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Barron, ME, Thies AB, Espinoza JA, Barott KL, Hamdoun A, Tresguerres M.  2018.  A vesicular Na+/Ca2+ exchanger in coral calcifying cells. PLOS ONE. 13: Public Library of Science   10.1371/journal.pone.0205367   Abstract

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.

Campanale, JP, Gokirmak T, Espinoza JA, Oulhen N, Wessel GM, Hamdoun A.  2014.  Migration of sea urchin primordial germ cells. Developmental Dynamics. 243:917-927.   10.1002/dvdy.24133   AbstractWebsite

Background: Small micromeres are produced at the fifth cleavage of sea urchin development. They express markers of primordial germ cells (PGCs), and are required for the production of gametes. In most animals, PGCs migrate from sites of formation to the somatic gonad. Here, we investigated whether they also exhibit similar migratory behaviors using live-cell imaging of small micromere plasma membranes. Results: Early in gastrulation, small micromeres transition from non-motile epithelial cells, to motile quasi-mesenchymal cells. Late in gastrulation, at 43 hr post fertilization (HPF), they are embedded in the tip of the archenteron, but remain motile. From 43-49 HPF, they project numerous cortical blebs into the blastocoel, and filopodia that contact ectoderm. By 54 HPF, they begin moving in the plane of the blastoderm, often in a directed fashion, towards the coelomic pouches. Isolated small micromeres also produced blebs and filopodia. Conclusions: Previous work suggested that passive translocation governs some of the movement of small micromeres during gastrulation. Here we show that small micromeres are motile cells that can traverse the archenteron, change position along the left-right axis, and migrate to coelomic pouches. These motility mechanisms are likely to play an important role in their left-right segregation. (C) 2014 Wiley Periodicals, Inc.

Cole, BJ, Hamdoun A, Epel D.  2013.  Cost, effectiveness and environmental relevance of multidrug transporters in sea urchin embryos. Journal of Experimental Biology. 216:3896-3905.   10.1242/jeb.090522   AbstractWebsite

ATP-binding cassette transporters protect cells via efflux of xenobiotics and endogenous byproducts of detoxification. While the cost of this ATP-dependent extrusion is known at the molecular level, i.e. the ATP used for each efflux event, the overall cost to a cell or organism of operating this defense is unclear, especially as the cost of efflux changes depending on environmental conditions. During prolonged exposure to xenobiotics, multidrug transporter activity could be costly and ineffective because effluxed substrate molecules are not modified in the process and could thus undergo repeated cycles of efflux and re-entry. Here we use embryos of the purple sea urchin, Strongylocentrotus purpuratus, as a model to determine transport costs and benefits under environmentally relevant xenobiotic concentrations. Strikingly, our results show that efflux transporter activity costs less than 0.2% of total ATP usage, as a proportion of oxygen consumption. The benefits of transport, defined as the reduction in substrate accumulation due to transporter activity, depended largely, but not entirely, on the rate of passive flux of each substrate across the plasma membrane. One of the substrates tested exhibited rapid membrane permeation coupled with high rates of efflux, thus inducing rapid and futile cycles of efflux followed by re-entry of the substrate. This combination significantly reduced transporter effectiveness as a defense and increased costs even at relatively low substrate concentrations. Despite these effects with certain substrates, our results show that efflux transporters are a remarkably effective and low-cost first line of defense against exposure to environmentally relevant concentrations of xenobiotics.

Bosnjak, I, Uhlinger KR, Heim W, Smital T, Franekic-Colic J, Coale K, Epel D, Hamdoun A.  2009.  Multidrug Efflux Transporters Limit Accumulation of Inorganic, but Not Organic, Mercury in Sea Urchin Embryos. Environmental Science & Technology. 43:8374-8380.   10.1021/es901677r   AbstractWebsite

Mercuric compounds are persistent global pollutants that accumulate in marine organisms and in humans who consume them. While the chemical cycles and speciation of mercury in the oceans are relatively well described, the cellular mechanisms that govern which forms of mercury accumulate in cells and why they persist are less understood. In this study we examined the role of multidrug efflux transport in the differential accumulation of inorganic (HgCl(2)) and organic (CH(3)HgCl) mercury in sea urchin (Strongylocentrotus purpuratus) embryos. We found that inhibition of MRP/ABCC-type transporters increases intracellular accumulation of inorganic mercury but had no effect on accumulation of organic mercury. Similarly, pharmacological inhibition of metal conjugating enzymes by ligands GST/GSH significantly increases this antimitotic potency of inorganic mercury, but had no effect on the potency of organic mercury. Our results point to MRP-mediated elimination of inorganic mercury conjugates as a cellular basis for differences in the accumulation and potency of the two major forms of mercury found in marine environments.

Epel, D, Luckenbach T, Stevenson CN, Macmanus-Spencer LA, Hamdoun A, Smital T.  2008.  Efflux transporters: Newly appreciated roles in protection against pollutants. Environmental Science & Technology. 42:3914-3920.   10.1021/es087187v   AbstractWebsite
Hamdoun, A, Epel D.  2007.  Embryo stability and vulnerability in an always changing world. Proceedings of the National Academy of Sciences of the United States of America. 104:1745-1750.   10.1073/pnas.0610108104   AbstractWebsite

Contrary to the view that embryos and larvae are the most fragile stages of life, development is stable under real-world conditions. Early cleavage embryos are prepared for environmental vagaries by having high levels of cellular defenses already present in the egg before fertilization. Later in development, adaptive responses to the environment either buffer stress or produce alternative developmental phenotypes. These buffers, defenses, and alternative pathways set physiological limits for development under expected conditions; teratology occurs when embryos encounter unexpected environmental changes and when stress exceeds these limits. Of concern is that rapid anthropogenic changes to the environment are beyond the range of these protective mechanisms.

Goldstone, JV, Hamdoun A, Cole BJ, Howard-Ashby M, Nebert DW, Scally M, Dean M, Epel D, Hahn ME, Stegeman JJ.  2006.  The chemical defensome: Environmental sensing and response genes in the Strongylocentrotus purpuratus genome. Developmental Biology. 300:366-384.   10.1016/j.ydbio.2006.08.066   AbstractWebsite

Metazoan genomes contain large numbers of genes that participate in responses to environmental stressors. We surveyed the sea urchin Strongylocentrotus purpuratus genome for homologs of gene families thought to protect against chemical stressors; these genes collectively comprise the 'chemical defensome.' Chemical defense genes include cytochromes P450 and other oxidases, various conjugating enzymes, ATP-dependent efflux transporters, oxidative detoxification proteins, and transcription factors that regulate these genes. Together such genes account for more than 400 genes in the sea urchin genome. The transcription factors include homologs of the aryl hydrocarbon receptor, hypoxia-inducible factor, nuclear factor erythroid-derived 2, heat shock factor, and nuclear hormone receptors, which regulate stress-response genes in vertebrates. Some defense gene families, including the ABCC, the UGT, and the CYP families, have undergone expansion in the urchin relative to other deuterostome genomes, whereas the stress sensor gene families do not show such expansion. More than half of the defense genes are expressed during embryonic or larval life stages, indicating their importance during development. This genome-wide survey of chemical defense genes in the sea urchin reveals evolutionary conservation of this network combined with lineage-specific diversification that together suggest the importance of these chemical stress sensing and response mechanisms in early deuterostomes. These results should facilitate future studies on the evolution of chemical defense gene networks and the role of these networks in protecting embryos from chemical stress during development. (c) 2006 Elsevier Inc. All rights reserved.

Epel, D, Cole B, Hamdoun A, Thurber RV.  2006.  The sea urchin embryo as a model for studying efflux transporters: Roles and energy cost. Marine Environmental Research. 62:S1-S4.   10.1016/j.marenvres.2006.04.062   AbstractWebsite

We describe the use of the sea urchin as a model for studying efflux transporters and estimating energy cost for the cytotoxin protective system provided by these transporters. The unfertilized egg has low transport activity, which increases to a new steady state shortly after fertilization. Activity results from p-glycoprotein (p-gp) and MRP type transporters which protect the embryo from cytotoxic drugs that can disrupt cell division or induce apoptosis. The energy cost is estimated from a novel use of calcein-AM as a substrate; keeping 0.25 mu M substrate levels out of the cell utilizes only 0.023% of steady state respiration. (c) 2006 Elsevier Ltd. All rights reserved.

Hamdoun, AM, Cherr GN, Roepke TA, Epel D.  2004.  Activation of multidrug efflux transporter activity at fertilization in sea urchin embryos (Strongylocentrotus purpuratus). Developmental Biology. 276:452-462.   10.1016/j.ydbio.2004.09.013   AbstractWebsite

This study presents functional and molecular evidence for acquisition of multidrug transporter-mediated efflux activity as a consequence of fertilization in the sea urchin. Sea urchin eggs and embryos express low levels of efflux transporter genes with homology to the multidrug resistance associated protein (mrp) and permeability glycoprotein (p-gp) families of ABC transporters. The corresponding efflux activity is low in unfertilized eggs but is dramatically upregulated within 25 min of fertilization; the expression of this activity does not involve de novo gene expression and is insensitive to inhibitors of transcription and translation indicating activation of pre-existing transporter protein. Our study, using specific inhibitors of efflux transporters, indicates that the major activity is from one or more mrp-like transporters. The expression of activity at fertilization requires microfilaments, suggesting that the transporters are in vesicles and moved to the surface after fertilization. Pharmacological inhibition of mrp-mediated efflux activity with MK571 sensitizes embryos to the toxic compound vinblastine, confirming that one role for the efflux transport activity is embryo protection from xenobiotics. In addition, inhibition of mrp activity with MK571 alone retards mitosis indicating that mrp-like activity may also be required for early cell divisions. (C) 2004 Elsevier Inc. All rights reserved.

Smital, T, Luckenbach T, Sauerborn R, Hamdoun AM, Vega RL, Epel D.  2004.  Emerging contaminants--pesticides, PPCPs, microbial degradation products and natural substances as inhibitors of multixenobiotic defense in aquatic organisms. Mutation research. 552:101-17.   10.1016/j.mrfmmm.2004.06.006   Abstract

The environmental presence of chemosensitizers or inhibitors of the multixenobiotic resistance (MXR) defense system in aquatic organisms could cause increase in intracellular accumulation and toxic effects of other xenobiotics normally effluxed by MXR transport proteins (P-glycoprotein (P-gps), MRPs). MXR inhibition with concomitant detrimental effects has been shown in several studies with aquatic organisms exposed to both model MXR inhibitors and environmental pollutants. The presence of MXR inhibitors has been demonstrated in environmental samples from polluted locations at concentrations that could abolish P-gp transport activity. However, it is not clear whether the inhibition observed after exposure to environmental samples is a result of saturation of MXR transport proteins by numerous substrates present in polluted waters or results from the presence of powerful MXR inhibitors. And are potent environmental MXR inhibitors natural or man-made chemicals? As a consequence of these uncertainties, no official action has been taken to monitor and control the release and presence of MXR inhibitors into aquatic environments. In this paper we present our new results addressing these critical questions. Ecotoxicological significance of MXR inhibition was supported in in vivo studies that demonstrated an increase in the production of mutagenic metabolites by mussels and an increase in the number of sea urchin embryos with apoptotic cells after exposure to model MXR inhibitors. We also demonstrated that MXR inhibitors are present among both conventional and emerging man-made pollutants: some pesticides and synthetic musk fragrances show extremely high MXR inhibitory potential at environmentally relevant concentrations. In addition, we emphasized the biological transformation of crude oil hydrocarbons into MXR inhibitors by oil-degrading bacteria, and the risk potentially caused by powerful natural MXR inhibitors produced by invasive species.