Gary W. Moy

Investigating the molecular basis of local adaptation to thermal stress: population differences in gene expression across the transcriptome of the copepod Tigriopus californicus

BackgroundGeographic variation in the thermal environment impacts a broad range of biochemical and physiological processes and can be a major selective force leading to local population adaptation. In the intertidal copepod Tigriopus californicus, populations along the coast of California show differences in thermal tolerance that are consistent with adaptation, i.e., southern populations withstand thermal stresses that are lethal to northern populations. To understand the genetic basis of these physiological differences, we use an RNA-seq approach to compare genome-wide patterns of gene expression in two populations known to differ in thermal tolerance.ResultsObserved differences in gene expression between the southern (San Diego) and the northern (Santa Cruz) populations included both the number of affected loci as well as the identity of these loci. However, the most pronounced differences concerned the amplitude of up-regulation of genes producing heat shock proteins (Hsps) and genes involved in ubiquitination and proteolysis. Among the hsp genes, orthologous pairs show markedly different thermal responses as the amplitude of hsp response was greatly elevated in the San Diego population, most notably in members of the hsp70 gene family. There was no evidence of accelerated evolution at the sequence level for hsp genes. Among other sets of genes, cuticle genes were up-regulated in SD but down-regulated in SC, and mitochondrial genes were down-regulated in both populations.ConclusionsMarked changes in gene expression were observed in response to acute sub-lethal thermal stress in the copepod T. californicus. Although some qualitative differences were observed between populations, the most pronounced differences involved the magnitude of induction of numerous hsp and ubiquitin genes. These differences in gene expression suggest that evolutionary divergence in the regulatory pathway(s) involved in acute temperature stress may offer at least a partial explanation of population differences in thermal tolerance observed in Tigriopus.

Extraordinary intraspecific diversity in oyster sperm bindin

In free-spawning invertebrates sperm–egg incompatibility is a barrier to mating between species, and divergence of gamete recognition proteins (GRPs) can result in reproductive isolation. Of interest are processes that create reproductive protein diversity within species, because intraspecific variants are potentially involved in mate choice and early speciation. Sperm acrosomes of the Pacific oyster Crassostrea gigas contain the protein bindin that bonds sperm to egg during fertilization. Oyster bindin is a single-copy gene encoding a diversity of protein variants. Oyster bindins have a conserved N-terminal region followed by one to five tandem fucose-binding lectin (F-lectin) domains. These repeats have diversified by positive selection at eight sites clustered on the F-lectins fucose binding face. Additional bindin variants result from recombination in an intron in each F-lectin repeat. Males also express alternatively spliced bindin cDNAs with one to five repeats, but typically translate only one or two isoforms into protein. Thus, positive selection, alternative splicing, and recombination can create thousands of bindin variants within C. gigas. Models of sexual conflict predict high male diversity when females are diverse and sexual conflict is strong. The amount of intraspecific polymorphism in male GRPs may be a consequence of the relative efficiency of local (molecular recognition) and global (electrical, cortical, and physical) polyspermy blocks that operate during fertilization.

Chapter 2 Immunoperoxidase Localization of Bindin During the Adhesion of Sperm to Sea Urchin Eggs

Publisher Summary This chapter focuses on the ultrastructural immunohistochemical localization of bindin. Sperm–egg adhesion during sea urchin fertilization is an ideal model system for studying the molecular basis of a specific intercellular adhesion. The biological significance of sperm–egg interaction is well established; the gametes are homogeneous populations of single cells that can be obtained in large quantity and the interaction of sperm and egg occurs with great synchrony in a time span of seconds. The apex of the sea urchin spermatozoan contains a Golgi-derived acrosome granule approximately 0.3 μm in diameter composed of a uniformly electron-dense granular material. Immediately before or during the contact of the sperm with the egg surface, exocytosis of the granule occurs. Bindin is a species-specific agglutin of unfertilized eggs. Experiments with isolated bindin support the concept that it is the ligand bonding sperm to eggs. The egg-agglutinating property of bindin is lost when it is mixed with glycopeptides produced by the trypsinization of unfertilized eggs and trypsinized eggs are not agglutinated by bindin. Failure of antibindin to react with sperm in which the plasma membrane and acrosomal granule membrane remain intact supports the theory that the protein is not exposed until the acrosome reaction has occurred.

Interpopulation patterns of divergence and selection across the transcriptome of the copepod Tigriopus californicus

The accumulation of genetic incompatibilities between isolated populations is thought to lead to the evolution of intrinsic postzygotic isolation. The molecular basis for these mechanisms, however, remains poorly understood. The intertidal copepod Tigriopus californicus provides unique opportunities for addressing mechanistic questions regarding the early stages of speciation; hybrids between highly divergent populations are fertile and viable, but exhibit reduced fitness at the F2 or later generations. Given the current scarcity of genomic information in taxa at incipient stages of reproductive isolation, we utilize high‐throughout 454 pyrosequencing to characterize a substantial fraction of protein‐coding regions (the transcriptome) of T. californicus. Our sequencing effort was divided equally between two divergent populations in order to estimate levels of divergence and to reveal patterns of selection across the transcriptome. Assembly of sequences generated over 40 000 putatively unique transcripts (unigenes) for each population, 19 622 of which were orthologous between populations. BLAST searches of public databases determined protein identity and functional features for 15 402 and 12 670 unigenes, respectively. Based on rates of nonsynonymous and synonymous substitutions in 5897 interpopulation orthologs (those >150 bp and with at least 2X coverage), we identified 229 potential targets of positive selection. Many of these genes are predicted to be involved in several metabolic processes, and to function in hydrolase, peptidase and binding activities. The library of T. californicus coding regions, annotated with their predicted functions and level of divergence, will serve as an invaluable resource for elucidating molecular mechanisms underlying the early stages of speciation.

The cytolytic isolation of the cortex of the sea urchin egg

Abstract When the vitelline layer of sea urchin eggs ( Lytechinus pictus ) is disrupted by trypsin or dithiothreitol and the eggs are placed in an isosmotic medium devoid of Ca 2+ , cytolysis of the eggs occurs. During lysis the entire egg cortex peels off in one piece. Lysis is temperature and pH dependent and is inhibited by cytochalasin B. Cortices from unfertilized eggs contain seven major macromolecular components. A 42K-dalton component is believed to be actin, representing between 12 and 27% of the total protein. Cortices from fertilized eggs may contain between 50 and 65% actin. The actin appears to increase the strength of its attachment to the cortex after fertilization. This method of isolating the entire cortex may be useful for studying structural and enzymatic changes which may occur in the cortex during the cell cycle.

Calcium-mediated release of glucanase activity from cortical granules of sea urchin eggs☆

Confluent monolayers of sea urchin eggs were bonded to culture dishes coated with protamine sulfate. The cytoplasm was then sheared away by a jet of isosmotic buffer. About 326,000 circular fragments of individual egg cortices (430 micrograms protein) remained bound to each dish. The fragments are composed of cortical granules (CG), plasma membrane, and vitelline layer. A single dish contains 7.7 X 10(8) CG and is referred to as a CG lawn (CGL). Ca2+-EGTA buffers of estimated free-Ca2+ concentrations (0.06-25.7 microM) were applied to CGL and samples removed and assayed for the CG marker enzyme exo-beta (1 leads to 3)-glucanohydrolase (glucanase). Estimated free-Ca2+ concentrations above 2.75 microM caused the total release of the glucanase to the supernatant within 4 min. The half-maximal rate of appearance of glucanase occurred in 2.5 microM Ca2+. At all Ca2+ concentrations tested, the appearance of enzyme activity exhibited sigmoidal kinetics. The visual disappearance of CG correlated with the appearance of glucanase in the Ca2+ buffer. In response to Ca2+ the CG probably lyse, fuse with adjacent CG, or fuse with the underlying plasma membrane. The calmodulin antagonist trifluoperazine inhibited Ca2+-mediated glucanase release from CGL (I50 8 microM). The sensitivity of the CGL to Ca2+ in the 1-10 microM range is rapidly lost during incubation of CGL in the isolation buffer. ATP and low temperature retard the rate of loss of Ca2+ sensitivity. These secretory granules are a model for studying the mechanism of Ca2+-induced secretion. In addition, they contain structural proteins and enzymes which function in the fertilization process. CGL preparations should be useful in studies dealing with the processing of CG components after their release in response to micromolar concentrations of Ca2+.

Isolation and characterization of a plasma membrane fraction from sea urchin sperm exhibiting species specific recognition of the egg surface.

A method is described for isolating preparative quantities of plasma membranes from sea urchin sperm. The final membrane fraction is homogeneous by sucrose density sedimentation and is enriched in adenylate cyclase as well as in the four glycoproteins accessible to radioiodination of intact sperm. The electrophoretic profiles of sperm membranes from three sea urchin species are very similar. The membrane preparation consists primarily of sealed vesicles which release carboxyfluorescein when exposed to detergents or distilled water. Ninety-two percent of the 125I-labeled vesicle material binds to wheat germ lectin columns, suggesting a right-side-out orientation. The isolated sperm membrane vesicles exhibit species specific adhesion to the surfaces of sea urchin eggs; this adhesion is blocked by pretreatment of the vesicles with trypsin or egg jelly. This method will be useful for isolating biologically active sperm membrane components involved in sperm-egg recognition during fertilization.

Molecular characterization of a novel intracellular ADP-ribosyl cyclase.

Background ADP-ribosyl cyclases are remarkable enzymes capable of catalyzing multiple reactions including the synthesis of the novel and potent intracellular calcium mobilizing messengers, cyclic ADP-ribose and NAADP. Not all ADP-ribosyl cyclases however have been characterized at the molecular level. Moreover, those that have are located predominately at the outer cell surface and thus away from their cytosolic substrates. Methodology/Principal Findings Here we report the molecular cloning of a novel expanded family of ADP-ribosyl cyclases from the sea urchin, an extensively used model organism for the study of inositol trisphosphate-independent calcium mobilization. We provide evidence that one of the isoforms (SpARC1) is a soluble protein that is targeted exclusively to the endoplasmic reticulum lumen when heterologously expressed. Catalytic activity of the recombinant protein was readily demonstrable in crude cell homogenates, even under conditions where luminal continuity was maintained. Conclusions/Significance Our data reveal a new intracellular location for ADP-ribosyl cyclases and suggest that production of calcium mobilizing messengers may be compartmentalized.

Localization and Substrate Selectivity of Sea Urchin Multidrug (MDR) Efflux Transporters

Background: MDR transporters are important for many human diseases, but their phylogenetic origins and diversity are poorly understood. Results: Sea urchin MDR transporters homologous to ABCB1, ABCC1, and ABCG2 were characterized. Conclusion: Substitutions in TMH6 tune substrate selectivity of ABCB1 in sea urchins. Significance: Polyspecific MDR transport is conserved despite fine-tuning of substrate selectivity in different clades. In this study, we cloned, expressed and functionally characterized Stronglycentrotus purpuratus (Sp) ATP-binding cassette (ABC) transporters. This screen identified three multidrug resistance (MDR) transporters with functional homology to the major types of MDR transporters found in humans. When overexpressed in embryos, the apical transporters Sp-ABCB1a, ABCB4a, and ABCG2a can account for as much as 87% of the observed efflux activity, providing a robust assay for their substrate selectivity. Using this assay, we found that sea urchin MDR transporters export canonical MDR susbtrates such as calcein-AM, bodipy-verapamil, bodipy-vinblastine, and mitoxantrone. In addition, we characterized the impact of nonconservative substitutions in the primary sequences of drug binding domains of sea urchin versus murine ABCB1 by mutation of Sp-ABCB1a and treatment of embryos with stereoisomeric cyclic peptide inhibitors (QZ59 compounds). The results indicated that two substitutions in transmembrane helix 6 reverse stereoselectivity of Sp-ABCB1a for QZ59 enantiomers compared with mouse ABCB1a. This suggests that subtle changes in the primary sequence of transporter drug binding domains could fine-tune substrate specificity through evolution.

Oyster sperm bindin is a combinatorial fucose lectin with remarkable intra-species diversity

Sperm of the oyster, Crassostrea gigas, have ring-shaped acrosomes that, after exocytosis, bind the sperm to the egg vitelline layer. Isolated acrosomal rings contain proteins of various sizes: 35-, 48-, 63-, 75- and 88-kDa. These proteins, called bindins, have identical 24-residue signal peptides and conserved 97-residue N-terminal sequences, and they differ in mass because of the presence of between 1 and 5 tandemly repeated 134-residue fucose-binding lectin (F-lectin) domains. Southern blots suggest that oyster bindin is a single copy gene, but F-lectin repeat number and sequence are variable within and between individuals. Eight residues in the F-lectin fucose-binding groove are subject to positive diversifying selection, indicating a history of adaptive evolution at the lectins active site. There is one intron in the middle of each F-lectin repeat, and recombination in this intron creates many combinations of repeat halves. Alternative splicing creates many additional size and sequence variants of the repeat array. Males contain full-length bindin cDNAs of all 5 possible sizes, but only one or two protein mass forms exist in each individual. Sequence analysis indicates that recombination and alternate splicing create hundreds, possibly thousands, of different bindin sequences in C. gigas. The extreme within-species sequence variation in the F-lectin sequence of oyster bindin is a novel finding; most male gamete-recognition proteins are much less variable. In experimental conditions oyster eggs have poor polyspermy blocks, and bindin diversity could be an evolutionary response by sperm to match egg receptors that have diversified to avoid being fertilized by multiple sperm.