Subcellular Partitioning Research Articles

Mitochondrial network reorganization and transient expansion during oligodendrocyte generation

Oligodendrocyte precursor cells (OPCs) give rise to myelinating oligodendrocytes of the brain. This process persists throughout life and is essential for recovery from neurodegeneration. To better understand the cellular checkpoints that occur during oligodendrogenesis, we determined the mitochondrial distribution and morphometrics across the oligodendrocyte lineage in mouse and human cerebral cortex. During oligodendrocyte generation, mitochondrial content expands concurrently with a change in subcellular partitioning towards the distal processes. These changes are followed by an abrupt loss of mitochondria in the oligodendrocyte processes and myelin, coinciding with sheath compaction. This reorganization and extensive expansion and depletion take 3 days. Oligodendrocyte mitochondria are stationary over days while OPC mitochondrial motility is modulated by animal arousal state within minutes. Aged OPCs also display decreased mitochondrial size, volume fraction, and motility. Thus, mitochondrial dynamics are linked to oligodendrocyte generation, dynamically modified by their local microenvironment, and altered in the aging brain.

Combined effects of cadmium and sulfamethoxazole on Eisenia fetida: Insights into accumulation, subcellular partitioning, biomarkers and toxicological responses

Cadmium (Cd) and sulfamethoxazole (SMX) frequently coexist in farmlands, yet their synergistic toxicological impacts on terrestrial invertebrates remain unexplored. In this study, earthworms were exposed to artificial soils percolated with Cd (5 mg/kg), SMX (5 mg/kg) or combination of them for 7 days, followed by a 12-day elimination phase in uncontaminated soil. The uptake of Cd and SMX by the earthworms, along with their subcellular distribution, was meticulously analyzed. Additionally, a suite of biomarkers—including superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA), and weight loss—were evaluated to assess the health status of the earthworms and the toxicological effects of the Cd and SMX mixture. Notably, the cotreatment with Cd and SMX resulted in a significantly higher weight loss in Eisenia fetida (41.25 %) compared to exposure to Cd alone (26.84 %). Moreover, the cotreatment group exhibited substantially higher concentrations of Cd in the total internal body, fraction C (cytosol), and fraction E (tissue fragments and cell membranes) in Eisenia fetida compared to Cd alone counterparts. The combined exposure also significantly elevated the SMX levels in the total body and fraction C compared with the SMX-only treated earthworms. Additionally, Eisenia fetida subjected to the combined treatment showed markedly increased activities of SOD, CAT, and MDA compared to those treated with Cd alone. The effect addition indices (EAIs), ranging from 1.00 to 2.23, unequivocally demonstrated a synergistic effect of the combined treatments. Interestingly, relocating the earthworms to clean soil did not mitigate the observed adverse effects. These findings underscore the increased risk posed by the Cd-SMX complex to terrestrial invertebrates in agricultural areas.

Methylmercury in northern pike (Esox lucius) liver and hepatic mitochondria is linked to lipid peroxidation

Methylmercury (MeHg) readily bioaccumulates and biomagnifies in aquatic food webs leading to elevated concentrations in fish and may thus induce toxicity. Oxidative stress is a suggested effect of MeHg bioaccumulation in fish. However, studies on how MeHg triggers oxidative stress in wild fish are scarce. The purpose of this study was to link the subcellular distribution of MeHg in the liver of northern pike from the St. Maurice River (Québec, Canada), affected by two run-of-river (RoR) dams, artificial wetlands, forest fires, and logging activity, to lipid peroxidation as an indicator of oxidative stress. We also evaluated the protective effects of the glutathione (GSH) system and selenium (Se), as they are known to alleviate MeHg toxicity. A customized subcellular partitioning protocol was used to separate the liver into metal-sensitive (mitochondria, microsome/lysosome and HDP – heat-denatured proteins) and metal-detoxified fractions (metal-rich granules and HSP – heat-stable proteins). We examined the relation among THg, MeHg, and Se concentration in livers and subcellular fractions, and the hepatic ratio of total GSH (GSHt) to oxidized glutathione (GSSG) on lipid peroxidation levels, using the concentrations of malondialdehyde (MDA), a product of lipid peroxidation. Results showed that hepatic MDA concentration was positively correlated with the combined MeHg and Se concentrations in northern pike liver (r2 = 0.88, p < 0.001) and that MDA concentrations were best predicted by MeHg associated with the mitochondria (r2 = 0.71, p < 0.001). This highlights the need for additional research on the MeHg influence on fish health and the interactions between Hg and Se in northern pike.

Distribution of metals in the queen scallop Aequipecten opercularis during a transplant experiment: Metal rich granules as drivers of Pb bioaccumulation

The queen scallop Aequipecten opercularis accumulates high concentrations of lead (Pb) in its tissues, what has led to the interruption of this fishery in some extraction areas in Galicia (NW Spain). This study follows the dynamics of bioaccumulation of Pb and other metals in this species, the tissue distribution and the subcellular partitioning in selected organs, in order to understand the mechanisms that provoke the high Pb levels reached in its tissues and to increase our knowledge about metal bioaccumulation dynamics in this species. Scallops originating from a clean area were exposed in cages in two places in the Ría de Vigo (one shipyard and a less impacted location) and 10 individuals were collected every month over a three months period. Metal bioaccumulation and metal distribution in several organs, including gills, digestive gland, kidneys, muscle, gonad and remaining tissues, was studied. The results showed that scallops accumulated similar levels of Cd, Pb and Zn at both sites, while Cu and Ni showed an opposite pattern at the shipyard, with Cu concentrations increasing around 10 times and Ni decreasing during the 3 months of exposure. The preferential organs for metal accumulation were the kidneys for Pb and Zn, the digestive gland for Cd, both organs for Cu and Ni, and the muscle for As. Subcellular partitioning of kidney samples additionally showed an extraordinary ability to accumulate Pb and Zn at very high concentrations in kidney granules, a fraction that accounted for 30 to 60 % of Pb in soft-tissues. It is concluded that Pb bioaccumulation in kidney granules is the mechanism responsible for the high levels of Pb observed in this species.

Joint toxicity of cadmium and fenpyroximate on two earthworms: Interspecific differences, subcellular partitioning and biomarker responses

Cadmium (Cd) and fenpyroximate are common soil contaminants found together in the field, but their combined toxicity to terrestrial invertebrates has not been studied. Therefore, earthworms Aporrectodea jassyensis and Eisenia fetida were exposed into Cd (5, 10, 50 and 100 μg/g) and fenpyroximate (0.1, 0.5, 1, and 1.5 μg/g) and their mixture, and multiple biomarker responses (mortality, catalase (CAT), superoxide dismutase (SOD), total antioxidant activity (TAC), lipid peroxidation (MDA), protein content, weight loss and subcellular partitioning) were determined to estimate health status and mixture effect. MDA, SOD, TAC, and weight loss were significantly correlated with Cd in total internal and debris (p < 0.01). Fenpyroximate altered the subcellular distribution of Cd. It appears that maintaining Cd in a non-toxic form was the earthworms' primary Cd detoxification strategy. CAT activity was inhibited by Cd, fenpyroximate, and their combined presence. BRI values for all treatments indicated a major and severe alteration in earthworm's health. The combined toxicity of Cd and fenpyroximate was greater than the toxicity of either substance alone. According to EAI, all combined treatments exhibited a clear antagonistic effect. In general, the sensitivity of A. jassyensis was greater than that of E. fetida.

Subcellular Accumulation and Depuration of Zinc in Periphytic Algae during Episodic and Continuous Exposures

As the severity of extreme precipitation events increases with global climate change, so will episodic pulses of contamination into lotic systems. Periphytic algae represents bioindicator species in most freshwater systems due to their rapid accumulation of toxicants; therefore, it is vital to understand how accumulation in this group differs across temporally variable exposure regimes. The ability to rapidly accrue contaminants has additional implications for the trophic transfer of metals to primary consumers. While dietary toxicity has been studied in algivorous consumers, techniques used to prepare contaminated periphytic algae for consumption have not been compared. This study used a modified subcellular fractionation method to compare the partitioning of zinc (Zn) in periphyton cultures exposed for various durations (cultured in the presence of Zn and 15 min, 24 h, and 48 h exposures). Three exposure groups were additionally depurated over a period of 24 h in order to compare retention of Zn, an important aspect of preparing diets used in dietary toxicity studies. The results not only provide evidence for increased retention by periphytic algae cultured in the presence of Zn but reveal relationships among treatments and subcellular partitioning that suggest time-dependent accumulation and detoxification. These relationships suggest that episodic exposure of periphytic algae to contaminants may pose a greater risk than that of chronic regimes. Based on these results, we additionally advocate for culturing periphytic algae in the presence of contamination to produce a more reliable diet for dietary exposure testing in algivorous organisms.

Nuclear accumulation of host transcripts during Zika Virus Infection.

Zika virus (ZIKV) infects fetal neural progenitor cells (NPCs) causing severe neurodevelopmental disorders in utero. Multiple pathways involved in normal brain development are dysfunctional in infected NPCs but how ZIKV centrally reprograms these pathways remains unknown. Here we show that ZIKV infection disrupts subcellular partitioning of host transcripts critical for neurodevelopment in NPCs and functionally link this process to the up-frameshift protein 1 (UPF1). UPF1 is an RNA-binding protein known to regulate decay of cellular and viral RNAs and is less expressed in ZIKV-infected cells. Using infrared crosslinking immunoprecipitation and RNA sequencing (irCLIP-Seq), we show that a subset of mRNAs loses UPF1 binding in ZIKV-infected NPCs, consistent with UPF1's diminished expression. UPF1 target transcripts, however, are not altered in abundance but in subcellular localization, with mRNAs accumulating in the nucleus of infected or UPF1 knockdown cells. This leads to diminished protein expression of FREM2, a protein required for maintenance of NPC identity. Our results newly link UPF1 to the regulation of mRNA transport in NPCs, a process perturbed during ZIKV infection.

Subcellular partitioning of protein kinase activity revealed by functional kinome profiling

Protein kinases and their substrates form signaling networks partitioned across subcellular compartments to facilitate critical biological processes. While the subcellular roles of many individual kinases have been elucidated, a comprehensive assessment of the synaptic subkinome is lacking. Further, most studies of kinases focus on transcript, protein, and/or phospho-protein expression levels, providing an indirect measure of protein kinase activity. Prior work suggests that gene expression levels are not a good predictor of protein function. Thus, we assessed global serine/threonine protein kinase activity profiles in synaptosomal, nuclear, and cytosolic fractions from rat frontal cortex homogenate using peptide arrays. Comparisons made between fractions demonstrated differences in overall protein kinase activity. Upstream kinase analysis revealed a list of cognate kinases that were enriched in the synaptosomal fraction compared to the nuclear fraction. We identified many kinases in the synaptic fraction previously implicated in this compartment, while also identifying other kinases with little or no evidence for synaptic localization. Our results show the feasibility of assessing subcellular fractions with peptide activity arrays, as well as suggesting compartment specific activity profiles associated with established and novel kinases.

Subcellular Partitioning of Arsenic Trioxide Revealed by Label-Free Imaging.

Subcellular partitioning of therapeutic agents is highly relevant to their interactions with target molecules and drug efficacy, but studying subcellular partitioning is an enormous challenge. Here, we describe the application of nanoscale secondary ion mass spectrometry (NanoSIMS) analysis to define the subcellular pharmacokinetics of a cytotoxic chemotherapy drug, arsenic trioxide (ATO). We reasoned that defining the partitioning of ATO would yield valuable insights into the mechanisms underlying ATO efficacy. NanoSIMS imaging made it possible to define the intracellular fate of ATO in a label-free manner─and with high resolution and high sensitivity. Our studies of ATO-treated cells revealed that arsenic accumulates in the nucleolus. After prolonged ATO exposure, ∼40 nm arsenic- and sulfur-rich protein aggregates appeared in the cell nucleolus, nucleus, and membrane-free compartments in the cytoplasm, and our studies suggested that the partitioning of nanoscale aggregates could be relevant to cell survival. All-trans retinoic acid increased intracellular ATO levels and accelerated the nanoscale aggregate formation in the nucleolus. This study yielded fresh insights into the subcellular pharmacokinetics of an important cancer therapeutic agent and the potential impact of drug partitioning and pharmacokinetics on drug activity.

KRAS protein expression becomes progressively restricted during embryogenesis and in adulthood.

KRAS mutants are common in many cancers and wild-type KRAS is essential in development as its absence causes embryonic lethality. Despite this critical role in development and disease, the normal expression pattern of KRAS protein is still largely unknown at the tissue level due to the lack of valid antibodies. To address this issue, we used the citrine-Kras mouse model in which the Citrine-KRAS (Cit-K) fusion protein functions as a validated surrogate of endogenous KRAS protein that can be detected on tissue sections by immunolabeling with a GFP antibody. In the embryo, we found expression of KRAS protein in a wide range of organs and tissues. This expression tends to decrease near birth, mainly in mesenchymal cells. During transition to the adult stage, the dynamics of KRAS protein expression vary among organs and detection of KRAS becomes restricted to specific cell types. Furthermore, we found that steady state KRAS protein expression is detectable at the cell membrane and in the cytoplasm and that this subcellular partitioning differed among cell types. Our results reveal hitherto unanticipated dynamics in developmental, tissular, cell-specific and subcellular expression of KRAS protein. They provide insight into the reason why specific cell-types are sensitive to KRAS mutations during cancer initiation.

Subcellular localization and compartment-specific toxicokinetics of cadmium, arsenic, and zinc in brandling worm Eisenia fetida

Awareness of toxicokinetics at the subcellular level is crucial to deciphering the underlying intoxication processes of metal(loid)s, although this information is often lacking. Here, the toxicokinetics of two non-essential metal(loid)s (Cd and As) and one essential metal (Zn) in both the whole body and subcellular fractions of earthworm (Eisenia fetida) were assessed. Earthworms were exposed to natural soils originating from a gradient of metal(loid) pollution for 14 days followed by a 14-day elimination phase in clean soil. Clearly distinct toxicokinetic patterns were found in the earthworms according to the metal(loid) considered. An obvious concentration-dependent increase was observed in earthworms or subcellular compartments where no equilibrium was reached (with slow or no elimination) for Cd and As throughout the experiment. As for Zn, the earthworms were able to retain a steady-state concentration of Zn in its body or each fraction without a clear intake behavior via the dynamic trade-off between uptake and elimination at different pollution levels. These differences in toxicokinetics at the subcellular level supported the observed differences in bioaccumulation patterns and were indicative of the strategy by which non-essential and essential elements are handled by earthworms. Notably, the concentration of Cd and As in subcellular compartments showed the same pattern as for Zn in the order of cellular cytosol > cellular debris > metal-rich granules, which might be associated with the binding of non-essential/essential elements with metallothionein enriched in the cytosol. Our findings enhance the understanding of the underlying mechanisms for metal(loid) accumulation kinetics in earthworms from the perspective of subcellular partitioning, and will be beneficial for accurate risk assessment of Cd, As, and Zn.

Subcellular Element Distribution in Shoots of Wheat Grown in an Acidic Soil with Native AMF Extraradical Mycelium

Soil acidity can reduce crop growth by increasing bioavailable soil Al, Fe, and/or Mn to toxic levels. The presence of an intact extraradical mycelium (ERM) of arbuscular mycorrhizal fungi (AMF), developed by the native Ornithopus compressus in the acidic soil, can increase wheat growth and prevent symptoms of Mn toxicity. To understand the protective effect of the intact ERM of this native plant on wheat element balance and distribution, in the present study, shoot Al, Fe, K, Zn, Na, and Si levels and their subcellular partitioning were determined by inductively coupled plasma mass spectrometry (ICP-MS), for the first time, for this system. In undisturbed soil, where an intact ERM structure is maintained, wheat shoot growth was promoted, probably due to faster root mycorrhizal colonization. The levels of potentially toxic Al and Fe were reduced, the proportions of the macronutrient K and micronutrient Zn were higher in the symplast, and the Na proportion increased in the vacuole, while Si increased in the apoplast. Overall, the undisturbed soil from O. compressus treatment appeared to influence the uptake and distribution of essential and beneficial elements, as a strategy to reduce the negative effect of soil acidity on wheat growth. Understanding the dynamics of element distribution influenced by stress-adapted AMF on wheat growth can provide more sustainable approaches to intensive agriculture.

Significance of chemical affinity on metal subcellular distribution in yellow perch (Perca flavescens) livers from Lake Saint-Pierre (QUEBEC, Canada)

The subcellular partitioning approach provides useful information on the location of metals within cells and is often used on organisms with high levels of bioaccumulation to establish relationships between the internal concentration and the potential toxicity of metals. Relatively little is known about the subcellular partitioning of metals in wild fish with low bioaccumulation levels in comparison with those from higher contaminated areas. This study aims to examine the subcellular partitioning of various metals considering their chemical affinity and essentiality at relatively low contamination levels. Class A (Y, Sr), class B (Cu, Cd, MeHg), and borderline (Fe, Mn) metal concentrations were measured in livers and subcellular fractions of yellow perch (n = 21) collected in Lake Saint-Pierre, QC, Canada. The results showed that all metals, apart from MeHg, were distributed among subcellular fractions according to their chemical affinity. More than 60% of Y, Sr, Fe, and Mn were found in the metal-sensitive fractions. Cd and Cu were largely associated with the metallothionein-like proteins and peptides (60% and 67% respectively) whereas MeHg was found mainly in the metal-sensitive fractions (86%). In addition, the difference between the subcellular distribution of Cu and other essential metals like Fe and Mn denotes that, although the essentiality of some metals is a determinant of their subcellular distribution, the chemical affinity of metals is also a key driver. The similarity of the subcellular partitioning results with previous studies on yellow perch and other fish species from higher contaminated areas supports the idea that metals are distributed in the cellular environment according to their chemical properties regardless of the bioaccumulation gradient.

Widespread hydroxylation of unstructured lysine-rich protein domains by JMJD6

The Jumonji domain-containing protein JMJD6 is a 2-oxoglutarate-dependent dioxygenase associated with a broad range of biological functions. Cellular studies have implicated the enzyme in chromatin biology, transcription, DNA repair, mRNA splicing, and cotranscriptional processing. Although not all studies agree, JMJD6 has been reported to catalyze both hydroxylation of lysine residues and demethylation of arginine residues. However, despite extensive study and indirect evidence for JMJD6 catalysis in many cellular processes, direct assignment of JMJD6 catalytic substrates has been limited. Examination of a reported site of proline hydroxylation within a lysine-rich region of the tandem bromodomain protein BRD4 led us to conclude that hydroxylation was in fact on lysine and catalyzed by JMJD6. This prompted a wider search for JMJD6-catalyzed protein modifications deploying mass spectrometric methods designed to improve the analysis of such lysine-rich regions. Using lysine derivatization with propionic anhydride to improve the analysis of tryptic peptides and nontryptic proteolysis, we report 150 sites of JMJD6-catalyzed lysine hydroxylation on 48 protein substrates, including 19 sites of hydroxylation on BRD4. Most hydroxylations were within lysine-rich regions that are predicted to be unstructured; in some, multiple modifications were observed on adjacent lysine residues. Almost all of the JMJD6 substrates defined in these studies have been associated with membraneless organelle formation. Given the reported roles of lysine-rich regions in subcellular partitioning by liquid-liquid phase separation, our findings raise the possibility that JMJD6 may play a role in regulating such processes in response to stresses, including hypoxia.

A campaign targeting a conserved Hsp70 binding site uncovers how subcellular localization is linked to distinct biological activities

The potential of small molecules to localize within subcellular compartments is rarely explored. To probe this question, we measured the localization of Hsp70 inhibitors using fluorescence microscopy. We found that even closely related analogs had dramatically different distributions, with some residing predominantly in the mitochondria and others in the ER. CRISPRi screens supported this idea, showing that different compounds had distinct chemogenetic interactions with Hsp70s of the ER (HSPA5/BiP) and mitochondria (HSPA9/mortalin) and their co-chaperones. Moreover, localization seemed to determine function, even for molecules with conserved binding sites. Compounds with distinct partitioning have distinct anti-proliferative activity in breast cancer cells compared with anti-viral activity in cellular models of Dengue virus replication, likely because different sets of Hsp70s are required in these processes. These findings highlight the contributions of subcellular partitioning and chemogenetic interactions to small molecule activity, features that are rarely explored during medicinal chemistry campaigns.

Salt tolerance in relation to elemental concentrations in leaf cell vacuoles and chloroplasts of a C4 monocotyledonous halophyte.

Halophytes accumulate and sequester high concentrations of salt in vacuoles while maintaining lower levels of salt in the cytoplasm. The current data on cellular and subcellular partitioning of salt in halophytes are, however, limited to only a few dicotyledonous C3 species. Using cryo‐scanning electron microscopy X‐ray microanalysis, we assessed the concentrations of Na, Cl, K, Ca, Mg, P and S in various cell types within the leaf‐blades of a monocotyledonous C4 halophyte, Rhodes grass (Chloris gayana). We also linked, for the first time, elemental concentrations in chloroplasts of mesophyll and bundle sheath cells to their ultrastructure and photosynthetic performance of plants grown in nonsaline and saline (200 mM NaCl) conditions. Na and Cl accumulated to the highest levels in xylem parenchyma and epidermal cells, but were maintained at lower concentrations in photosynthetically active mesophyll and bundle sheath cells. Concentrations of Na and Cl in chloroplasts of mesophyll and bundle sheath cells were lower than in their respective vacuoles. No ultrastructural changes were observed in either mesophyll or bundle sheath chloroplasts, and photosynthetic activity was maintained in saline conditions. Salinity tolerance in Rhodes grass is related to specific cellular Na and Cl distributions in leaf tissues, and the ability to regulate Na and Cl concentrations in chloroplasts.

CLAVATA3 mediated simultaneous control of transcriptional and post-translational processes provides robustness to the WUSCHEL gradient

Regulation of the homeodomain transcription factor WUSCHEL concentration is critical for stem cell homeostasis in Arabidopsis shoot apical meristems. WUSCHEL regulates the transcription of CLAVATA3 through a concentration-dependent activation-repression switch. CLAVATA3, a secreted peptide, activates receptor kinase signaling to repress WUSCHEL transcription. Considering the revised regulation, CLAVATA3 mediated repression of WUSCHEL transcription alone will lead to an unstable system. Here we show that CLAVATA3 signaling regulates nuclear-cytoplasmic partitioning of WUSCHEL to control nuclear levels and its diffusion into adjacent cells. Our work also reveals that WUSCHEL directly interacts with EXPORTINS via EAR-like domain which is also required for destabilizing WUSCHEL in the cytoplasm. We develop a combined experimental and computational modeling approach that integrates CLAVATA3-mediated transcriptional repression of WUSCHEL and post-translational control of nuclear levels with the WUSCHEL concentration-dependent regulation of CLAVATA3. We show that the dual control by the same signal forms a seamless connection between de novo WUSCHEL synthesis and sub-cellular partitioning in providing robustness to the WUSCHEL gradient.

Early Response to Dehydration Six-Like Transporter Family: Early Origin in Streptophytes and Evolution in Land Plants.

Carbon management by plants involves the activity of many sugar transporters, which play roles in sugar subcellular partitioning and reallocation at the whole organism scale. Among these transporters, the early response to dehydration six-like (ESL) monosaccharide transporters (MSTs) are still poorly characterized although they represent one of the largest sugar transporter subfamilies. In this study, we used an evolutionary genomic approach to infer the evolutionary history of this multigenic family. No ESL could be identified in the genomes of rhodophytes, chlorophytes, and the brown algae Ectocarpus siliculosus, whereas one ESL was identified in the genome of Klebsormidium nitens providing evidence for the early emergence of these transporters in Streptophytes. A phylogenetic analysis using the 519 putative ESL proteins identified in the genomes of 47 Embryophyta species and being representative of the plant kingdom has revealed that ESL protein sequences can be divided into three major groups. The first and second groups originated in the common ancestor of all spermaphytes [ζ: 340 million years ago (MYA)] and of angiosperms (ε: 170–235 MYA), respectively, and the third group originated before the divergence of rosids and asterids (γ/1R: 117 MYA). In some eudicots (Vitales, Malpighiales, Myrtales, Sapindales, Brassicales, Malvales, and Solanales), the ESL family presents remarkable expansions of gene copies associated with tandem duplications. The analysis of non-synonymous and synonymous substitutions for the dN/dS ratio of the ESL copies of the genus Arabidopsis has revealed that ESL genes are evolved under a purifying selection even though the progressive increase of dN/dS ratios in the three groups suggests subdiversification phenomena. To further explore the possible acquisition of novel functions by ESL MSTs, we identified the gene structure and promoter cis-acting elements for Arabidopsis thaliana ESL genes. The expression profiling of Arabidopsis ESL unraveled some gene copies that are almost constitutively expressed, whereas other gene copies display organ-preferential expression patterns. This study provides an evolving framework to better understand the roles of ESL transporters in plant development and response to environmental constraints.

Delineation of the exposure-response causality chain of chronic copper toxicity to the zebra mussel, Dreissena polymorpha, with a TK-TD model based on concepts of biotic ligand model and subcellular metal partitioning model

A toxicokinetic-toxicodynamic model was constructed to delineate the exposure–response causality. The model could be used: to predict metal accumulation considering the influence of water chemistry and biotic ligand characteristics; to simulate the dynamics of subcellular partitioning considering metabolism, detoxification, and elimination; and to predict chronic toxicity as represented by biomarker responses from the concentration of metals in the fraction of potentially toxic metal. The model was calibrated with data generated from an experiment in which the Zebra mussel Dreissena polymorpha was exposed to Cu at nominal concentrations of 25 and 50 μg/L and with varied Na+ concentrations in water up to 4.0 mmol/L for 24 days. Data used in the calibration included physicochemical conditions of the exposure environment, Cu concentrations in subcellular fractions, and oxidative stress-induced responses, i.e. glutathione-S-transferase activity and lipid peroxidation. The model explained the dynamics of subcellular Cu partitioning and the effect mechanism reasonably well. With a low affinity constant for Na + binding to Cu2+ uptake sites, Na + had limited influence on Cu2+ uptake at low Na+ concentrations in water. Copper was taken up into the metabolically available pool (MAP) at a largely higher rate than into the cellular debris. Similar Cu concentrations were found in these two fractions at low exposure levels, which could be attributed to sequestration pathways (metabolism, detoxification, and elimination) in the MAP. However, such sequestration was inefficient as shown by similar Cu concentrations in detoxified fractions with increasing exposure level accompanied by the increasing Cu concentration in the MAP.

Brassinosteroids inhibit miRNA-mediated translational repression by decreasing AGO1 on the endoplasmic reticulum.

Translational repression is a conserved mechanism in microRNA (miRNA)-guided gene silencing. In Arabidopsis, ARGONAUTE1 (AGO1), the major miRNA effector, localizes in the cytoplasm for mRNA cleavage and at the endoplasmic reticulum (ER) for translational repression of target genes. However, the mechanism underlying miRNA-mediated translational repression is poorly understood. In particular, how the subcellular partitioning of AGO1 is regulated is largely unexplored. Here, we show that the plant hormone brassinosteroids (BRs) inhibit miRNA-mediated translational repression by negatively regulating the distribution of AGO1 at the ER in Arabidopsis thaliana. We show that the protein levels rather than the transcript levels of miRNA target genes were reduced in BR-deficient mutants but increased under BR treatments. The localization of AGO1 at the ER was significantly decreased under BR treatments while it was increased in the BR-deficient mutants. Moreover, ROTUNDIFOLIA3 (ROT3), an enzyme involved in BR biosynthesis, co-localizes with AGO1 at the ER and interacts with AGO1 in a GW motif-dependent manner. Complementation analysis showed that the AGO1-ROT3 interaction is necessary for the function of ROT3. Our findings provide new clues to understand how miRNA-mediated gene silencing is regulated by plant endogenous hormones.