Characteristics of SP600125 Induced Tetraploid Cells in Comparison With Diploid and Tetraploid Cells of Fish

In our previous research, SP600125 (Anthrapyrazolone) was used to induce autotetraploid of crucian carp cells (SP4N cells), and tetraploid fry was generated from the SP4N cells by somatic cell nuclear transfer technique. However, it is still unclear about biological characteristics of the SP4N cells. In this article, the cytological characteristic and gene expression profiles of the SP4N cells are investigated in comparison with the crucian carp cells (2N cells) and the tetraploid crucian carp cells (CC4N cells). The SP4N cells have tetraploid characteristics in terms of morphology and DNA ploidy levels, and their chromosome behavior is stable during the cell proliferation. The migration ability and the mtDNA copy number of SP4N cells are both lower than those in the CC4N cells and the 2N cells, but there exist giant mitochondria in the SP4N cells. The similar expression trends in the cell cycle regulation genes of the SP4N cells and 2N cells, while the corresponding expression profiles are clearly different between the SP4N cells and the CC4N cells. Moreover, the significant difference genes are associated with energy metabolism pathways among the SP4N cells, 2N cells and CC4N cells. These results can provide deeper understanding of SP600125 induction, as well as finding applications in polyploidization breeding of fish species.

Existence of giant mitochondria-containing sheet structures lacking cristae and matrix in the etiolated cotyledon of Arabidopsis thaliana

Mitochondria are essential organelles involved in the production and supply of energy in eukaryotic cells. Recently, the use of serial section scanning electron microscopy (S3EM) has allowed accurate three-dimensional (3D) reconstructed images of even complex organelle structures. Using this method, ultrathin sections of etiolated cotyledons were observed 4 days after germination of Arabidopsis thaliana in the dark, and giant mitochondria were found. To exclude the possibility of chemical fixation artifacts, this study confirmed the presence of giant mitochondria in high-pressure frozen samples. The 3D reconstructed giant mitochondria had a complex structure that included not only the elongated region but also the flattened shape of a disk. It contained the characteristic sheet structure, and the sheet lacked cristae and matrix but consisted of outer and inner membranes. Whether this phenomenon could be observed in living cells was investigated using the transformant with mitochondrial matrix expressing green fluorescent protein. Small globular mitochondria observed in light-treated samples were also represented in etiolated cotyledons. Although no giant mitochondria were observed in light-treated samples, they were found in the dark 3 days after germination and rapidly increased in number on the fourth day. Therefore, giant mitochondria were observed only in dark samples. These findings were supported by electron microscopy results.

Three-dimensional ultrastructure of giant mitochondria in human non-alcoholic fatty liver disease

Giant mitochondria are peculiarly shaped, extremely large mitochondria in hepatic parenchymal cells, the internal structure of which is characterised by atypically arranged cristae, enlarged matrix granules and crystalline inclusions. The presence of giant mitochondria in human tissue biopsies is often linked with cellular adversity, caused by toxins such as alcohol, xenobiotics, anti-cancer drugs, free-radicals, nutritional deficiencies or as a consequence of high fat Western diets. To date, non-alcoholic fatty liver disease is the most prevalent liver disease in lipid dysmetabolism, in which mitochondrial dysfunction plays a crucial role. It is not well understood whether the morphologic characteristics of giant mitochondria are an adaption or caused by such dysfunction. In the present study, we employ a complementary multimodal imaging approach involving array tomography and transmission electron tomography in order to comparatively analyse the structure and morphometric parameters of thousands of normal- and giant mitochondria in four patients diagnosed with non-alcoholic fatty liver disease. In so doing, we reveal functional alterations associated with mitochondrial gigantism and propose a mechanism for their formation based on our ultrastructural findings.

Impaired turnover of hyperfused mitochondria in severe axonal neuropathy due to a novel DRP1 mutation

Mitochondria undergo continuous cycles of fusion and fission in response to physiopathological stimuli. The key player in mitochondrial fission is dynamin-related protein 1 (DRP1), a cytosolic protein encoded by dynamin 1-like (DNM1L) gene, which relocalizes to the outer mitochondrial membrane, where it assembles, oligomerizes and drives mitochondrial division upon guanosine-5′-triphosphate (GTP) hydrolysis. Few DRP1 mutations have been described so far, with patients showing complex and variable phenotype ranging from early death to encephalopathy and/or optic atrophy. The disease is the consequence of defective mitochondrial fission due to faulty DRP1 function. However, the underlying molecular mechanisms and the functional consequences at mitochondrial and cellular level remain elusive. Here we report on a 5-year-old girl presenting psychomotor developmental delay, global hypotonia and severe ataxia due to axonal sensory neuropathy harboring a novel de novo heterozygous missense mutation in the GTPase domain of DRP1 (NM_012062.3:c.436G>A, NP_036192.2: p.D146N variant in DNM1L). Patient’s fibroblasts show hyperfused/balloon-like giant mitochondria, highlighting the importance of D146 residue for DRP1 function. This dramatic mitochondrial rearrangement phenocopies what observed overexpressing DRP1-K38A, a well-known experimental dominant negative version of DRP1. In addition, we demonstrated that p.D146N mutation has great impact on peroxisomal shape and function. The p.D146N mutation compromises the GTPase activity without perturbing DRP1 recruitment or assembly, causing decreased mitochondrial and peroxisomal turnover. In conclusion, our findings highlight the importance of sensory neuropathy in the clinical spectrum of DRP1 variants and, for the first time, the impact of DRP1 mutations on mitochondrial turnover and peroxisomal functionality.

Chromatin streaming from giant polyploid nuclei in Ishikawa endometrial hollow spheroids results in the amitotic proliferation of nuclei that fill the spheroid envelope

This paper describes the amitotic proliferation of nuclei that fill the envelope of Ishikawa hollow spheroids. The presence of hollow spheroids in malignant ascites fluid has intrigued cancer researchers, but little is understood about how they form. Observations in Ishikawa endometrial cell cultures demonstrate that nuclei filling the spheroid envelope are generated amitotically by the same mechanism responsible for cell formation in domes. Transient structures of aggregated chromatin surrounded by fused giant mitochondria, the initiating structure for dome formation, are also the starting point for the differentiation of unicellular polyploid hollow spheroids. Nuclei from monolayer cells are aggregated in a single enlarged cell where they become surrounding by giant fused mitochondria. A gaseous vacuole forms inside the mitonucleon extending it so that all of the cell material, including nuclei is pressed against the cell membrane. The resulting unicellular hollow spheroid detaches from the colony, capable of migration from the site of its formation. Ultimately, pressure on the aggregated chromatin results in the release of streams of chromatin granules that initially travel as if guided by microtubules through the shell of the hollow spheroid. Granules dissolve into filaments and, as initially described in dome formation, this material self-assembles into clusters of nuclei. Nuclei move out of these clusters into a regular array within the spheroid envelope, with formation of cell membranes as the final step in the creation of multicellular hollow spheroids. The curved membrane characteristic of domes and spheroids, as well as colonies of nuclei produced by amitosis have been identified in tumor tissue that survives chemotherapy, suggesting that amitotic cell proliferation may at least partially explain the population of cancer tumor cells in humans that are resistant to chemotherapy.

Chromatin streaming from giant polyploid nuclei in Ishikawa endometrial hollow spheroids results in the amitotic proliferation of nuclei that fill the spheroid envelope

This paper describes the amitotic proliferation of nuclei that fill the envelope of Ishikawa hollow spheroids. The presence of hollow spheroids in malignant ascites fluid has intrigued cancer researchers, but little is understood about how they form. Observations in Ishikawa endometrial cell cultures demonstrate that nuclei filling the spheroid envelope are generated amitotically by the same mechanism responsible for cell formation in domes. Transient structures of aggregated chromatin surrounded by fused giant mitochondria, the initiating structure for dome formation, are also the starting point for the differentiation of unicellular polyploid hollow spheroids. Nuclei from monolayer cells are aggregated in a single enlarged cell where they become surrounding by giant fused mitochondria. A gaseous vacuole forms inside the mitonucleon extending it so that all of the cell material, including nuclei is pressed against the cell membrane. The resulting unicellular hollow spheroid detaches from the colony, capable of migration from the site of its formation. Ultimately, pressure on the aggregated chromatin results in the release of streams of chromatin granules that initially travel as if guided by microtubules through the shell of the hollow spheroid. Granules dissolve into filaments and, as initially described in dome formation, this material self-assembles into clusters of nuclei. Nuclei move out of these clusters into a regular array within the spheroid envelope, with formation of cell membranes as the final step in the creation of multicellular hollow spheroids. The curved membrane characteristic of domes and spheroids, as well as colonies of nuclei produced by amitosis have been identified in tumor tissue that survives chemotherapy, suggesting that amitotic cell proliferation may at least partially explain the population of cancer tumor cells in humans that are resistant to chemotherapy.