A small protein coded within the mitochondrial canonical gene nd4 regulates mitochondrial bioenergetics

BACKGROUND: Mitochondria have a central role in cellular functions, aging, and in certain diseases. They possess their own genome, a vestige of their bacterial ancestor. Over the course of evolution, most of the genes of the ancestor have been lost or transferred to the nucleus. In humans, the mtDNA is a very small circular molecule with a functional repertoire limited to only 37 genes. Its extremely compact nature with genes arranged one after the other and separated by short non-coding regions suggests that there is little room for evolutionary novelties. This is radically different from bacterial genomes, which are also circular but much larger, and in which we can find genes inside other genes. These sequences, different from the reference coding sequences, are called alternatives open reading frames or altORFs, and they are involved in key biological functions. However, whether altORFs exist in mitochondrial protein-coding genes or elsewhere in the human mitogenome has not been fully addressed. RESULTS: We found a downstream alternative ATG initiation codon in the + 3 reading frame of the human mitochondrial nd4 gene. This newly characterized altORF encodes a 99-amino-acid-long polypeptide, MTALTND4, which is conserved in primates. Our custom antibody, but not the pre-immune serum, was able to immunoprecipitate MTALTND4 from HeLa cell lysates, confirming the existence of an endogenous MTALTND4 peptide. The protein is localized in mitochondria and cytoplasm and is also found in the plasma, and it impacts cell and mitochondrial physiology. CONCLUSIONS: Many human mitochondrial translated ORFs might have so far gone unnoticed. By ignoring mtaltORFs, we have underestimated the coding potential of the mitogenome. Alternative mitochondrial peptides such as MTALTND4 may offer a new framework for the investigation of mitochondrial functions and diseases

In vitro proliferation of Mytilus edulis male germ cell progenitors.

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

Average number of cells per microscopic frame (0.3 mm²) from dissociated male and female mantles and gills after the indicated number of days in culture.

5 microscope fields were counted from three replicates from 10 males and 10 females. L-15 medium dissolved in artificial sea water supplemented with antibiotics and enriched with 20% BCS and 0.1% YE was used as a culture medium, changed every four days. Within each box, bold horizontal lines denote median values; boxes extend from the 25th to the 75th percentile of each group’s distribution of values; vertical extending lines denote the 5th and 95th percentiles. The number of cells for each individual also appears as small lighter dots.</p

Different cell morphologies and expression of DDX4 and cytokeratin in cells from male mantle after 5 days in culture.

(A-D) Phase contrast and immunofluorescence staining for DDX4 (cyan) and cytokeratin (magenta) showing cells positive for both antigens (type A cells, large arrows), DDX4 only (type B cells, small arrows), cytokeratin only (type C, filled arrowheads), or unlabelled cells visible by weak autofluorescence (type D, empty arrowheads). Nuclei were counterstained with DAPI (coloured in yellow). (A) High magnification of typical type A and type B cells of approximately 2.5 μm in diameter and positive for both antibodies or DDX4 only. (B) Similar rounded cells weakly positive for both antibodies and DDX4 only, a larger cell (5.5 μm) and two elongated cells (~7.5 μm in length) negative for both antibodies. (C) Small rounded cells of each type. (D) Lower magnification showing small, rounded cells and one bigger, oval cell (~5 μm) positive for both antibodies, one large (~5 μm) cell positive for DDX4 only, and one unlabelled elongated cell (11 μm). Scale bar represents 5 μm in A,B and C and 10 μm in D.</p

Statistical analysis of cell number in different media.

A) GLM model with a confidence interval for cell number in different media. There is no overlap of the confidence intervals at 25°C, showing that cell growth is positive and significantly different in L15_BCS and L15+BCS+YE compared to L15 alone. Growth is negative in all other tested conditions. B) Tukey’s comparison of mean cell number at different times for all media tested at 25°C. Significance is shown only for consecutive measures. ns non significant, *p (PDF)</p

Change in marker expression over time in cultures originating from mantle and gill cells.

The number of each cell type over time as determined by detection of DDX4 and cytokeratin immunofluorescence in cultures of (A) male mantle cells, (B) female mantle cells, (C) male gill cells and (D) female gill cells. The number of unstained cells was obtained by counting the DAPI stained nuclei. Cells were counted from at least 10 captured frames at 100X magnification in 3 independent experiments (2 for male samples on Day 5).</p

Different cell morphologies and expression of DDX4 and cytokeratin in cells cultured from female mantle after 5 days in culture.

(A-C) Phase contrast and immunofluorescence staining for DDX4 (cyan) and cytokeratin (magenta), with nuclei counterstained with DAPI (yellow). (A) Small ovoid cell positive for DDX4 and cytokeratin. (B) Small ovoid cell positive for cytokeratin only. Note the debris surrounding the cell that was often present in cultures from female samples. (C) Large, elongated cells negative for both antibodies. Scale bar represents 5 μm in A and B, and 7.5 μm in C.</p

Statistical analysis of cell number from different tissues.

A) GLM model with a confidence interval for cell number and growth obtained from different tissues. Cells from male and female mantle increased more over time than cells from male and female gills. There is no overlap in the models, meaning that all cultures behaved differently. B) Tukey’s comparison of mean cell number at different times for all tissues tested. Significance is shown only for consecutive measures. ns non significant, *p (PDF)</p

Male mantle cell viability and proliferation in different culture media.

A) Examples of images obtained for counting cells from one culture at the indicated time point. Nuclei were stained with Hoescht dye and cells were counted using automatic detection. Five frames from triplicate cultures were counted for every sample. For space concerns, only a fraction of the complete image is shown. Scale bar = 25μm. B, C) Relative number of cells per frame compared to the number of cells that remained attached after the first change of media (Day 1) in each medium at 15 (B) and 25°C (C) after the indicated number of days in culture. Within each box, bold horizontal lines denote median values; boxes extend from the 25th to the 75th percentile of each group’s distribution of values; vertical extending lines denote the 5th and 95th percentiles.</p

Media composition.

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.</div