Anton Strunov

Spatial and temporal distribution of pathogenic Wolbachia strain wMelPop in Drosophila melanogaster central nervous system under different temperature conditions

The pathogenic Wolbachia strain, wMelPop, of Drosophila melanogaster is propagated in the flys brain and muscles. To determine how wMelPop spreads in the hosts central nervous system (CNS) during its life cycle, we used whole-mount fluorescent in situ hybridization to demonstrate the spatial distribution of wMelPop in D.melanogaster larvae and adults. To assess the effect of temperature on the pattern of wMelPop spread, we performed this analysis under moderate (25°C) and high (29°C) temperature conditions. Wolbachia distribution pattern in the third instar larva and adult brain was similar at both temperatures. wMelPop was generally localized to the subesophageal ganglion and the central brain of the host, whereas optic lobe anlagen cells of third instar larvae and cells of the optic lobe, lamina and retina of adult flies were mostly free of bacteria. Interestingly, high temperature had no significant effect on wMelPop titer or localization in the brain during larval development, but considerably altered it in adults immediately after eclosion. At both temperatures and within all tested stages of the life cycle, the bacterial titer varied only slightly between individuals. The observed differences in wMelPop titers in the central brain, subesophageal ganglion and optic lobe anlagen cells of third instar larvas CNS, together with the observation that these patterns are conserved in the adult brain, suggest that Wolbachia distribution is determined during fly embryogenesis.

Drosophila melanogaster brain invasion pathogenic Wolbachia in central nervous system of the fly

The pathogenic Wolbachia strain wMelPop rapidly over‐replicates in the brain, muscles, and retina of Drosophila melanogaster, causing severe tissue degeneration and premature death of the host. The unique features of this endosymbiont make it an excellent tool to be used for biological control of insects, pests, and vectors of human diseases. To follow the dynamics of bacterial morphology and titer in the nerve cells we used transmission electron microscopy of 3‐d‐old female brains. The neurons and glial cells from central brain of the fly had different Wolbachia titers ranging from single bacteria to large accumulations, tearing cell apart and invading extracellular space. The neuropile regions of the brain were free of wMelPop. Wolbachia tightly interacted with host cell organelles and underwent several morphological changes in nerve cells. Based on different morphological types of bacteria described we propose for the first time a scheme of wMelPop dynamics within the somatic tissue of the host.

The Drosophila orthologue of the INT6 onco-protein regulates mitotic microtubule growth and kinetochore structure

INT6/eIF3e is a highly conserved component of the translation initiation complex that interacts with both the 26S proteasome and the COP9 signalosome, two complexes implicated in ubiquitin-mediated protein degradation. The INT6 gene was originally identified as the insertion site of the mouse mammary tumor virus (MMTV), and later shown to be involved in human tumorigenesis. Here we show that depletion of the Drosophila orthologue of INT6 (Int6) results in short mitotic spindles and deformed centromeres and kinetochores with low intra-kinetochore distance. Poleward flux of microtubule subunits during metaphase is reduced, although fluorescence recovery after photobleaching (FRAP) demonstrates that microtubules remain dynamic both near the kinetochores and at spindle poles. Mitotic progression is delayed during metaphase due to the activity of the spindle assembly checkpoint (SAC). Interestingly, a deubiquitinated form of the kinesin Klp67A (a putative orthologue of human Kif18A) accumulates near the kinetochores in Int6-depleted cells. Consistent with this finding, Klp67A overexpression mimics the Int6 RNAi phenotype. Furthermore, simultaneous depletion of Int6 and Klp67A results in a phenotype identical to RNAi of just Klp67A, which indicates that Klp67A deficiency is epistatic over Int6 deficiency. We propose that Int6-mediated ubiquitination is required to control the activity of Klp67A. In the absence of this control, excess of Klp67A at the kinetochore suppresses microtubule plus-end polymerization, which in turn results in reduced microtubule flux, spindle shortening, and centromere/kinetochore deformation.

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

Graphical abstract

First evidence of Wolbachia infection in populations of grasshopper Podisma sapporensis (Orthoptera: Acrididae)

The brachypterous grasshopper Podisma sapporensis (Orthoptera: Acrididae) is distributed throughout the Sakhalin, Kunashir and Hokkaido Islands. Karyotypes of this species consist of two major chromosomal races with different sex chromosome systems, XO/XX and XY/XX. Molecular phylogeographic analysis of the chromosome races and subraces confirms the genetic divergence of the races and subraces in P. sapporensis. Here we first report that P. sapporensis is infected with Wolbachia consisting of three variants on wsp locus, while gatB locus was monomorphic. Furthermore, observation of cell tissue of P. sapporensis using electron microscopy confirmed the infection of Wolbachia that was inferred from polymerase chain reaction and revealed the distribution of the bacteria in the head, thorax and abdomen of P. sapporensis embryos. Our finding may shed new light on Wolbachia as a possible agent causing hybrid dysfunction resulting from experimental crosses between chromosome races or subraces of P. sapporensis.

Imaging yeast NPCs: from classical electron microscopy to Immuno-SEM.

Electron microscopy (EM) has been used extensively for the study of nuclear transport as well as the structure of the nuclear pore complex (NPC) and nuclear envelope. However, there are specific challenges faced when carrying out EM in one of the main model organisms used: the yeast, Saccharomyces cerevisiae. These are due to the presence of a cell wall, vacuoles, and a densely packed cytoplasm which, for transmission EM (TEM), make fixation, embedding, and imaging difficult. These also present problems for scanning EM (SEM) because cell wall removal and isolation of nuclei can easily damage the relatively fragile NPCs. We present some of the protocols we use to prepare samples for TEM and SEM to provide information about yeast NPC ultrastructure and the location of nucleoporins and transport factors by immunogold labeling within that ultrastructure.

Ultrastructural analysis of mitotic Drosophila S2 cells identifies distinctive microtubule and intracellular membrane behaviors

BackgroundS2 cells are one of the most widely used Drosophila melanogaster cell lines. A series of studies has shown that they are particularly suitable for RNAi-based screens aimed at the dissection of cellular pathways, including those controlling cell shape and motility, cell metabolism, and host–pathogen interactions. In addition, RNAi in S2 cells has been successfully used to identify many new mitotic genes that are conserved in the higher eukaryotes, and for the analysis of several aspects of the mitotic process. However, no detailed and complete description of S2 cell mitosis at the ultrastructural level has been done. Here, we provide a detailed characterization of all phases of S2 cell mitosis visualized by transmission electron microscopy (TEM).ResultsWe analyzed by TEM a random sample of 144 cells undergoing mitosis, focusing on intracellular membrane and microtubule (MT) behaviors. This unbiased approach provided a comprehensive ultrastructural view of the dividing cells, and allowed us to discover that S2 cells exhibit a previously uncharacterized behavior of intracellular membranes, involving the formation of a quadruple nuclear membrane in early prometaphase and its disassembly during late prometaphase. After nuclear envelope disassembly, the mitotic apparatus becomes encased by a discontinuous network of endoplasmic reticulum membranes, which associate with mitochondria, presumably to prevent their diffusion into the spindle area. We also observed a peculiar metaphase spindle organization. We found that kinetochores with attached k-fibers are almost invariably associated with lateral MT bundles that can be either interpolar bundles or k-fibers connected to a different kinetochore. This spindle organization is likely to favor chromosome alignment at metaphase and subsequent segregation during anaphase.ConclusionsWe discovered several previously unknown features of membrane and MT organization during S2 cell mitosis. The genetic determinants of these mitotic features can now be investigated, for instance by using an RNAi-based approach, which is particularly easy and efficient in S2 cells.

Ultrastructural analysis of intracellular membrane and microtubule behavior during mitosis of Drosophila S2 cells

S2 cells are one of the most widely used Drosophila melanogaster cell lines for molecular dissection of mitosis using RNA interference (RNAi). However, a detailed and complete description of S2 cell mitosis at the ultrastructural level is still missing. Here, we analyzed by transmission electron microscopy (TEM) a random sample of 144 cells undergoing mitosis, focusing on intracellular membrane and microtubule (MT) behavior. This unbiased approach allowed us to discover that S2 cells exhibit a characteristic behavior of intracellular membranes, involving the formation of a quadruple nuclear membrane in early prometaphase and its disassembly during late prometaphase. After nuclear envelope disassembly, the mitotic apparatus becomes encased by a discontinuous network of ER membranes that associate with mitochondria preventing their diffusion into the spindle area. We also observed a peculiar metaphase spindle organization. We found that kinetochores with attached k-fibers are almost invariably associated with lateral MT bundles that can be either interpolar bundles or k-fibers connected to a different kinetochore. This spindle organization is likely to favor chromosome alignment at metaphase and subsequent segregation during anaphase. In summary, we describe several previously unknown features of membrane and microtubule organization during S2 cell mitosis. The genetic determinants of these mitotic features of can now be investigated using an RNAi-based approach, which is particularly easy and efficient in S2 cells