A simple and effective method for ultrastructural analysis of mitosis in Drosophila S2 cells

Strunov, Anton; Boldyreva, Lidiya V.; Pavlova, Gera A.; Pindyurin, Alexey V.; Gatti, Maurizio; Киселева, Елена

doi:10.1016/j.mex.2016.10.003

Cited by 11 publications

(7 citation statements)

References 24 publications

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“…Preparation of erythrocytes and fibroblasts for TEM was made using the protocol described in detail by (54). Briefly, cell were pelleted by mild centrifugation (200g) and fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer for 1 h at room temperature, post-fixed for 1 h in 1% aqueous solution of osmium tetroxide containing a few crystals of potassium ferricyanide (K3[Fe(CN)6]) and then incubated overnight at 4°C in 1% aqueous solution of uranyl acetate.…”

Section: Methodsmentioning

confidence: 99%

3D organization of chicken genome demonstrates evolutionary conservation of topologically associated domains and highlights unique architecture of erythrocytes’ chromatin

Fishman

Battulin

Nuriddinov

et al. 2018

Nucleic Acids Research

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101

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How chromosomes are folded, spatially organized and regulated in three dimensions inside the cell nucleus are among the longest standing questions in cell biology. Genome-wide chromosome conformation capture (Hi-C) technique allowed identifying and characterizing spatial chromatin compartments in several mammalian species. Here, we present the first genome-wide analysis of chromatin interactions in chicken embryonic fibroblasts (CEF) and adult erythrocytes. We showed that genome of CEF is partitioned into topologically associated domains (TADs), distributed in accordance with gene density, transcriptional activity and CTCF-binding sites. In contrast to mammals, where all examined somatic cell types display relatively similar spatial organization of genome, chicken erythrocytes strongly differ from fibroblasts, showing pronounced A- and B- compartments, absence of typical TADs and formation of long-range chromatin interactions previously observed on mitotic chromosomes. Comparing mammalian and chicken genome architectures, we provide evidence highlighting evolutionary role of chicken TADs and their significance in genome activity and regulation.

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Section: Methodsmentioning

confidence: 99%

3D organization of chicken genome demonstrates evolutionary conservation of topologically associated domains and highlights unique architecture of erythrocytes’ chromatin

Fishman

Battulin

Nuriddinov

et al. 2018

Nucleic Acids Research

Self Cite

101

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“…This study is limited by a single focus on cell ultrastructure. Prediction of cell behavior based on ultrastructural morphology is well documented (Klymkowsky et al 1983; Strunov et al 2016). Additionally, the cell behaviors in 2D culture were recapitulated in a 3D environment.…”

Section: Discussionmentioning

confidence: 99%

Ultrastructural morphology is distinct among primary progenitor cell isolates from normal, inflamed, and cryopreserved equine hoof tissue and CD105+K14+ progenitor cells

Yang

Lopez

2019

In Vitro Cell.Dev.Biol.-Animal

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The equine hoof dermal-epidermal interface requires progenitor cells with distinct characteristics. This study was designed to provide accurate ultrastructural depictions of progenitor cells isolated from inflamed tissue and normal tissue before and after cryopreservation and following selection of cells expressing both keratin (K) 14 (ectodermal) and cluster of differentiation (CD) 105 (mesodermal). Passage 3 cell ultrastructure was assessed following 2D culture and after 3D culture on decellularized hoof tissue scaffolds. Outcome measures included light, transmission electron, and scanning electron microscopy, immunocytochemistry, and CD105 + K14 + cell trilineage plasticity. Cells from normal tissue had typical progenitor cell characteristics. Those from inflamed tissue had organelles and morphology consistent with catabolic activities including lysosomes, irregular rough endoplasmic reticulum, and fewer vacuoles and early endosomes than those from normal tissue. Cryopreserved tissue cells appeared apoptotic with an irregular cell membrane covered by cytoplasmic protrusions closely associated with endocytic and exocytic vesicles, chromatin aggregated on the nuclear envelop, abundant, poorly organized rough endoplasmic reticulum, and plentiful lysosomes. Cells that were CD105 + K14 + were distinguishable from heterogenous cells by infrequent microvilli on the cell surface, sparse endosomes and vesicles, and desmosomes between cells. Cells expressed ectodermal (K15) and mesodermal (CD105) proteins in 2D and 3D cultures. Inflamed and cryopreserved tissue isolates attached poorly to tissue scaffold while normal tissue cells attached well, but only CD105 + K14 + cells produced extracellular matrix after 4 d. The CD105 + K14 + cells exhibited osteoblastic, adipocytic, and neurocytic differentiation. Ultrastructural information provided by this study contributes to understanding of equine hoof progenitor cells to predict their potential contributions to tissue maintenance, healing, and damage as well post-implantation behavior.

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“…S2 cell culture and RNA interference (RNAi). S2 cells (for details, see Strunov et al, 2016) were cultured at 25 °C in 39.4 g/L Shields and Sang M3 Insect medium (Sigma, S8398) supplemented with 0.5 g/L KHCO 3 and 20 % heat-inactivated fetal bovine serum (FBS) (Thermo Scientific, 10270106). RNAi treatments were carried out as described previously (Somma et al, 2008), with the following modifications: 25 μg of dsRNA was added to the cells three times (on the first, the third and the fifth day of incubation), and cells were harvested for analyses after 7 days of RNAi.…”

Section: Methodsmentioning

confidence: 99%

Non3 is an essential Drosophila gene required for proper nucleolus assembly

et al. 2019

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The nucleolus is a dynamic non-membrane-bound nuclear organelle, which plays key roles not only in ribosome biogenesis but also in many other cellular processes. Consistent with its multiple functions, the nucleolus has been implicated in many human diseases, including cancer and degenerative pathologies of the nervous system and heart. Here, we report the characterization of the Drosophila Non3 (Novel nucleolar protein 3) gene, which encodes a protein homologous to the human Brix domain-containing Rpf2 that has been shown to control ribosomal RNA (rRNA) processing. We used imprecise P-element excision to generate four new mutant alleles in the Non3 gene. Complementation and phenotypic analyses showed that these Non3 mutations can be arranged in an allelic series that includes both viable and lethal alleles. The strongest lethal allele (Non3∆600) is a genetically null allele that carries a large deletion of the gene and exhibits early lethality when homozygous. Flies heterozygous for Non3∆600 occasionally exhibit a mild reduction in the bristle size, but develop normally and are fertile. However, heteroallelic combinations of viable Non3 mutations (Non3197, Non3310 and Non3259) display a Minute-like phenotype, consisting in delayed development and short and thin bristles, suggesting that they are defective in ribosome biogenesis. We also demonstrate that the Non3 protein localizes to the nucleolus of larval brain cells and it is required for proper nucleolar localization of Fibrillarin, a protein important for post-translational modification and processing of rRNAs. In summary, we generated a number of genetic and biochemical tools that were exploited for an initial characterization of Non3, and will be instrumental for future functional studies on this gene and its protein product.

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A simple and effective method for ultrastructural analysis of mitosis in Drosophila S2 cells

Abstract: Graphical abstract

Cited by 11 publications

References 24 publications

3D organization of chicken genome demonstrates evolutionary conservation of topologically associated domains and highlights unique architecture of erythrocytes’ chromatin

3D organization of chicken genome demonstrates evolutionary conservation of topologically associated domains and highlights unique architecture of erythrocytes’ chromatin

Ultrastructural morphology is distinct among primary progenitor cell isolates from normal, inflamed, and cryopreserved equine hoof tissue and CD105+K14+ progenitor cells

Non3 is an essential Drosophila gene required for proper nucleolus assembly

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