L. V. Omelyanchuk

The role of Drosophila Merlin in spermatogenesis

BackgroundDrosophila Merlin, the homolog of the human Neurofibromatosis 2 (NF2) gene, is important for the regulation of cell proliferation and receptor endocytosis. Male flies carrying a Mer3 allele, a missense mutation (Met177→Ile) in the Merlin gene, are viable but sterile; however, the cause of sterility is unknown.ResultsTestis examination reveals that hemizygous Mer3 mutant males have small seminal vesicles that contain only a few immotile sperm. By cytological and electron microscopy analyses of the Mer3, Mer4 (Gln170→stop), and control testes at various stages of spermatogenesis, we show that Merlin mutations affect meiotic cytokinesis of spermatocytes, cyst polarization and nuclear shaping during spermatid elongation, and spermatid individualization. We also demonstrate that the lethality and sterility phenotype of the Mer4 mutant is rescued by the introduction of a wild-type Merlin gene. Immunostaining demonstrates that the Merlin protein is redistributed to the area associated with the microtubules of the central spindle in telophase and its staining is less in the region of the contractile ring during meiotic cytokinesis. At the onion stage, Merlin is concentrated in the Nebenkern of spermatids, and this mitochondrial localization is maintained throughout sperm formation. Also, Merlin exhibits punctate staining in the acrosomal region of mature sperm.ConclusionMerlin mutations affect spermatogenesis at multiple stages. The Merlin protein is dynamically redistributed during meiosis of spermatocytes and is concentrated in the Nebenkern of spermatids. Our results demonstrated for the first time the mitochondrial localization of Merlin and suggest that Merlin may play a role in mitochondria formation and function during spermatogenesis.

The role of Drosophila hyperplastic discs gene in spermatogenesis

In Drosophila, the ubiquitin ligase Hyd (hyperplastic disc) is required for regulation of cell proliferation during development [ Martin et al. (1977) Dev Biol 55, 213–232; Mansfield et al. (1994) Dev Biol 165, 507–526]. Earlier, we demonstrated that the Drosophila tumour suppressor Merlin participates not only in imaginal discs proliferation control, but also performs a separate Nebenkern structural function in Drosophila spermatogenesis [ Dorogova et al. (2008) BMC Cell Biol 9, 1. Here, we show that the hyd mutants also have spermatogenesis defects: chromosome condensation and attachment to the spindle, centrosome behaviour and cytokinesis in meiosis. The process of spermatid elongation was also greatly affected: nuclei were scattered along the cyst and had an abnormal shape, Nebenkern–axoneme angular relation and attachment was distorted, axonemes themselves lost correct structure. Since Hyd and pAbp protein families share a common PABC [poly(A)‐binding protein C‐terminal] protein domain, we also studied spermatogenesis in pAbp homozygotes and found defects in cytokinesis and spermatid elongation. However, our study of hyd and pAbp genetic interaction revealed only the phenotype of defective nuclei shape at the final stage of spermatid differentiation. So, the PABC domain is unlikely to be responsible for meiotic defects. Thus, our data document that, in addition to the tumour suppressor Merlin, another tumour suppressor, Hyd, also has a function in spermatogenesis.

DNA content in nuclei of Cyclops kolensis and Cyclops insignis (Crustacea, Copepoda)

Chromatin diminution (CD) in two Cyclopoida species, Cyclops kolensis and Cyclops insignis, was studied by static digital Feulgen cytophotometry. DNA content (pg/cell) was evaluated with standard dependences constructed by amounts of DNA in the blood cells of five organisms with known DNA contents of 1.25–14.7 pg. It was found that the C. kolensis diploid genome had about 40 pg DNA before CD and 1.8–2.0 pg DNA after CD. These values are similar both for Moscow and Baikal population of C. kolensis and exceed previous estimates by six to ten times (Grishanin, 2008). Our data confirm that CD reaches 94–96% of DNA content in C. kolensis. In mitotic cells of C. insignis DNA content was about 7.5 pg in both early and late embryos; CD was not revealed for this species. The data obtained show that the DNA content in the C. kolensis genome before CD is highest among the examined Cyclopoides.

Feed-back regulation of successive meiotic cytokinesis

The paper considers a number of abnormal phenotypes with impaired temporal regulation of cytokinesis during the meiotic division of pollen mother cells. The phenomenon of “non‐stop” cytokinesis with blocked arrest of the phragmoplast centrifugal motion and cell plate growth as well as incomplete and premature cytokinesis are described. The obtained data suggested a model for regulation of the processes involved in the arrest of the main cytokinesis processes during its completion in the plant meiosis.

A GFP trap study uncovers the functions of Gilgamesh protein kinase in Drosophila melanogaster spermatogenesis

The function of the gene gilgamesh (89B9‐12) encoding a casein kinase in Drosophila spermatogenesis was studied. The chimeric Gilgamesh–GFP protein in spermatocytes is cortically located. In the polar and apolar spermatocytes, it concentrates at the terminal ends of the fusome, the organelle that passes through the system of ring canals of the spermatocyte cyst. At the stage of spermatid elongation, the protein associates with the nucleus. A spot of the highest Gilgamesh–GFP concentration in the nucleus co‐localizes with γ‐tubulin in the basal body. At later stages, Gilgamesh is localized to the individualization complex (IC), leaving the nuclei somewhat before the IC investment cones, as detected by actin binding. The sterile mutation due to the gilgamesh gene leads to the phenotype of scattered nuclei and altered structure of actin cones in the individualizing spermatid cyst. Ultrastructural evidence confirmed defective spermatid individualization due to the mutation. The phylogenetic origin of the protein, and the connection between vesicular trafficking and spermatid individualization, are discussed.

Drosophila nervous system as a target of aging and anti-aging interventions.

Nervoussystemregulateshomeostasisandadaptationtoenvironmentalchangesofawholeorgan-ism, thus deregulation of nervous processes accelerates aging (Alcedo et al., 2013a,b). The agingprocess in different models is associated with progressive degeneration of the nervous system (Leeet al., 2000) and progression of age-related neurodegenerative diseases such as Alzheimer’s dis-ease, Parkinson’s disease, Huntington’s disease, and amyotrophic lateral sclerosis (Boerrigter et al.,1992; Coppede and Migliore, 2010). The neurodegeneration also characterizes the progeroid syn-dromes,includingHutchinson-GilfordsyndromeandWerner’ssyndrome(CoppedeandMigliore,2010).Drosophila melanogaster is a good model organism to study age-related neurodegenerativechanges (Lu and Vogel, 2009). Enrichment in mutants with neurodegeneration among flies withshortened lifespan has been reported (Buchanan and Benzer, 1993; Kretzschmar et al., 1997). Thebrain from old flies demonstrates the ultrastructural neurodegenerative changes such as reductionin the number of synapses, defects in mitochondria, and increase in neuronal apoptosis (Had-dadietal.,2014).However,anti-aginginterventionsmaypostponetheneurodegeneration(Bgatovaet al., 2015).Here we consider molecular genetic changes in the Drosophilaaging brain and the bases forapplying the brain as a target for anti-aging intervention.

Gadd45 expression correlates with age dependent neurodegeneration in Drosophila melanogaster

The neurodegeneration is one of the features of aging and age-related disorders. Yet, only several antiaging interventions are known to affect the processes of neurodegeneration. Here we show that overexpression of the pro-longevity gene D-GADD45 in Drosophila neurons leads to a postponed manifestation of histological and ultrastructural features of age-dependent neurodegeneration, such as decrease in the packing density of neurons, increasing the degree of neuron cytoplasmic vacuolization, and morphological defects of mitochondrial cristae. Thus, the previously observed (Plyusnina, Biogerontology 12: 211–226, 2011) life extending effect of D-GADD45 overexpression in the nervous system is associated with delayed neurodegeneration.

[Dynamics of the spatial organization of the chromosome set in cells of Drosophila melanogaster imaginal disks normally and under the action of the tumor-inducing mutation Merlin].

Fluorescence of H3-p histone and DAPI was studi ed at different stages of interphase and mitosis in cells of imaginal disks of third-instar Drosophila melanogaster larvae. Three stages differing in the spatial organization of the chromosome set in mitosis were revealed. At the first stage (prophase, prometaphase), the histone 3 phosphorylation level rises, and the volume occupied by the chromosome set in the nucleus increases. The distinctive features of the second stage (metaphase) are a gradual decrease in the histone 3 phosphorylation (the density of phosphorylation remaining constant) and a reduction of the volume occupied by the chromosome set. At the third stage (anaphase, telophase), the intensity and density of the signal from H3-p histone decrease, and the volume occupied by the chromosome set reduces. At this stage, in Mer4 larvae, in contrast to the control strain, the cells prematurely pass from anaphase into telophase. In addition, a subpopulation of cells with an abnormally large volume of nuclear DNA during the G1 period was revealed in Mer4 larvae. The cells of this subpopulation do not enter into the DNA synthesis and quit the cycle.

Key Events of the Cell Cycle: Regulation and Organization

The review surveys the studies of molecular genetic mechanisms of the cell cycle control on various eukaryotic models. The major cell cycle phenomena are considered: (1) checkpoints and their role in preserving DNA integrity and fidelity of mitosis, (2) the cell oscillator model, and (3) the role of cyclins in timing of cell division and coordination of mitotic events. The main classes of regulatory proteins involved in the cell cycle are discussed in detail.

Cytophotometric determination of genome size in two species of Cyclops of Lake Baikal (Crustacea: Copepoda, Cyclopoida) in ontogenetic development

The genome size of Cyclops in cells at early stages of cleavage (up to the fifth division) and in somatic cells was estimated by static digital Feulgen cytophotometry in order to study quantitative changes in DNA content during chromatin diminution. Described here cytophotometric method was approbated on five different digital-imaging systems in blood cells of four vertebrate species. In all cases, we observed a direct correlation between the data obtained with known from the literature on genome size and high reproducibility, which will allow these systems to be used in future work. We also optimized the conditions for DNA hydrolysis of both blood smears and for two species of Cyclops from the Moscow population as 30 min in 5 N HCl at 24°C. Here, we first revealed chromatin diminution in two endemic Baikal species of Cyclopoida: Acanthocyclops incolotaenia and Diacyclops galbinus. We estimated the extent of chromatin diminution in Diacyclops galbinus as 95.5–96.2%. Cytometric analysis of the third species, Mesocyclops leuckarti, did not reveal obvious chromatin diminution.