Marine pollutants bioaccumulate at high trophic levels of marine food webs and are transferred to humans through consumption of apex species. Yellowfin tuna (Thunnus albacares) are marine predators, and one of largest commercial fisheries in the world. Previous studies have shown that yellowfin tuna can accumulate high levels of persistent organic pollutants, including Transporter Interfering Chemicals (TICs), which are chemicals shown to bind to mammalian xenobiotic transporters and interfere with their function. Here, we examined the extent to which these same compounds might interfere with the activity of the yellowfin tuna (Thunnus albacares) ortholog of this transporter. To accomplish this goal we identified, expressed, and functionally assayed tuna ABCB1. The results demonstrated a common mode of vertebrate ABCB1 interaction with TICs that predicts effects across these species, based on high conservation of specific interacting residues. Importantly several TICs showed potent inhibition of Ta-ABCB1, such as the organochlorine pesticides Endrin (EC50 = 1.2 ± 0.2 μM) and Mirex (EC50 = 2.3 ± 0.9 μM). However, unlike the effects observed on mouse ABCB1, low concentrations of the organochlorine pesticide TICs p,p'-DDT and its metabolite p,p'-DDD co-stimulated verapamil-induced Ta-ABCB1 ATPase activity possibly suggesting a low transport activity for these ligands in tuna. These results provide a mechanistic basis for understanding the potential vulnerability of tuna to these ubquitous pollutants.

Fertilization changes the structure and function of the cell surface. In sea urchins, these changes include polymerization of cortical actin and a coincident, switch-like increase in the activity of the multidrug efflux transporter ABCB1a. However, it is not clear how cortical reorganization leads to changes in membrane transport physiology. In this study, we used three-dimensional superresolution fluorescence microscopy to resolve the fine-scale movements of the transporter along polymerizing actin filaments, and we show that efflux activity is established after ABCB1a translocates to the tips of the microvilli. Inhibition of actin polymerization or bundle formation prevents tip localization, resulting in the patching of ABCB1a at the cell surface and decreased efflux activity. In contrast, enhanced actin polymerization promotes tip localization. Finally, interference with Rab11, a regulator of apical recycling, inhibits activation of efflux activity in embryos. Together our results show that actin-mediated, short-range traffic and positioning of transporters at the cell surface regulates multidrug efflux activity and highlight the multifaceted roles of microvilli in the spatial distribution of membrane proteins.

Although persistent, bioaccumulative and toxic pollutants (PBTs) are well-studied individually, their distribution and variability on a global scale are largely unknown, particularly in marine fish. Using 2,662 measurements collected from peer-reviewed literature spanning 1969-2012, we examined variability of five classes of PBTs, considering effects of geography, habitat, and trophic level on observed concentrations. While we see large-scale spatial patterning in some PBTs (chlordanes, polychlorinated biphenyls), habitat type and trophic level did not contribute to significant patterning, with the exception of mercury. We further examined patterns of change in PBT concentration as a function of sampling year. All PBTs showed significant declines in concentration levels through time, ranging from 15-30% reduction per decade across PBT groups. Despite consistent evidence of reductions, variation in pollutant concentration remains high, indicating ongoing consumer risk of exposure to fish with pollutant levels exceeding EPA screening values. The temporal trends indicate that mitigation programs are effective, but that global levels decline slowly. In order for monitoring efforts to provide more targeted assessments of risk to PBT exposure, these data highlight an urgent need for improved replication and standardization of pollutant monitoring protocols for marine finfish.

ATP-binding cassette (ABC) transporters are membrane proteins that regulate intracellular concentrations of myriad compounds and ions. There are >100 ABC transporter predictions in the Strongylocentrotus purpuratus genome, including 40 annotated ABCB, ABCC, and ABCG "multidrug efflux" transporters. Despite the importance of multidrug transporters for protection and signaling, their expression patterns have not been characterized in deuterostome embryos.

Fish are a source of persistent organic pollutants (POPs) in the human diet. Although species, trophic level, and means of production are typically considered in predicting fish pollutant load, and thus recommendations of consumption, capture location is usually not accounted for.

The world's oceans are a global reservoir of persistent organic pollutants to which humans and other animals are exposed. Although it is well known that these pollutants are potentially hazardous to human and environmental health, their impacts remain incompletely understood. We examined how persistent organic pollutants interact with the drug efflux transporter P-glycoprotein (P-gp), an evolutionarily conserved defense protein that is essential for protection against environmental toxicants. We identified specific congeners of organochlorine pesticides, polychlorinated biphenyls, and polybrominated diphenyl ethers that inhibit mouse and human P-gp, and determined their environmental levels in yellowfin tuna from the Gulf of Mexico. In addition, we solved the cocrystal structure of P-gp bound to one of these inhibitory pollutants, PBDE (polybrominated diphenyl ether)-100, providing the first view of pollutant binding to a drug transporter. The results demonstrate the potential for specific binding and inhibition of mammalian P-gp by ubiquitous congeners of persistent organic pollutants present in fish and other foods, and argue for further consideration of transporter inhibition in the assessment of the risk of exposure to these chemicals.

Tissue morphogenesis is intimately linked to the changes in shape and organisation of individual cells. In curved epithelia, cells can intercalate along their own apicobasal axes adopting a shape named "scutoid" that allows energy minimization in the tissue. Although several geometric and biophysical factors have been associated with this 3D reorganisation, the dynamic changes underlying scutoid formation in 3D epithelial packing remain poorly understood. Here we use live-imaging of the sea star embryo coupled with deep learning-based segmentation, to dissect the relative contributions of cell density, tissue compaction, and cell proliferation on epithelial architecture. We find that tissue compaction, which naturally occurs in the embryo, is necessary for the appearance of scutoids. Physical compression experiments identify cell density as the factor promoting scutoid formation at a global level. Finally, the comparison of the developing embryo with computational models indicates that the increase in the proportion of scutoids is directly associated with cell divisions. Our results suggest that apico-basal intercalations appearing just after mitosis may help accommodate the new cells within the tissue. We propose that proliferation in a compact epithelium induces 3D cell rearrangements during development.

The calcium carbonate skeletons of corals provide the underlying structure of coral reefs; however, the cellular mechanisms responsible for coral calcification remain poorly understood. In osteoblasts from vertebrate animals, a Na+/Ca2+ exchanger (NCX) present in the plasma membrane transports Ca2+ to the site of bone formation. The aims of this study were to establish whether NCX exists in corals and its localization within coral cells, which are essential first steps to investigate its potential involvement in calcification. Data mining identified genes encoding for NCX proteins in multiple coral species, a subset of which were more closely related to NCXs from vertebrates (NCXA). We cloned NCXA from Acropora yongei (AyNCXA), which, unexpectedly, contained a peptide signal that targets proteins to vesicles from the secretory pathway. AyNCXA subcellular localization was confirmed by heterologous expression of fluorescently tagged AyNCXA protein in sea urchin embryos, which localized together with known markers of intracellular vesicles. Finally, immunolabeling of coral tissues with specific antibodies revealed AyNCXA was present throughout coral tissue. AyNCXA was especially abundant in calcifying cells, where it exhibited a subcellular localization pattern consistent with intracellular vesicles. Altogether, our results demonstrate AyNCXA is present in vesicles in coral calcifying cells, where potential functions include intracellular Ca2+ homeostasis and Ca2+ transport to the growing skeleton as part of an intracellular calcification mechanism.

Directed intercellular movement of diverse small molecules, including metabolites, signal molecules and xenobiotics, is a key feature of multicellularity. Networks of small molecule transporters (SMTs), including several ATP Binding Cassette (ABC) transporters, are central to this process. While small molecule transporters are well described in differentiated organs, little is known about their patterns of expression in early embryogenesis. Here we report the pattern of ABC-type SMT expression and activity during the early development of sea urchins. Of the six major ABCs in this embryo (ABCB1, -B4, -C1, -C4, -C5 and -G2), three expression patterns were observed: 1) ABCB1 and ABCC1 are first expressed ubiquitously, and then become enriched in endoderm and ectoderm-derived structures. 2) ABCC4 and ABCC5 are restricted to a ring of mesoderm in the blastula and ABCC4 is later expressed in the coelomic pouches, the embryonic niche of the primordial germ cells. 3) ABCB4 and ABCG2 are expressed exclusively in endoderm-fated cells. Assays with fluorescent substrates and inhibitors of transporters revealed a ring of ABCC4 efflux activity emanating from ABCC4+ mesodermal cells. Similarly, ABCB1 and ABCB4 efflux activity was observed in the developing gut, prior to the onset of feeding. This study reveals the early establishment of unique territories of small molecule transport during embryogenesis. A pattern of ABCC4/C5 expression is consistent with signaling functions during gut invagination and germ line development, while a later pattern of ABCB1/B4 and ABCG2 is consistent with roles in the embryonic gut. This work provides a conceptual framework with which to examine the function and evolution of SMT networks and to define the specific developmental pathways that drive the expression of these genes.

The ABC transporter ABCB1 plays an important role in the disposition of xenobiotics. Embryos of most species express high levels of this transporter in early development as a protective mechanism, but its native substrates are not known. Here, we used larvae of the sea urchin Strongylocentrotus purpuratus to characterize the early life expression and role of Sp-ABCB1a, a homolog of ABCB1. The results indicate that while Sp-ABCB1a is initially expressed ubiquitously, it becomes enriched in the developing gut. Using optimized CRISPR/Cas9 gene editing methods to achieve high editing efficiency in the F0 generation, we generated ABCB1a crispant embryos with significantly reduced transporter efflux activity. When infected with the opportunistic pathogen Vibrio diazotrophicus, Sp-ABCB1a crispant larvae demonstrated significantly stronger gut inflammation, immunocyte migration and cytokine Sp-IL-17 induction, as compared with infected control larvae. The results suggest an ancestral function of ABCB1 in host-microbial interactions, with implications for the survival of invertebrate larvae in the marine microbial environment.

Sea urchins are premier model organisms for the study of early development. However, the lengthy generation times of commonly used species have precluded application of stable genetic approaches. Here, we use the painted sea urchin Lytechinus pictus to address this limitation and to generate a homozygous mutant sea urchin line. L. pictus has one of the shortest generation times of any currently used sea urchin. We leveraged this advantage to generate a knockout mutant of the sea urchin homolog of the drug transporter ABCB1, a major player in xenobiotic disposition for all animals. Using CRISPR/Cas9, we generated large fragment deletions of ABCB1 and used these readily detected deletions to rapidly genotype and breed mutant animals to homozygosity in the F2 generation. The knockout larvae are produced according to expected Mendelian distribution, exhibit reduced xenobiotic efflux activity and can be grown to maturity. This study represents a major step towards more sophisticated genetic manipulation of the sea urchin and the establishment of reproducible sea urchin animal resources.