Cellular Stoichiometry Research Articles

Direct and indirect cumulative effects of temperature, nutrients, and light on phytoplankton growth.

Temperature and resource availability are pivotal factors influencing phytoplankton community structures. Numerous prior studies demonstrated their significant influence on phytoplankton stoichiometry, cell size, and growth rates. The growth rate, serving as a reflection of an organism's success within its environment, is linked to stoichiometry and cell size. Consequently, alterations in abiotic conditions affecting cell size or stoichiometry also exert indirect effects on growth. However, such results have their limitations, as most studies used a limited number of factors and factor levels which gives us limited insights into how phytoplankton respond to environmental conditions, directly and indirectly. Here, we tested for the generality of patterns found in other studies, using a combined multiple-factor gradient design and two single species with different size characteristics. We used a structural equation model (SEM) that allowed us to investigate the direct cumulative effects of temperature and resource availability (i.e., light, N and P) on phytoplankton growth, as well as their indirect effects on growth through changes in cell size and cell stoichiometry. Our results mostly support the results reported in previous research thus some effects can be identified as dominant effects. We identified rising temperature as the dominant driver for cell size reduction and increase in growth, and nutrient availability (i.e., N and P) as dominant factor for changes in cellular stoichiometry. However, indirect effects of temperature and resources (i.e., light and nutrients) on species' growth rates through cell size and cell stoichiometry differed across the two species suggesting different strategies to acclimate to its environment.

Biogenic Phosphonate Utilization by Globally Distributed Diatom Thalassiosira pseudonana.

Phosphonates are a class of organic phosphorus (P) compounds that contribute ~25% of dissolved organic P. Recent studies reveal the important role of phosphonates mediated by prokaryotes in the marine P redox cycle. However, its bioavailability by eukaryotic phytoplankton is under debate. 2-Aminoethylphosphonic acid (2-AEP) and 2-amino-3-phosphonopropionic acid (2-AP3) are two biogenic phosphonates in the marine environment. Here, Thalassiosira pseudonana, a common diatom species in the ocean, is able to recover growth from P starvation when provided with 2-AEP and 2-AP3. Moreover, 2-AEP cultures exhibited a more similar growth rate at 12 °C than at 25 °C when compared with inorganic P cultures. The cellular stoichiometry of 2-AEP groups was further determined, the values of which are in-between the P-depleted and DIP-replete cultures. This study provides evidence that biogenic phosphonates could be adopted as alternative P sources to support diatom growth and may provide physiological adaptation.

Divergent roles of the regulatory subunits of class IA PI3K.

The regulatory subunit of phosphatidylinositol 3-kinase (PI3K), known as p85, is a critical component in the insulin signaling pathway. Extensive research has shed light on the diverse roles played by the two isoforms of p85, namely p85α and p85β. The gene pik3r1 encodes p85α and its variants, p55α and p50α, while pik3r2 encodes p85β. These isoforms exhibit various activities depending on tissue types, nutrient availability, and cellular stoichiometry. Whole-body or liver-specific deletion of pik3r1 have shown to display increased insulin sensitivity and improved glucose homeostasis; however, skeletal muscle-specific deletion of p85α does not exhibit any significant effects on glucose homeostasis. On the other hand, whole-body deletion of pik3r2 shows improved insulin sensitivity with no significant impact on glucose tolerance. Meanwhile, liver-specific double knockout of pik3r1 and pik3r2 leads to reduced insulin sensitivity and glucose tolerance. In the context of obesity, upregulation of hepatic p85α or p85β has been shown to improve glucose homeostasis. However, hepatic overexpression of p85α in the absence of p50α and p55α results in increased insulin resistance in obese mice. p85α and p85β have distinctive roles in cancer development. p85α acts as a tumor suppressor, but p85β promotes tumor progression. In the immune system, p85α facilitates B cell development, while p85β regulates T cell differentiation and maturation. This review provides a comprehensive overview of the distinct functions attributed to p85α and p85β, highlighting their significance in various physiological processes, including insulin signaling, cancer development, and immune system regulation.

Present and future potential role of toxin-producing Synechococcus in the tropical region

As anthropogenic induced temperature rises and nutrient loadings increase in fresh and brackish environments, the ecological function of the phytoplankton community is expected to favour the picocyanobacteria, of the genus Synechococcus. Synechococcus is already a ubiquitous cyanobacterium found in both freshwater and marine environments, notwithstanding that the toxigenic species still remains unexplored in many freshwaters. Their fast growth rate and their ability to produce toxins make Synechococcus a potential dominant player in harmful algal blooms under climate change scenarios. This study examines the responses of a novel toxin-producing Synechococcus (i.e., one belonging to a freshwater clade; the other belonging to a brackish clade) to environmental changes that reflect climate change effects. We conducted a series of controlled experiments under present and predicted future temperatures, as well as under various N and P nutrients loadings.Our findings highlight how Synechococcus can be altered by the differing reactions to increasing temperature and nutrients, which resulted in considerable variations in cell abundance, growth rate, death rate, cellular stoichiometry and toxin production. Synechococcus had the highest growth observed at 28 °C, and further increases in temperature resulted in a decline for both fresh and brackish waters. Cellular stoichiometry was also altered, where more nitrogen (N) per cell was required, and the plasticity of N:P was more severe for the brackish clade. However, Synechococcus become more toxic under future scenario. Anatoxin-a (ATX) saw the greatest spike when temperature was at 34 °C especially under P-enrichment conditions. In contrast, Cylindrospermopsin (CYN) was promoted at the lowest tested temperature (25 °C) and under N-limitation. Overall, both temperature and external nutrients are the dominant control over Synechococcus toxins production. A model was also created to assess Synechococcus toxicity to zooplankton grazing. Zooplankton grazing was reduced by two folds under nutrient limitation, but temperature accounted for very insignificant change.

Proteome deconvolution of liver biopsies reveals hepatic cell composition as an important marker of fibrosis

Human liver tissue is composed of heterogeneous mixtures of different cell types and their cellular stoichiometry can provide information on hepatic physiology and disease progression. Deconvolution algorithms for the identification of cell types and their proportions have recently been developed for transcriptomic data. However, no method for the deconvolution of bulk proteomics data has been presented to date. Here, we show that proteomes, which usually contain less data than transcriptomes, can provide useful information for cell type deconvolution using different algorithms. We demonstrate that proteomes from defined mixtures of cell lines, isolated primary liver cells, and human liver biopsies can be deconvoluted with high accuracy. In contrast to transcriptome-based deconvolution, liver tissue proteomes also provided information about extracellular compartments. Using deconvolution of proteomics data from liver biopsies of 56 patients undergoing Roux-en-Y gastric bypass surgery we show that proportions of immune and stellate cells correlate with inflammatory markers and altered composition of extracellular matrix proteins characteristic of early-stage fibrosis. Our results thus demonstrate that proteome deconvolution can be used as a molecular microscope for investigations of the composition of cell types, extracellular compartments, and for exploring cell-type specific pathological events. We anticipate that these findings will allow the refinement of retrospective analyses of the growing number of proteome datasets from various liver disease states and pave the way for AI-supported clinical and preclinical diagnostics.

Diminished carbon and nitrate assimilation drive changes in diatom elemental stoichiometry independent of silicification in an iron-limited assemblage

In the California Current Ecosystem, upwelled water low in dissolved iron (Fe) can limit phytoplankton growth, altering the elemental stoichiometry of the particulate matter and dissolved macronutrients. Iron-limited diatoms can increase biogenic silica (bSi) content >2-fold relative to that of particulate organic carbon (C) and nitrogen (N), which has implications for carbon export efficiency given the ballasted nature of the silica-based diatom cell wall. Understanding the molecular and physiological drivers of this altered cellular stoichiometry would foster a predictive understanding of how low Fe affects diatom carbon export. In an artificial upwelling experiment, water from 96 m depth was incubated shipboard and left untreated or amended with dissolved Fe or the Fe-binding siderophore desferrioxamine-B (+DFB) to induce Fe-limitation. After 120 h, diatoms dominated the communities in all treatments and displayed hallmark signatures of Fe-limitation in the +DFB treatment, including elevated particulate Si:C and Si:N ratios. Single-cell, taxon-resolved measurements revealed no increase in bSi content during Fe-limitation despite higher transcript abundance of silicon transporters and silicanin-1. Based on these findings we posit that the observed increase in bSi relative to C and N was primarily due to reductions in C fixation and N assimilation, driven by lower transcript expression of key Fe-dependent genes.

Selective Feeding of a Mixotrophic Dinoflagellate (Lepidodinium sp.) in Response to Experimental Warming and Inorganic Nutrient Imbalance.

Mixotrophic protists are widely observed in the aquatic ecosystems, while how they respond to inorganic nutrient imbalance and ocean warming remains understudied. We conducted a series of experiments on a mixotrophic dinoflagellate Lepidodinium sp. isolated from subtropical coastal waters to investigate the combined effect of temperature and medium nitrate to phosphate ratio (N:P ratio) on the ingestion activities of mixotrophic protists. We found Lepidodinium sp. displayed selective feeding behaviour with a higher ingestion rate on high-N prey (N-rich Rhodomonas salina) when the ambient inorganic N:P ratio was equal to or below the Redfield ratio. The Chesson selectivity index α increased with increasing temperature, suggesting that warming exacerbated the selective feeding of Lepidodinium sp. Under inorganic nitrogen sufficient conditions (N:P ratio = 64), no selective feeding was observed at 25 and 28°C, while it occurs at 31°C, which also indicates that warming alters the feeding behaviour of Lepidodinium sp. In addition, our results revealed that the total ingestion rate of Lepidodinium sp. under the condition with normal inorganic nutrients (Redfield ratio) was significantly lower than that under nutrient-imbalanced conditions, which indicates that Lepidodinium sp. developed compensatory feeding to balance their cellular stoichiometry and satisfy their growth. Our study is the first attempt on revealing the selective feeding behaviours of mixotrophic protists on prey under different inorganic nutrient environments and rising temperatures, which will contribute to our understanding of the response of marine plankton food web to projected climate changes.

Phytoplankton antioxidant systems and their contributions to cellular elemental stoichiometry

AbstractOxidative stress plays a role in many aspects of cellular metabolism, and as a result, antioxidants have the potential to impact cellular stoichiometry and biogeochemical cycles. We reviewed how antioxidant systems influence macronutrient and micronutrient stoichiometry in marine phytoplankton and identified that antioxidant systems have important implications for micronutrient stoichiometry. By leveraging diatom proteomic data, we empirically estimated the level of micronutrient quota variation that can be attributed to antioxidant systems. Fe‐containing antioxidant expression may contribute to 3.3–10 μmol : mol variation in Fe : C, and superoxide dismutases appear to be important contributors to variation in Mn, Ni, Zn, and Cu quotas in phytoplankton. Critical next steps for the study of phytoplankton antioxidant systems are to (1) distinguish between oxidative stress and redox‐based gene regulation and (2) determine how antioxidants influence variation or consistency in micronutrient quotas under various environmental conditions.

From adverse to beneficial: Contrasting dietary effects of freshwater mixotrophs on zooplankton

Abstract The importance of mixotrophic algae as key bacterivores in microbial food webs is increasingly acknowledged, but their effects on the next trophic level remain poorly understood. Their high stoichiometric food quality is contrasted by anti‐grazing strategies. We tested the quality of freshwater mixotrophs as prey for zooplankton, using four non‐colonial chrysophyte species and a cryptophyte as a high quality reference food. We (1) analyzed the stoichiometric and biochemical (fatty acid) composition of the mixotrophs, and (2) quantified their dietary effects on Daphnia longispina survival. Survival of D. longispina significantly depended on the identity of species provided as food, ranging from higher to lower as compared to starvation. This was not reflected in differences in cellular stoichiometry or fatty acid profiles of the mixotrophs. We suggest that toxicity may be the driver for the observed differences. Generalization of the dietary effects of mixotrophic chrysophytes does not appear straightforward. Besides fundamental species‐specific differences, potential toxic effects may vary depending on environmental cues or physiological strategies. Notably in our study, Ochromonas tuberculata, a species previously reported to be deleterious, turned out to be a beneficial food source in terms of enabling high survival of D. longispina. We challenge the generality of the assumption that chrysophytes are of low value as food for zooplankton. We recommend that future studies test how environmental conditions and physiological strategies shape the quality of mixotrophs as food for consumers at higher trophic levels, specifically focusing on effects of dietary toxicity.

The effects of absolute and relative nutrient concentrations (N/P) on phytoplankton in a subtropical reservoir

Abstract The elemental composition of phytoplankton is a critical factor for primary production and nutrient recycling. The increase anthropogenic nutrient input into freshwater ecosystems is affecting phytoplankton assemblage structure and its stoichiometry. Reservoirs of South China generally show low level of phosphate and it is not clear how phytoplankton can grow and occasionally bloom in such conditions. Therefore, an indoor experiment was conducted to investigate the response of natural phytoplankton communities to 25 levels of supplied nitrogen to phosphorus ratios (N/P), arising from the combination of 5 levels of N and P. Our aim was to check the effects of absolute and relative N and P on phytoplankton growth and structure. We assumed that Alkaline Phosphatase (APA) provides a way to use alternative P resource. Our hypotheses include: (1) phytoplankton stoichiometry would be in homeostasis (sensu Sterner & Elser, 2002) under different N and P treatments; (2) absolute nutrient values rather than its ratio matters for phytoplankton assemblage; and (3) phytoplankton cell size declines to lower P requirements facing P limitation. Results showed that phytoplankton in this subtropical reservoir use alternative P sources via APA to support its growth. The absolute values of N and P instead and not their ratios were important for phytoplankton. The N/P ratio cannot be a reliable indicator for nutrient limitation or phytoplankton shift. During the fast-growing phase, their elemental contents were not sensitive to supplied nutrients, and their stoichiometry was more constrained compared to that in slow-growing phase. The plasticity of cellular stoichiometry was mainly due to the variations of cellular P. Phytoplankton stoichiometry was weakly homeostatic, and this mechanism provides a strategy to keep growth relatively stable in a variable nutrient environment.

Intact myocardial preparations reveal intrinsic transmural heterogeneity in cardiac mechanics

Determining transmural mechanical properties in the heart provides a foundation to understand physiological and pathophysiological cardiac mechanics. Although work on mechanical characterisation has begun in isolated cells and permeabilised samples, the mechanical profile of living individual cardiac layers has not been examined. Myocardial slices are 300 μm-thin sections of heart tissue with preserved cellular stoichiometry, extracellular matrix, and structural architecture. This allows for cardiac mechanics assays in the context of an intact in vitro organotypic preparation. In slices obtained from the subendocardium, midmyocardium and subepicardium of rats, a distinct pattern in transmural contractility is found that is different from that observed in other models. Slices from the epicardium and midmyocardium had a higher active tension and passive tension than the endocardium upon stretch. Differences in total myocyte area coverage, and aspect ratio between layers underlined the functional readouts, while no differences were found in total sarcomeric protein and phosphoprotein between layers. Such intrinsic heterogeneity may orchestrate the normal pumping of the heart in the presence of transmural strain and sarcomere length gradients in the in vivo heart.

Biological Stoichiometry Regulates Toxin Production in Microcystis aeruginosa (UTEX 2385).

Harmful algal blooms (HABs) are increasing in magnitude, frequency, and duration globally. Even though a limited number of phytoplankton species can be toxic, they are becoming one of the greatest water quality threats to public health and ecosystems due to their intrinsic toxicity to humans and the numerous interacting factors that undermine HAB forecasting. Here, we show that the carbon:nitrogen:phosphorus (C:N:P) stoichiometry of a common toxic phytoplankton species, Microcystis, regulates toxin quotas during blooms through a tradeoff between primary and secondary metabolism. Populations with optimal C:N (< 8) and C:P (< 200) cellular stoichiometry consistently produced more toxins than populations exhibiting stoichiometric plasticity. Phosphorus availability in water exerted a strong control on population biomass and C:P stoichiometry, but N availability exerted a stronger control on toxin quotas by regulating population biomass and C:N:P stoichiometry. Microcystin-LR, like many phytoplankton toxins, is an N-rich secondary metabolite with a C:N stoichiometry that is similar to the optimal growth stoichiometry of Microcystis. Thus, N availability relative to P and light provides a dual regulatory mechanism that controls both biomass production and cellular toxin synthesis. Overall, our results provide a quantitative framework for improving forecasting of toxin production during HABs and compelling support for water quality management that limit both N and P inputs from anthropogenic sources.

The impacts of iron limitation and ocean acidification on the cellular stoichiometry, photophysiology, and transcriptome of Phaeocystis antarctica

Abstract Phaeocystis antarctica is an integral player of the phytoplankton community of the Southern Ocean (SO), the world's largest high‐nutrient low‐chlorophyll region, and faces chronic iron (Fe) limitation. As the SO is responsible for 40% of anthropogenic CO2 uptake, P. antarctica must also deal with ocean acidification (OA). However, mechanistic studies investigating the effects of Fe limitation and OA on trace metal (TM) stoichiometry, transcriptomic, and photophysiological responses of this species, as well as on the Fe chemistry, are lacking. This study reveals that P. antarctica responded strongly to Fe limitation by reducing its growth rate and particulate organic carbon (POC) production. Cellular concentrations of all TMs, not just Fe, were greatly reduced, suggesting that Fe limitation may drive cells into secondary limitation by another TM. P. antarctica was able to adjust its photophysiology in response to Fe limitation, resulting in similar absolute electron transport rates across PSII. Even though OA‐stimulated growth in Fe‐limited and ‐replete treatments, the slight reduction in cellular POC resulted in no net effect on POC production. In addition, relatively few genes were differentially expressed due to OA. Finally, this study demonstrates that, under our culture conditions, OA did not affect inorganic Fe or humic‐acid‐like substances in seawater but triggered the production of humic‐acid‐like substances by P. antarctica. This species is well adapted to OA under all Fe conditions, giving it a competitive advantage over more sensitive species in a future ocean.

Abstract 5650: Functional proteomics for precise cancer diagnosis and therapy using the MSK Quantitative Cancer Proteomics Atlas

Abstract The advent of molecular biology and molecular profiling in clinical medicine has transformed our understanding of the molecular basis of human cancer. As a result, we are increasingly improving the classification of human tumors based on their specific genetic and molecular mechanisms of pathogenesis. However, currently only a small number of mutant alleles guide treatment decisions, while most observed mutations remain of unknown pathologic and clinical significance. In addition, even for recently approved drugs, such as those targeting activated kinase signaling, clinical efficacy is highly varied, with no currently satisfactory means to identify molecular markers of response and resistance. To address this need, and broadly enable transformative future advances in precision oncology and patient outcomes, we have now developed the Quantitative Cancer Proteomics Atlas (QCPA). This technology permits highly multiplexed, quantitative analysis of the expression and biochemical activity of hundreds of proteins, covering most recurrently mutated and known pathogenic pathways in cancer cells, and designed to be applied to clinically accessible patient specimens. QCPA enables concomitant measurement of cellular concentration and stoichiometry of regulatory post-translational modification of key proteins mediating aberrant survival and proliferation. The activation status of these proteins is then used as a proxy to infer regulatory status of relevant biochemical pathways. These sentinel proteins include 467 known effectors of kinase regulatory signaling, apoptosis, stress response, and proliferation. QCPA measurements can be made using conventional parallel reaction monitoring mass spectrometry, or using an improved method termed accumulated ion monitoring (Cifani &amp; Kentsis 2017). We established analytical metrics of QCPA assays in cancer cell lines, and demonstrated sensitivity and quantification accuracy amenable to measure endogenous chemically modified proteins from microgram specimens. We then demonstrated the ability of QCPA profiling to detect differential biochemical activities in response to defined stimuli in vitro. Next, we deployed QCPA profiling to reveal functional differences between primary human acute myeloid leukemia specimens and CD34+ myeloid progenitors, measuring specific activation of biochemical pathways in cancer cells. Finally, we tested the clinical utility of functional proteomic profiling by determining molecular markers of response and resistance to ERK pathway inhibitors in patient specimens. Thus, QCPA functional proteomic profiling provides a tool to achieve precise diagnosis and rational targeted cancer therapies. Citation Format: Paolo Cifani, Ahmet Dogan, David B. Solit, Alex Kentsis. Functional proteomics for precise cancer diagnosis and therapy using the MSK Quantitative Cancer Proteomics Atlas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5650.

High Variability in Cellular Stoichiometry of Carbon, Nitrogen, and Phosphorus Within Classes of Marine Eukaryotic Phytoplankton Under Sufficient Nutrient Conditions.

Current hypotheses suggest that cellular elemental stoichiometry of marine eukaryotic phytoplankton such as the ratios of cellular carbon:nitrogen:phosphorus (C:N:P) vary between phylogenetic groups. To investigate how phylogenetic structure, cell volume, growth rate, and temperature interact to affect the cellular elemental stoichiometry of marine eukaryotic phytoplankton, we examined the C:N:P composition in 30 isolates across 7 classes of marine phytoplankton that were grown with a sufficient supply of nutrients and nitrate as the nitrogen source. The isolates covered a wide range in cell volume (5 orders of magnitude), growth rate (<0.01–0.9 d−1), and habitat temperature (2–24°C). Our analysis indicates that C:N:P is highly variable, with statistical model residuals accounting for over half of the total variance and no relationship between phylogeny and elemental stoichiometry. Furthermore, our data indicated that variability in C:P, N:P, and C:N within Bacillariophyceae (diatoms) was as high as that among all of the isolates that we examined. In addition, a linear statistical model identified a positive relationship between diatom cell volume and C:P and N:P. Among all of the isolates that we examined, the statistical model identified temperature as a significant factor, consistent with the temperature-dependent translation efficiency model, but temperature only explained 5% of the total statistical model variance. While some of our results support data from previous field studies, the high variability of elemental ratios within Bacillariophyceae contradicts previous work that suggests that this cosmopolitan group of microalgae has consistently low C:P and N:P ratios in comparison with other groups.

Cellular Stoichiometry of Methyl-Accepting Chemotaxis Proteins in Sinorhizobium meliloti.

The chemosensory system in Sinorhizobium meliloti has several important deviations from the widely studied enterobacterial paradigm. To better understand the differences between the two systems and how they are optimally tuned, we determined the cellular stoichiometry of the methyl-accepting chemotaxis proteins (MCPs) and the histidine kinase CheA in S. meliloti Quantitative immunoblotting was used to determine the total amount of MCPs and CheA per cell in S. meliloti The MCPs are present in the cell in high abundance (McpV), low abundance (IcpA, McpU, McpX, and McpW), and very low abundance (McpY and McpZ), whereas McpT was below the detection limit. The approximate cellular ratio of these three receptor groups is 300:30:1. The chemoreceptor-to-CheA ratio is 23.5:1, highly similar to that seen in Bacillus subtilis (23:1) and about 10 times higher than that in Escherichia coli (3.4:1). Different from E. coli, the high-abundance receptors in S. meliloti are lacking the carboxy-terminal NWETF pentapeptide that binds the CheR methyltransferase and CheB methylesterase. Using transcriptional lacZ fusions, we showed that chemoreceptors are positively controlled by the master regulators of motility, VisNR and Rem. In addition, FlbT, a class IIA transcriptional regulator of flagellins, also positively regulates the expression of most chemoreceptors except for McpT and McpY, identifying chemoreceptors as class III genes. Taken together, these results demonstrate that the chemosensory complex and the adaptation system in S. meliloti deviates significantly from the established enterobacterial paradigm but shares some similarities with B. subtilisIMPORTANCE The symbiotic soil bacterium Sinorhizobium meliloti is of great agricultural importance because of its nitrogen-fixing properties, which enhances growth of its plant symbiont, alfalfa. Chemotaxis provides a competitive advantage for bacteria to sense their environment and interact with their eukaryotic hosts. For a better understanding of the role of chemotaxis in these processes, detailed knowledge on the regulation and composition of the chemosensory machinery is essential. Here, we show that chemoreceptor gene expression in S. meliloti is controlled through the main transcriptional regulators of motility. Chemoreceptor abundance is much lower in S. meliloti than in Escherichia coli and Bacillus subtilis Moreover, the chemoreceptor-to-kinase CheA ratio is different from that of E. coli but similar to that of B. subtilis.

Thermal constraints on growth, stoichiometry and lipid content of different groups of microalgae with bioenergy potential

Temperature has a range of effects on population demographic rates and physiology of microalgae for potential bioenergy application. The changes in microalgal growth, stoichiometry and cellular lipid contents of 14 different microalgae species with high bioenergy potentials in response to different temperature between 10 and 35 °C were investigated. For most of the chosen microalgae, the specific growth rate increased with temperature over most of the range. On the other hand, the biomass density of most of the selected species declined with temperature at stationary phase. Certain cyanobacteria failed to grow below 20 °C, while eukaryotes had a wider temperature range. Lipid content, by contrast, showed different responses to temperature among the 14 species. Nine of the species showed a positive response with increased temperature, while others showed neural or unimodal response. Cellular C:N and C:P ratios were mostly unimodally related to temperature. Some eukaryotic species showed the lowest total lipid contents but the highest C:N and C:P ratios, the fastest growth, and the highest biomass density at their stationary phases. The results indicated that the responses of the life history and cellular stoichiometry to temperature were more consistent across microalgae than that of total lipid content.

Seasonal phosphorus and carbon dynamics in a temperate shelf sea (Celtic Sea)

The seasonal cycle of resource availability in shelf seas has a strong selective pressure on phytoplankton diversity and the biogeochemical cycling of key elements, such as carbon (C) and phosphorus (P). Shifts in carbon consumption relative to P availability, via changes in cellular stoichiometry for example, can lead to an apparent ‘excess’ of carbon production. We made measurements of inorganic P (Pi) uptake, in parallel to C-fixation, by plankton communities in the Celtic Sea (NW European Shelf) in spring (April 2015), summer (July 2015) and autumn (November 2014). Short-term (<8 h) Pi-uptake coupled with dissolved organic phosphorus (DOP) release, in parallel to net (24 h) primary production (NPP), were all measured across an irradiance gradient designed to typify vertically and seasonally varying light conditions. Rates of Pi-uptake were highest during spring and lowest in the low light conditions of autumn, although biomass-normalised Pi-uptake was highest in the summer. The release of DOP was highest in November and declined to low levels in July, indicative of efficient utilization and recycling of the low levels of Pi available. Examination of daily turnover times of the different particulate pools, including estimates of phytoplankton and bacterial carbon, indicated a differing seasonal influence of autotrophs and heterotrophs in P-dynamics, with summer conditions associated with a strong bacterial influence and the early spring period with fast growing phytoplankton. These seasonal changes in autotrophic and heterotrophic influence, coupled with changes in resource availability (Pi, light) resulted in seasonal changes in the stoichiometry of NPP to daily Pi-uptake (C:P ratio); from relatively C-rich uptake in November and late April, to P-rich uptake in early April and July. Overall, these results highlight the seasonally varying influence of both autotrophic and heterotrophic components of shelf sea ecosystems on the relative uptake of C and P.

Sm-ChIPi: Single-Molecule Chromatin Immunoprecipitation Imaging.

Epigenetic complexes regulate chromatin dynamics via binding to and assembling on chromatin. However, the mechanisms of chromatin binding and assembly of epigenetic complexes within cells remain incompletely understood, partly due to technical challenges. Here, we present a new approach termed single-molecule chromatin immunoprecipitation imaging (Sm-ChIPi) that enables to assess the cellular assembly stoichiometry of epigenetic complexes on chromatin. Sm-ChIPi was developed based on chromatin immunoprecipitation followed by single-molecule fluorescence microscopy imaging. In this method, an epigenetic complex subunit fused with a gene coding for a fluorescent protein is stably expressed in its corresponding knockout cells. Nucleosomes associated with epigenetic complexes are isolated from cells at native conditions and incubated with biotinylated antibodies. The resulting complexes are immobilized on a quartz slide that had been passivated and functionalized with NeutrAvidin. Image stacks are then acquired by using single-molecule TIRF microscopy. The individual spots imaged by TIRF microscopy represent single protein-nucleosome complexes. The number of copies of the protein complexes on a nucleosome is inferred from the fluorescence photobleaching measurements. Sm-ChIPi is a sensitive and direct method that can quantify the cellular assembly stoichiometry of epigenetic complexes on chromatin.

Full-length cellular β-secretase has a trimeric subunit stoichiometry, and its sulfur-rich transmembrane interaction site modulates cytosolic copper compartmentalization

The β-secretase (BACE1) initiates processing of the amyloid precursor protein (APP) into Aβ peptides, which have been implicated as central players in the pathology of Alzheimer disease. BACE1 has been described as a copper-binding protein and its oligomeric state as being monomeric, dimeric, and/or multimeric, but the native cellular stoichiometry has remained elusive. Here, by using single-molecule fluorescence and in vitro cross-linking experiments with photo-activatable unnatural amino acids, we show that full-length BACE1, independently of its subcellular localization, exists as trimers in human cells. We found that trimerization requires the BACE1 transmembrane sequences (TMSs) and cytoplasmic domains, with residues Ala463 and Cys466 buried within the trimer interface of the sulfur-rich core of the TMSs. Our 3D model predicts that the sulfur-rich core of the trimeric BACE1 TMS is accessible to metal ions, but copper ions did not trigger trimerization. The results of functional assays of endogenous BACE1 suggest that it has a role in intracellular copper compartmentalization by transferring cytosolic copper to intracellular compartments, while leaving the overall cellular copper concentration unaltered. Adding to existing physiological models, our results provide novel insight into the atypical interactions between copper and BACE1 and into its non-enzymatic activities. In conclusion, therapeutic Alzheimer disease prevention strategies aimed at decreasing BACE1 protein levels should be regarded with caution, because adverse effects in copper homeostasis may occur.