6 is enough to obscure most of the sample peaks. The ESI spray appears to be good, and the Orbitrap was recently tuned and serviced by a ThermoFisher technician. Running samples that previously were of high quality shows the same problem, indicating a problem with the instrument, not the sample preparation. Any advice would be greatly appreciated.
Question
We are using NuPAGE gels to resolve protein samples, and the included protocol calls for the addition of 0.5mL "antioxidant" to the inner gel chamber. I understand the purpose of the reagent, but is anyone aware of a recipe to prepare an appropriate substitute?
Question
We are considering quantitative shotgun proteomics experiments on cultured marine algae using ESI-LC MS/MS followed by analysis by Mascot. It is not overly difficult to culture cells in a 99% 13C medium, so we can expect high rates of 13C protein enrichment. The included metabolic quantification configuration files for Mascot Distiller, however, they use only 15N labeling or 15N combined with 13C. Has a 13C-only configuration file been developed?
Question
We are attempting non-quantitative shotgun proteomics of unicellular algal culture cell lysates using LC-ESI-MS/MS Orbitrap, but are encountering the issue of only detecting a few dozen very high-abundance, mostly chloroplast-associated proteins (light harvesting proteins, rubisco, etc.). Other than separating the chloroplast from the sample (not possible in these samples), how can we deplete these high-abundance proteins so we can detect lower-abundance proteins in our sample? Is fractionation by SDS-PAGE acceptable?

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