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Nicole King - One of the best experts on this subject based on the ideXlab platform.
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Evidence for Sex and Recombination in the Choanoflagellate Salpingoeca rosetta
Current Biology, 2013Co-Authors: Tera C. Levin, Nicole KingAbstract:Summary Nearly all animals reproduce sexually through the production and fusion of sperm and egg cells, yet little is known about the ancestry of animal sexual reproduction. Moreover, the sexual cycle of the closest living relatives of animals, the choanoflagellates [1, 2], remains completely unknown. The choanoflagellate Monosiga brevicollis possesses a "meiotic toolkit" of genes [3], but the lack of polymorphisms detected during genome sequencing precluded inferences about its ploidy or sexual cycle [1]. Here, we report that a related choanoflagellate, Salpingoeca rosetta [4, 5], has a sexual life cycle and transitions between haploid and diploid states. Haploid cultures of S. rosetta became diploid in response to nutrient limitation. This ploidy shift coincided with anisogamous mating, during which small flagellated cells fused with larger flagellated cells. Distributions of polymorphisms in laboratory strains of S. rosetta provided independent evidence of historical recombination and mating. The ability of S. rosetta to produce morphologically differentiated gametes and to engage in sexual reproduction has implications for both reconstructing the evolution of sex in the progenitors of animals and establishing classical genetics in choanoflagellates.
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Premetazoan Ancestry of the Myc-Max Network
Molecular Biology and Evolution, 2011Co-Authors: Susan L Young, Iñaki Ruiz-trillo, Daniel Diolaiti, Maralice Conacci-sorrell, Robert N. Eisenman, Nicole KingAbstract:The origin of metazoans required the evolution of mechanisms for maintaining differentiated cell types within a multicellular individual, in part through spatially differentiated patterns of gene transcription. The unicellular ancestor of metazoans was presumably capable of regulating gene expression temporally in response to changing environmental conditions, and spatial cell differentiation in metazoans may represent a co-option of preexisting regulatory mechanisms. Myc is a critical regulator of cell growth, proliferation, and death that is found in all metazoans but absent in other multicellular lineages, including fungi and plants. Homologs of Myc and its binding partner, Max, exist in two of the closest living relatives of animals, the choanoflagellate Monosiga brevicollis (Mb) and Capsaspora owczarzaki, a unicellular opisthokont that is closely related to metazoans and choanoflagellates. We find that Myc and Max from M. brevicollis heterodimerize and bind to both canonical and noncanonical E-boxes, the DNA-binding sites through which metazoan Myc proteins act. Moreover, in M. brevicollis, MbMyc protein can be detected in nuclear and flagellar regions. Like metazoan Max proteins, MbMax can form homodimers that bind to E-boxes. However, cross-species dimerization between Mb and human Myc and Max proteins was not observed, suggesting that the binding interface has diverged. Our results reveal that the Myc/Max network arose before the divergence of the choanoflagellate and metazoan lineages. Furthermore, core features of metazoan Myc function, including heterodimerization with Max, binding to E-box sequences in DNA, and localization to the nucleus, predate the origin of metazoans.
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Sterols in a unicellular relative of the metazoans
Proceedings of the National Academy of Sciences of the United States of America, 2008Co-Authors: Robin B. Kodner, Nicole King, Roger E. Summons, Ann Pearson, Andrew H. KnollAbstract:Molecular clocks suggest that animals originated well before they first appear as macroscopic fossils, but geologic tests of these hypotheses have been elusive. A rare steroid hydrocarbon, 24-isopropylcholestane, has been hypothesized to be a biomarker for sponges or their immediate ancestors because of its relatively high abundance in pre-Ediacaran to Early Cambrian sedimentary rocks and oils. Biolipid precursors of this sterane have been reported to be prominent in several demosponges. Whether 24-isopropylcholestane can be interpreted as a sponge (and, hence, animal) biomarker, and so provide clues about early metazoan history, depends on an understanding of the distribution of sterol biosynthesis among animals and their protistan relatives. Accordingly, we characterized the sterol profile of the choanoflagellate Monosiga brevicollis, a representative of the unicellular sister group of animals. M. brevicollis does not produce a candidate sterol precursor for 24-isopropylcholestane under our experimental growth conditions. It does, however, produce a number of other sterols, and comparative genomics confirms its biosynthetic potential to produce the full suite of compounds recovered. Consistent with the phylogenetic position of choanoflagellates, the sterol profile and biosynthetic pathway of M. brevicollis display characteristics of both fungal and poriferan sterol biosynthesis. This is an example in which genomic and biochemical information have been used together to investigate the taxonomic specificity of a fossil biomarker.
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Signaling properties of a non-metazoan Src kinase and the evolutionary history of Src negative regulation.
Journal of Biological Chemistry, 2008Co-Authors: Wanqing Li, Nicole King, Susan L Young, W. Todd MillerAbstract:Choanoflagellates, unicellular organisms that are closely related to metazoans, possess cell adhesion and signaling proteins previously thought to be unique to animals, suggesting that these components may have played roles in the evolution of metazoan multicellularity. We have cloned, expressed, and purified the nonreceptor tyrosine kinase MbSrc1 from the choanoflagellate Monosiga brevicollis. The kinase has the same domain arrangement as mammalian Src kinases, and we find that the individual Src homology 3 (SH3), SH2, and catalytic domains have similar functions to their mammalian counterparts. In contrast to mammalian c-Src, the SH2 and catalytic domains of MbSrc1 do not appear to be functionally coupled. We cloned and expressed the M. brevicollis homolog of c-Src C-terminal kinase (MbCsk) and showed that it phosphorylates the C terminus of MbSrc1, yet this phosphorylation does not inhibit MbSrc to the same degree seen in the mammalian Src/Csk pair. Thus, Src autoinhibition likely evolved more recently within the metazoan lineage, and it may have played a role in the establishment of intercellular signaling in metazoans.
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The Premetazoan Ancestry of Cadherins
Science, 2008Co-Authors: Monika Abedin, Nicole KingAbstract:Cadherin-mediated cell adhesion and signaling is essential for metazoan development and yet is absent from all other multicellular organisms. We found cadherin genes at numbers similar to those observed in complex metazoans in one of the closest single-celled relatives of metazoans, the choanoflagellate Monosiga brevicollis. Because the evolution of metazoans from a single-celled ancestor required novel cell adhesion and signaling mechanisms, the discovery of diverse cadherins in choanoflagellates suggests that cadherins may have contributed to metazoan origins.
Pawel Burkhardt - One of the best experts on this subject based on the ideXlab platform.
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Choanoflagellates and the ancestry of neurosecretory vesicles
bioRxiv, 2020Co-Authors: Ronja Gohde, Benjamin H Cooper, Cordelia Imig, Frederique Varoqueaux, Dirk Fasshauer, Benjamin Nauman, Davis Laundon, Kent L. Mcdonald, Pawel BurkhardtAbstract:Neurosecretory vesicles are highly specialized trafficking organelles important for metazoan cell-cell signalling. Despite the high anatomical and functional diversity of neurons in metazoans, the protein composition of neurosecretory vesicles in bilaterians appears to be similar. This similarity points towards a common evolutionary origin. Moreover, many key neurosecretory vesicle proteins predate the origin of the first neurons and some even the origin of the first animals (metazoans). However, little is known about the molecular toolkit of these vesicles in non-bilaterian metazoans and their closest unicellular relatives, making inferences about the evolutionary origin of neurosecretory vesicles extremely difficult. By comparing 28 proteins of the core neurosecretory vesicle proteome in 13 different species, we demonstrate that most of the proteins are already present in unicellular organisms. Surprisingly, we find that the vesicle residing SNARE protein synaptobrevin is localized to the vesicle-rich apical and basal pole in the choanoflagellate Salpingoeca rosetta. Our 3D vesicle reconstructions reveal that the choanoflagellates Salpingoeca rosetta and Monosiga brevicollis exhibit a polarized and diverse vesicular landscape. This study sheds light on the ancestral molecular machinery of neurosecretory vesicles and provides a framework to understand the origin and evolution of secretory cells, synapses, and neurons.
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Animal evolution coincides with a novel degree of freedom in exocytic transport processes
bioRxiv, 2019Co-Authors: Martin Kollmar, Pawel Burkhardt, Tobias Welz, Felix Straub, Noura Alzahofi, Klas Hatje, Deborah A. Briggs, Annette Samol-wolf, Alistair N. Hume, Eugen KerkhoffAbstract:Abstract Exocytic transport of transmembrane receptors and secreted ligands provides the basis for cellular communication in animals. The RAB8/RAB3/RAB27 trafficking regulators function in transport processes towards the cell membrane. The small G-proteins recruit a diversity of effectors that mediate transport along microtubule and actin tracks, as well as membrane tethering and fusion. SPIRE actin nucleators organise local actin networks at exocytic vesicle membranes. By complex formation with class-5 myosins, vesicle transport track generation and motor protein activation are coordinated. Our phylogenetic analysis traced the onset of SPIRE function back to the origin of the Holozoa. We have identified SPIRE in the closest unicellular relatives of animals, the choanoflagellates, and the more distantly related ichthyosporeans. The discovery of a SPIRE-like protein encoding a KIND and tandem-WH2 domains in the amoebozoan Physarum polycephalum suggests that the SPIRE-type actin nucleation mechanism originated even earlier. Choanoflagellate SPIRE interacts with RAB8, the sole choanoflagellate representative of the metazoan RAB8/RAB3/RAB27 family. Major interactions including MYO5, FMN-subgroup formins and vesicle membranes are conserved between the choanoflagellate and mammalian SPIRE proteins and the choanoflagellate Monosiga brevicollis SPIRE protein can rescue mouse SPIRE1/2 function in melanosome transport. Genome duplications generated two mammalian SPIRE genes (SPIRE1 and SPIRE2) and allowed for the separation of SPIRE protein function in terms of tissue expression and RAB GTPase binding. SPIRE1 is highest expressed in the nervous system and interacts with RAB27 and RAB8. SPIRE2 shows high expression in the digestive tract and specifically interacts with RAB8. We propose that at the dawn of the animal kingdom a new transport mechanism came into existence, which bridges microtubule tracks, detached vesicles and the cellular actin cytoskeleton by organising actin/myosin forces directly at exocytic vesicle membranes. The new degree of freedom in transport may reflect the increased demands of the sophisticated cellular communications in animals.
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choanoflagellate models Monosiga brevicollis and salpingoeca rosetta
Current Opinion in Genetics & Development, 2016Co-Authors: Tarja T Hoffmeyer, Pawel BurkhardtAbstract:Choanoflagellates are the closest single-celled relatives of animals and provide fascinating insights into developmental processes in animals. Two species, the choanoflagellates Monosiga brevicollis and Salpingoeca rosetta are emerging as promising model organisms to reveal the evolutionary origin of key animal innovations. In this review, we highlight how choanoflagellates are used to study the origin of multicellularity in animals. The newly available genomic resources and functional techniques provide important insights into the function of choanoflagellate pre- and postsynaptic proteins, cell-cell adhesion and signaling molecules and the evolution of animal filopodia and thus underscore the relevance of choanoflagellate models for evolutionary biology, neurobiology and cell biology research.
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primordial neurosecretory apparatus identified in the choanoflagellate Monosiga brevicollis
Proceedings of the National Academy of Sciences of the United States of America, 2011Co-Authors: Pawel Burkhardt, Christian Stegmann, Benjamin H Cooper, Tobias H Kloepper, Cordelia Imig, Frederique Varoqueaux, Markus C Wahl, Dirk FasshauerAbstract:Abstract SNARE protein-driven secretion of neurotransmitters from synaptic vesicles is at the center of neuronal communication. In the absence of the cytosolic protein Munc18-1, synaptic secretion comes to a halt. Although it is believed that Munc18-1 orchestrates SNARE complexes, its mode of action is still a matter of debate. In particular, it has been challenging to clarify the role of a tight Munc18/syntaxin 1 complex, because this interaction interferes strongly with syntaxin's ability to form a SNARE complex. In this complex, two regions of syntaxin, the N-peptide and the remainder in closed conformation, bind to Munc18 simultaneously. Until now, this binary complex has been reported for neuronal tissues only, leading to the hypothesis that it might be a specialization of the neuronal secretion apparatus. Here we aimed, by comparing the core secretion machinery of the unicellular choanoflagellate Monosiga brevicollis with that of animals, to reconstruct the ancestral function of the Munc18/syntaxin1 complex. We found that the Munc18/syntaxin 1 complex from M. brevicollis is structurally and functionally highly similar to the vertebrate complex, suggesting that it constitutes a fundamental step in the reaction pathway toward SNARE assembly. We thus propose that the primordial secretion machinery of the common ancestor of choanoflagellates and animals has been co-opted for synaptic roles during the rise of animals.
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Vergleichende Untersuchungen zur Regulation der SNARE-Komplexbildung durch Sec1/Munc18-Proteine.
2009Co-Authors: Pawel BurkhardtAbstract:Membrane fusion events between various intracellular compartments in eukaryotic cells are mediated by two conserved protein families: SNARE- and Sec1/Munc18 (SM)-proteins. The SNARE proteins syntaxin1, SNAP-25, and synaptobrevin2 play a central role during fusion of synaptic vesicles with the plasma membrane. Syntaxin1 and SNAP-25 are located in the plasma membrane, whereas synaptobrevin2 resides on synaptic vesicles. Their assembly into a membrane-bridging ternary SNARE complex is believed to drive fusion. SM-proteins interact with SNARE-proteins but the molecular basis for this interaction is not entirely understood. Munc18a binds the cytosolic domain of syntaxin1a in a so called closed conformation. A binding mode distinct from that of Munc18a/syntaxin1a appears to govern the interaction of several other SM-proteins (i.e. Munc18c, Sly1, Vps45) with their cognate syntaxins. In these complexes the far N-terminal region of syntaxin binds to the SM-protein. In the first part of this thesis it is shown that Munc18a binds simultaneously to the closed conformation of syntaxin1a and to the N-terminal peptide of syntaxin1a by ITC. In addition, residual electron density on the outer surface of domain 1 of Munc18a was uncovered, a finding that had been overlooked until now. Re-refinement of the Munc18a/syntaxin1a-structure improved the electron density within this region, and residues 2 9 of syntaxin1a could be modelled. A second SM-protein/syntaxin-pair was also investigated. It was observed that the N-terminal peptide of syntaxin 16 binds to the SM-protein Vps45, while the remainder of syntaxin 16, probably in a closed conformation strongly enhances the affinity of the interaction. Collectively these data indicate that SM-proteins interact with their cognate syntaxins via a conserved binding mechanism. Two regions of syntaxin appear to cooperatively bind Munc18a. Next, the role of Munc18a in controlling SNARE complex assembly was investigated. It has previously been established, that Munc18a binds syntaxin1a and thereby blocks SNARE-complex assembly in vitro. However, how syntaxin1a can escape the tight grip of Munc18a, thus enabling its participation in SNARE complex formation remains unclear. Here it is demonstrated, that the interaction of Munc18a with the N-terminal peptide of syntaxin1a is essential for the inhibition of SNARE-complex formation. Removal of the N-terminal peptide of syntaxin1a, and either point mutations in the peptide, or in the Munc18a binding site, allowed for SNARE complex formation of Munc18a-bound syntaxin1a. These results suggest a conformational change in the Munc18a/syntaxin1a-complex. In the third section, the interaction of Munc18 and syntaxin1 and the role of Munc18 in SNARE-complex assembly in the choanoflagellate Monosiga brevicollis, a unicellular organism believed to be the closest known relative of animals, was investigated. The results indicate that not only is the binding of Munc18 to the N-terminal peptide and the closed conformation of syntaxin1 conserved, but also the regulative function of Munc18 in controlling SNARE-complex assembly in chaonoflagellates. Furthermore using light- and electron microscopy it was shown that Monosiga brevicollis appears to possess a secretion apparatus localized to the apical region of the cell.
Susan L Young - One of the best experts on this subject based on the ideXlab platform.
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The unicellular ancestry of the proto-oncogene Myc
2013Co-Authors: Susan L YoungAbstract:The origin of metazoans required the evolution of mechanisms of cell-cell adhesion, coordination and communication among neighboring cells, and the establishment of differentiated cell types. Did the molecular building blocks of metazoan multicellularity exist in their single celled ancestors, or are they unique metazoan innovations? To address this question we sequenced and analyzed the genome of the unicellular marine choanoflagellate Monosiga brevicollis . Choanoflagellates, a phylum of flagellated unicellular and colonial eukaryotes found in diverse aqueous habitats around the globe, are among the closest living relatives of metazoans. The roughly 46 million base pair genome of M. brevicollis contains approximately 9,200 unexpectedly intron-rich genes, including a number of genes that encode cell adhesion and signaling protein domains that are otherwise restricted to metazoans. The physical linkages among these domains often differ between M. brevicollis and metazoans suggesting that abundant domain shuffling followed the separation and subsequent diversification of the choanoflagellate and metazoan lineages. Metazoans also have a richer diversity of transcription factors than does M. brevicollis , indicating that the evolution of early metazoans may have involved an increase in the sophistication of transcriptional regulation. Nonetheless, a few metazoan-type transcription factors were identified in M. brevicollis : members of the p53, Myc, and Sox/TCF transcription factor families. Myc is a developmentally critical transcription factor that plays roles in the most fundamental of cellular processes: cell growth, proliferation, and death. Investigating the function of Myc in choanoflagellates promises to delineate the role of Myc before the origin of animals and may inform how the strict regulation of cell life and death in metazoans arose from a unicellular context. Here, we demonstrate M. brevicollis Myc heterodimerizes with M. brevicollis MAX and localizes to the nucleus and cytoplasm of choanoflagellate cells in varying intensity. We further show that the tyrosine kinase (TK) inhibitor genistein reduces the expression of MbMyc, suggesting that TK signaling regulates MbMyc. Because metazoans Mycs are also known to be regulated by TK signaling, we hypothesize that an emergent network of TK signaling and transcriptional regulation was present in the unicellular ancestor of animals.
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Premetazoan Ancestry of the Myc-Max Network
Molecular Biology and Evolution, 2011Co-Authors: Susan L Young, Iñaki Ruiz-trillo, Daniel Diolaiti, Maralice Conacci-sorrell, Robert N. Eisenman, Nicole KingAbstract:The origin of metazoans required the evolution of mechanisms for maintaining differentiated cell types within a multicellular individual, in part through spatially differentiated patterns of gene transcription. The unicellular ancestor of metazoans was presumably capable of regulating gene expression temporally in response to changing environmental conditions, and spatial cell differentiation in metazoans may represent a co-option of preexisting regulatory mechanisms. Myc is a critical regulator of cell growth, proliferation, and death that is found in all metazoans but absent in other multicellular lineages, including fungi and plants. Homologs of Myc and its binding partner, Max, exist in two of the closest living relatives of animals, the choanoflagellate Monosiga brevicollis (Mb) and Capsaspora owczarzaki, a unicellular opisthokont that is closely related to metazoans and choanoflagellates. We find that Myc and Max from M. brevicollis heterodimerize and bind to both canonical and noncanonical E-boxes, the DNA-binding sites through which metazoan Myc proteins act. Moreover, in M. brevicollis, MbMyc protein can be detected in nuclear and flagellar regions. Like metazoan Max proteins, MbMax can form homodimers that bind to E-boxes. However, cross-species dimerization between Mb and human Myc and Max proteins was not observed, suggesting that the binding interface has diverged. Our results reveal that the Myc/Max network arose before the divergence of the choanoflagellate and metazoan lineages. Furthermore, core features of metazoan Myc function, including heterodimerization with Max, binding to E-box sequences in DNA, and localization to the nucleus, predate the origin of metazoans.
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the protist Monosiga brevicollis has a tyrosine kinase signaling network more elaborate and diverse than found in any known metazoan
Proceedings of the National Academy of Sciences of the United States of America, 2008Co-Authors: Gerard Manning, Susan L Young, Todd W Miller, Yufeng ZhaiAbstract:Tyrosine kinase signaling has long been considered a hallmark of intercellular communication, unique to multicellular animals. Our genomic analysis of the unicellular choanoflagellate Monosiga brevicollis discovers a remarkable count of 128 tyrosine kinases, 38 tyrosine phosphatases, and 123 phosphotyrosine (pTyr)-binding SH2 proteins, all higher counts than seen in any metazoan. This elaborate signaling network shows little orthology to metazoan counterparts yet displays many innovations reminiscent of metazoans. These include extracellular domains structurally related to those of metazoan receptor kinases, alternative methods for membrane anchoring and phosphotyrosine interaction in cytoplasmic kinases, and domain combinations that link kinases to small GTPase signaling and transcription. These proteins also display a wealth of combinations of known signaling domains. This uniquely divergent and elaborate signaling network illuminates the early evolution of pTyr signaling, explores innovative ways to traverse the cellular signaling circuitry, and shows extensive convergent evolution, highlighting pervasive constraints on pTyr signaling.
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Signaling properties of a non-metazoan Src kinase and the evolutionary history of Src negative regulation.
Journal of Biological Chemistry, 2008Co-Authors: Wanqing Li, Nicole King, Susan L Young, W. Todd MillerAbstract:Choanoflagellates, unicellular organisms that are closely related to metazoans, possess cell adhesion and signaling proteins previously thought to be unique to animals, suggesting that these components may have played roles in the evolution of metazoan multicellularity. We have cloned, expressed, and purified the nonreceptor tyrosine kinase MbSrc1 from the choanoflagellate Monosiga brevicollis. The kinase has the same domain arrangement as mammalian Src kinases, and we find that the individual Src homology 3 (SH3), SH2, and catalytic domains have similar functions to their mammalian counterparts. In contrast to mammalian c-Src, the SH2 and catalytic domains of MbSrc1 do not appear to be functionally coupled. We cloned and expressed the M. brevicollis homolog of c-Src C-terminal kinase (MbCsk) and showed that it phosphorylates the C terminus of MbSrc1, yet this phosphorylation does not inhibit MbSrc to the same degree seen in the mammalian Src/Csk pair. Thus, Src autoinhibition likely evolved more recently within the metazoan lineage, and it may have played a role in the establishment of intercellular signaling in metazoans.
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the genome of the choanoflagellate Monosiga brevicollis and the origin of metazoans
Nature, 2008Co-Authors: Nicole King, Jody M Westbrook, Susan L Young, Monika Abedin, Jarrod Chapman, Stephen R Fairclough, Uffe Hellsten, Yoh Isogai, Ivica Letunic, Michael T MarrAbstract:Choanoflagellates are the closest known relatives of metazoans. To discover potential molecular mechanisms underlying the evolution of metazoan multicellularity, we sequenced and analysed the genome of the unicellular choanoflagellate Monosiga brevicollis. The genome contains approximately 9,200 intron-rich genes, including a number that encode cell adhesion and signalling protein domains that are otherwise restricted to metazoans. Here we show that the physical linkages among protein domains often differ between M. brevicollis and metazoans, suggesting that abundant domain shuffling followed the separation of the choanoflagellate and metazoan lineages. The completion of the M. brevicollis genome allows us to reconstruct with increasing resolution the genomic changes that accompanied the origin of metazoans.
Martin Carr - One of the best experts on this subject based on the ideXlab platform.
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A genomic survey of transposable elements in the choanoflagellate Salpingoeca rosetta reveals selection on codon usage
Mobile Dna, 2019Co-Authors: Jade Southworth, Alan O Marron, C. Alastair Grace, Nazeefa Fatima, Martin CarrAbstract:Unicellular species make up the majority of eukaryotic diversity, however most studies on transposable elements (TEs) have centred on multicellular host species. Such studies may have therefore provided a limited picture of how transposable elements evolve across eukaryotes. The choanoflagellates, as the sister group to Metazoa, are an important study group for investigating unicellular to multicellular transitions. A previous survey of the choanoflagellate Monosiga brevicollis revealed the presence of only three families of LTR retrotransposons, all of which appeared to be active. Salpingoeca rosetta is the second choanoflagellate to have its whole genome sequenced and provides further insight into the evolution and population biology of transposable elements in the closest relative of metazoans. Screening the genome revealed the presence of a minimum of 20 TE families. Seven of the annotated families are DNA transposons and the remaining 13 families are LTR retrotransposons. Evidence for two putative non-LTR retrotransposons was also uncovered, but full-length sequences could not be determined. Superfamily phylogenetic trees indicate that vertical inheritance and, in the case of one family, horizontal transfer have been involved in the evolution of the choanoflagellates TEs. Phylogenetic analyses of individual families highlight recent element activity in the genome, however six families did not show evidence of current transposition. The majority of families possess young insertions and the expression levels of TE genes vary by four orders of magnitude across families. In contrast to previous studies on TEs, the families present in S. rosetta show the signature of selection on codon usage, with families favouring codons that are adapted to the host translational machinery. Selection is stronger in LTR retrotransposons than DNA transposons, with highly expressed families showing stronger codon usage bias. Mutation pressure towards guanosine and cytosine also appears to contribute to TE codon usage. S. rosetta increases the known diversity of choanoflagellate TEs and the complement further highlights the role of horizontal gene transfer from prey species in choanoflagellate genome evolution. Unlike previously studied TEs, the S. rosetta families show evidence for selection on their codon usage, which is shown to act via translational efficiency and translational accuracy.
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Patterns of Ancestral Animal Codon Usage Bias Revealed through Holozoan Protists.
Molecular Biology and Evolution, 2018Co-Authors: Jade Southworth, Paul Armitage, Brandon Fallon, Holly Dawson, Jarosław Bryk, Martin CarrAbstract:: Choanoflagellates and filastereans are the closest known single celled relatives of Metazoa within Holozoa and provide insight into how animals evolved from their unicellular ancestors. Codon usage bias has been extensively studied in metazoans, with both natural selection and mutation pressure playing important roles in different species. The disparate nature of metazoan codon usage patterns prevents the reconstruction of ancestral traits. However, traits conserved across holozoan protists highlight characteristics in the unicellular ancestors of Metazoa. Presented here are the patterns of codon usage in the choanoflagellates Monosiga brevicollis and Salpingoeca rosetta, as well as the filasterean Capsaspora owczarzaki. Codon usage is shown to be remarkably conserved. Highly biased genes preferentially use GC-ending codons, however there is limited evidence this is driven by local mutation pressure. The analyses presented provide strong evidence that natural selection, for both translational accuracy and efficiency, dominates codon usage bias in holozoan protists. In particular, the signature of selection for translational accuracy can be detected even in the most weakly biased genes. Biased codon usage is shown to have coevolved with the tRNA species, with optimal codons showing complementary binding to the highest copy number tRNA genes. Furthermore, tRNA modification is shown to be a common feature for amino acids with higher levels of degeneracy and highly biased genes show a strong preference for using modified tRNAs in translation. The translationally optimal codons defined here will be of benefit to future transgenics work in holozoan protists, as their use should maximise protein yields from edited transgenes.
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Conserved meiotic genes point to sex in the choanoflagellates.
Journal of Eukaryotic Microbiology, 2009Co-Authors: Martin Carr, Barry S C Leadbeater, Sandra L BaldaufAbstract:The choanoflagellates are a widespread group of heterotrophic aquatic nanoflagellates, which have recently been confirmed as the sister-group to Metazoa. Asexual reproduction is the only mode of cell division that has been observed within the group; at present the range of reproductive modes, as well as the ploidy level, within choanoflagellates are unknown. The recent discovery of long terminal repeat retrotransposons within the genome of Monosiga brevicollis suggests that this species also has sexual stages in its life cycle because asexual organisms cannot tolerate retrotransposons due to the rapid accumulation of deleterious mutations caused by their transposition. We screened the M. brevicollis genome for known eukaryotic meiotic genes, using a recently established "meiosis detection toolkit" of 19 genes. Eighteen of these genes were identified, none of which appears to be a pseudogene. Four of the genes were also identified in expressed sequence tag data from the distantly related Monosiga ovata. The presence of these meiosis-specific genes provides evidence for meiosis, and by implication sex, within this important group of protists.
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three families of ltr retrotransposons are present in the genome of the choanoflagellate Monosiga brevicollis
Protist, 2008Co-Authors: Martin Carr, Michaela Nelson, Barry S C Leadbeater, Sandra L BaldaufAbstract:The choanoflagellates are a ubiquitous group of nanoflagellates and the sister group of Metazoa. Examination of the initial draft version of the first choanoflagellate genome, that of Monosiga brevicollis, reveals the presence of three novel families of long terminal repeat (LTR) retrotransposons and an apparent absence of non-LTR retrotransposons and transposons. One of the newly discovered LTR families falls in the chromovirus clade of the Ty3/gypsy group while the other two families are closely related members of the Ty1/copia group. Examination of EST sequences and nucleotide analyses show that all three families are transcriptionally active and potentially functional within the genome of M. brevicollis.
W. Todd Miller - One of the best experts on this subject based on the ideXlab platform.
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Temperature sensitivities of metazoan and pre-metazoan Src kinases.
Biochemistry and biophysics reports, 2020Co-Authors: W. Todd MillerAbstract:Abstract Homologous enzymes from different species display functional characteristics that correlate with the physiological and environmental temperatures encountered by the organisms. In this study, we have investigated the temperature sensitivity of the nonreceptor tyrosine kinase Src. We compared the temperature dependencies of c-Src and two Src kinases from single-celled eukaryotes, the choanoflagellate Monosiga brevicollis and the filasterean Capsaspora owczarzaki. Metazoan c-Src exhibits temperature sensitivity, with high activity at 30 °C and 37 °C. This sensitivity is driven by changes in substrate binding as well as maximal velocity, and it is dependent on the amino acid sequence surrounding tyrosine in the substrate. When tested with a peptide that displays temperature-dependent phosphorylation by c-Src, the enzymatic rates for the unicellular Src kinases show much less variation over the temperatures tested. The data demonstrate that unicellular Src kinases are temperature compensated relative to metazoan c-Src, consistent with an evolutionary adaptation to their environments.
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Constitutive Activity in an Ancestral Form of Abl Tyrosine Kinase.
PLOS ONE, 2015Co-Authors: Saadat Aleem, Barbara P Craddock, W. Todd MillerAbstract:The c-abl proto-oncogene encodes a nonreceptor tyrosine kinase that is found in all metazoans, and is ubiquitously expressed in mammalian tissues. The Abl tyrosine kinase plays important roles in the regulation of mammalian cell physiology. Abl-like kinases have been identified in the genomes of unicellular choanoflagellates, the closest relatives to the Metazoa, and in related unicellular organisms. Here, we have carried out the first characterization of a premetazoan Abl kinase, MbAbl2, from the choanoflagellate Monosiga brevicollis. The enzyme possesses SH3, SH2, and kinase domains in a similar arrangement to its mammalian counterparts, and is an active tyrosine kinase. MbAbl2 lacks the N-terminal myristoylation and cap sequences that are critical regulators of mammalian Abl kinase activity, and we show that MbAbl2 is constitutively active. When expressed in mammalian cells, MbAbl2 strongly phosphorylates cellular proteins on tyrosine, and transforms cells much more potently than mammalian Abl kinase. Thus, MbAbl2 appears to lack the autoinhibitory mechanism that tightly constrains the activity of mammalian Abl kinases, suggesting that this regulatory apparatus arose more recently in metazoan evolution.
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Metazoan-like signaling in a unicellular receptor tyrosine kinase.
BMC Biochemistry, 2013Co-Authors: Kira P. Schultheiss, Barbara P Craddock, Michael Tong, Markus A. Seeliger, W. Todd MillerAbstract:Background Receptor tyrosine kinases (RTKs) are crucial components of signal transduction systems in multicellular animals. Surprisingly, numerous RTKs have been identified in the genomes of unicellular choanoflagellates and other protists. Here, we report the first biochemical study of a unicellular RTK, namely RTKB2 from Monosiga brevicollis.
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Metazoan-like signaling in a unicellular receptor tyrosine kinase
BMC Biochemistry, 2013Co-Authors: Kira P. Schultheiss, Barbara P Craddock, Michael Tong, Markus A. Seeliger, W. Todd MillerAbstract:Background Receptor tyrosine kinases (RTKs) are crucial components of signal transduction systems in multicellular animals. Surprisingly, numerous RTKs have been identified in the genomes of unicellular choanoflagellates and other protists. Here, we report the first biochemical study of a unicellular RTK, namely RTKB2 from Monosiga brevicollis . Results We cloned, expressed, and purified the RTKB2 kinase, and showed that it is enzymatically active. The activity of RTKB2 is controlled by autophosphorylation, as in metazoan RTKs. RTKB2 possesses six copies of a unique domain (designated RM2) in its C-terminal tail. An isolated RM2 domain (or a synthetic peptide derived from the RM2 sequence) served as a substrate for RTKB2 kinase. When phosphorylated, the RM2 domain bound to the Src homology 2 domain of MbSrc1 from M. brevicollis . NMR structural studies of the RM2 domain indicated that it is disordered in solution. Conclusions Our results are consistent with a model in which RTKB2 activation stimulates receptor autophosphorylation within the RM2 domains. This leads to recruitment of Src-like kinases (and potentially other M. brevicollis proteins) and further phosphorylation, which may serve to increase or dampen downstream signals. Thus, crucial features of signal transduction circuitry were established prior to the evolution of metazoans from their unicellular ancestors.
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Evidence for convergent evolution in the signaling properties of a choanoflagellate tyrosine kinase.
Biochemistry, 2009Co-Authors: Wanqing Li, Suzanne Scarlata, W. Todd MillerAbstract:Until recently, phosphotyrosine signaling was thought to be restricted to multicellular animals. Surprisingly, the unicellular choanoflagellate Monosiga brevicollis contains a number and diversity of tyrosine kinases that exceeds that of any metazoan, including humans. Many of these M. brevicollis tyrosine kinases possess combinations of signaling domains that do not occur in metazoans. One such kinase, the Src-like protein MbSrc4, contains a lipid-binding C2 domain N-terminal to the conserved SH3-SH2-kinase domains. Here, we report that the enzyme is highly active as a tyrosine kinase and that the targeting functions of the C2, SH3, and SH2 domains are similar to the mammalian counterparts. The membrane-binding activity of the C2 domain is functionally equivalent to the myristoylation signal of c-Src, suggesting that it is an example of convergent evolution. When expressed in mammalian cells, full-length MbSrc4 displays low activity toward endogenous proteins, and it cannot functionally substitute for mammalian c-Src in a reporter gene assay. Removal of the MbSrc4 C2 domain leads to increased phosphorylation of cellular proteins. Thus, in contrast to the related M. brevicollis Src-like kinase MbSrc1, MbSrc4 is not targeted properly to mammalian Src substrates, suggesting that the C2 domain plays a specific role in M. brevicollis signaling.