Annie Angers

Lineage-specific genes, or orphan genes, are increasingly recognized as important drivers of evolutionary innovation, yet their functional characterization remains largely restricted to nuclear genomes of model organisms. Whether lineage-specific genes encoded by mitochondrial DNA contribute to phenotypic diversification remains virtually unexplored. Here we investigate MORF, a rapidly evolving ORFan protein encoded by the paternally transmitted mitochondrial genome of bivalves with doubly uniparental inheritance (DUI). Combining immunolocalization, functional neutralization, protein interaction assays and proteomics in the distantly related bivalves Mytilus edulis and Venustaconcha ellipsiformis , we characterize MORF localization, function and molecular associations during male germ cell development. MORF localizes to developing sperm cells and mature sperm mitochondria in both species and is additionally detected in the acrosome of M. edulis . In cultured M. edulis germ cells, antibody-mediated neutralization of MORF increases the frequency of spermatids displaying four rather than the canonical five mitochondria, providing functional evidence that MORF contributes to mitochondrial organization during spermatogenesis. Comparative proteomic analyses identify MORF-associated proteins involved in cytoskeletal organization, protein homeostasis and extracellular matrix organization. The association between MORF and laminin is independently supported by western blotting in both species. Despite extensive sequence divergence, the two MORF proteins display overlapping functional and molecular features. Together, these findings uncover a functional role for a rapidly evolving mitochondrial orphan protein and point to mitochondrial orphan genes as a potentially overlooked source of evolutionary novelty.

Doubly uniparental inheritance is a phenomenon unique to bivalve mollusks characterized by the inheritance of mitochondria from both parents by male embryos and the maintenance of paternal mitochondria in male gonads. This lineage-specific mechanism has been described in several bivalve species, where male mitochondrial DNA is also often found in somatic tissues. We exploited the development of a primary cell culture model for the blue mussel Mytilus edulis to investigate mitochondrial DNA dynamics at the cellular level and over time, using cells dissociated from male and female mantle (gonadal) and gill (somatic) tissues. We employed quantitative polymerase chain reactions to monitor fluctuations in the relative abundance of male and female mitochondrial DNAs. In male mantle cells, the amount of male mtDNA increased over time in culture across all tested samples. In male gill cells, male mtDNA was detectable in approximately 50% of the samples at the initial time point and generally decreased over time. No heteroplasmic female could be detected in this study. Illumina sequencing, comparing the proportion of the sex-linked mtDNAs between male mantle tissue and derived cultured cells, revealed a sharp increase in the proportion of male mtDNA in cultured mantle cells. Finally, immunofluorescence experiments using antibodies against open reading frames (ORFs) encoded by male or female mtDNA genomes showed that these peptides are present in the mitochondria of all male mantle cells after 10 days in culture. Overall, these findings support the idea that heteroplasmy is maintained in male germ cells in culture, whereas heteroplasmic cells in somatic tissues tend to be scarce and are lost over time.

Mitochondrial alternative open reading frames (ORFs) substantially broaden the functional scope traditionally attributed to mitochondrial DNA, encoding peptides and proteins that participate in diverse cellular processes. These newly identified ORFs are embedded within annotated sequences, both coding and non-coding, and reveal layers of overlapping genetic information. We report the discovery of MTALTCO1, a 259 amino-acid protein, the longest mitochondrial alternative protein identified to date, encoded by an ORF located within the human cytochrome oxidase 1 gene, in the +3 reading frame. We confirm the expression and mitochondrial origin of MTALTCO1 through multiple independent lines of evidence, including a custom-designed antibody, mass spectrometry-derived peptides, sequence analysis and inhibitors of mitochondrial expression. Despite encoding AGR codons as arginine, contrary to the prevailing view that these function invariably as stop codons in the vertebrate mitochondrial genetic code, MTALTCO1 shows strong evidence of mitochondrial translation, challenging established models of mitochondrial codon usage and gene expression. Co-immunoprecipitations and pulldown assays delineate MTALTCO1’s interaction landscape across major cellular pathways. Finally, we present the first in-depth analysis of conservation for a mitochondrial alternative ORF overlapping a reference protein-coding gene and discuss the results in light of MTALTCO1’s suggested role in protein scaffolding. This article is part of the theme issue ‘Evolutionary genetics of mitochondria: on diverse and common evolutionary constraints across eukarya’.

Strict maternal inheritance of mitochondria is known to be the rule in animals, but over 100 species across six orders of bivalves possess doubly uniparental inheritance (DUI) of mitochondria. Under DUI, two distinctive sex-specific mitogenomes coexist. In marine and freshwater mussels, each mitogenome has an additional protein-coding gene, called female- and male-specific open reading frame or forf and morf , respectively. The function(s) of the associated FORF and MORF proteins remain unknown. Herein, we show that these proteins present similar tissue expression patterns in two distantly related DUI species: MORF was only expressed in male gonads, whereas FORF was expressed in all tissues of both sexes in the marine mussel Mytilus edulis and the freshwater mussel Venustaconcha ellipsiformis . Moreover, MORF was only expressed during the reproductive season, while FORF presented no clear seasonality pattern in M. edulis . Immunocytochemistry revealed the presence of both proteins in mitochondria and acrosomes of late spermatids and mature sperm. We hypothesize that MORF has a key function in spermatogenesis, while FORF has a more general function in both sexes. We also propose that both proteins may be involved in the fertilization process. The involvement of MORF in paternal mitochondrial transmission is also discussed.

Receptor tyrosine kinases are activated by binding to their ligands, which trigger modifications in their cytoplasmic domains to initiate signal transduction. Control mechanisms to modulate the signaling of growth factor receptors are essential for proper signaling and require several levels of regulation. Post-translational modifications play crucial roles in intracellular trafficking through mechanisms that are not fully understood. Ubiquitylation is recognized as an essential signal in establishing molecular networks controlling receptor internalization and trafficking at the membrane and in sorting endosomes. In turn, receptor trafficking influences how the signaling networks are activated. Recent advances show how receptor targeting to clathrin-coated pits and internalization influences signaling by allowing specific target activation. At the same time, progress has been made in showing how membrane proteins are organized to facilitate the recruitment of activated receptors to clathrin-coated pits and how this whole process depends on the ubiquitylation of the receptors and endocytosis accessory proteins. Here, we review recent advances in the role of ubiquitylation in receptor internalization and trafficking.

Mitochondrial alternative Open Reading Frames (ORFs) substantially broaden the functional scope traditionally attributed to mitochondrial DNA, encoding peptides and proteins that participate in diverse cellular processes. These newly identified ORFs are embedded within annotated sequences, both coding and non-coding, and reveal layers of overlapping genetic information. We here report the discovery of MTALTCO1, a 259 amino-acid protein, the longest mitochondrial alternative protein identified to date, encoded by an ORF located within the human cytochrome oxidase 1 gene, in the +3 reading frame. We confirm the expression and mitochondrial origin of MTALTCO1 through multiple independent lines of evidence, including a custom-designed antibody, mass spectrometry-derived peptides, sequence analysis, and inhibitors of mitochondrial expression. Despite encoding AGR codons as arginine, contrary to the prevailing view that these function as stop codons in the vertebrate mitochondrial genetic code, MTALTCO1 shows strong evidence of mitochondrial translation, challenging established models of mitochondrial codon usage and gene expression. Co-immunoprecipitations and pull-down assays delineate MTALTCO1’s interaction landscape across major cellular pathways. Lastly, we present the first in-depth analysis of conservation for a mitochondrial alternative ORF overlapping a reference protein-coding gene and discuss the results in light of MTALTCO1’s suggested role in protein scaffolding.

Our understanding of basic cellular processes has mostly been provided by mammalian cell culture, and by some non-mammalian vertebrate and few invertebrate cell culture models. Developing reliable culture conditions for non-model organisms is essential to allow investigation of more unusual cellular processes. Here, we investigate how cells isolated from different tissues of the marine mussel Mytilus edulis thrive and survive in vitro in the hope of establishing a suitable laboratory model for the investigation of cellular mechanisms specific to these bivalve mollusks. We found that cells dissociated from mantle tissue attached to the culture vessels and proliferated well in vitro, whereas cells isolated from gills, although remaining viable, did not maintain divisions over three to four weeks in culture. We used antibodies against the germ-line marker DEAD-box helicase 4 (DDX4), also known as VASA, and the epithelial cell marker cytokeratin to distinguish different cell types in culture. DDX4-positive cells were predominant in 25-day-old cultures from male mantles. Cells from other tissues remained in low numbers and did not seem to change in composition over time. Overall, the culture conditions described here allow an efficient selection of male germ cells that could be used to study specific cellular mechanisms in vitro.

Our understanding of basic cellular processes has mostly been provided by mammalian cell culture, and by some non-mammalian vertebrate and few invertebrate cell culture models. Developing reliable culture conditions for non-model organisms is essential to allow investigation of more unusual cellular processes. Here, we investigate how cells isolated from different tissues of the marine mussel Mytilus edulis thrive and survive in vitro in the hope of establishing a suitable laboratory model for the investigation of cellular mechanisms specific to these bivalve mollusks. We found that cells dissociated from male mantle tissue attached to the culture vessels and proliferated well in vitro , whereas all other cell types, although remaining viable, did not maintain divisions over three to four weeks in culture. We used antibodies against the germ-line marker DEAD-box helicase 4 (DDX4), also known as VASA, and the epithelial cell marker cytokeratin to distinguish different cell types in culture. DDX4-positive cells were the only cells that significantly proliferated in culture, and only from male tissue samples. Overall, the culture conditions described here allow an efficient selection of male germ cells that could be used to study specific cellular mechanisms in vitro .