Nuclear lipid droplets identified by electron microscopy of serial sections.

International audienceBACKGROUND: Recent studies have suggested that nuclear lipid droplets (LDs) are organized into domains similar to those of cytoplasmic LDs. As cytoplasmic LDs are formed at the endoplasmic reticulum (ER) membrane, which is structurally continuous with the nuclear envelope, it could be suggested however that nuclear LDs are cytoplamic LDs trapped within an invagination of the nuclear envelope. The resolution of fluorescence confocal microscopy is not sufficiently high to exclude this hypothesis. FINDINGS: We therefore addressed this question by electron microscopy (EM) of serial sections. In human liver tissue, we observed some cytoplamic LDs partly surrounded by the nuclear compartment, but we were also able to identify LDs residing in the nuclear compartment that were not connected to the nuclear envelope. CONCLUSION: These findings indicate that nuclear LDs constitute specific subdomains of the nuclear compartment probably involved in nuclear lipid homeostasis

Clockwise or anticlockwise? Turning the centriole triplets in the right direction!

International audienceCentrosomes are small cytoplasmic macromolecular assemblies composed from two major components, centrioles and pericentriolar material, each with its own complex architecture. This organelle is of interest because it plays a role in a number of fundamental cellular processes and defects in these processes have recently been correlated with variety of human disease. Increasingly, what is known about the structure of this organelle has been overshadowed by the increasing wealth of information on its biochemistry. In this short review, we highlight some of the common centriole structural errors found in the literature and define a set of rules that define centriole structure

Ultrastructural and quantitative analysis of the lipid droplet clustering induced by hepatitis C virus core protein.: HCV-induced lipid droplet clustering

International audienceHepatitis C virus (HCV) release is linked to the formation of lipid droplet (LD) clusters in the perinuclear area of infected cells, induced by the core protein. We used electron microscopy (EM) to monitor and compare the number and size of LD in cells producing the mature and immature forms of the HCV core protein, and 3D EM to reconstruct whole cells producing the mature core protein. Only the mature protein coated the LD and induced their clustering and emergence from endoplasmic reticulum membranes enriched in this protein. We found no particular association between LD clusters and the centrosome in reconstructed cells. The LD clustering induced by the mature core protein was associated with an increase in LD synthesis potentially due, at least in part, to the ability of this protein to coat the LD. These observations provide useful information for further studies of the mechanisms involved in HCV-induced steatosis

Sequential biogenesis of host cell membrane rearrangements induced by hepatitis C virus infection.: HCV-induced membrane rearrangements

International audienceLike most positive-strand RNA viruses, hepatitis C virus (HCV) forms a membrane-associated replication complex consisting of replicating RNA, viral and host proteins anchored to altered cell membranes. We used a combination of qualitative and quantitative electron microscopy (EM), immuno-EM, and the 3D reconstruction of serial EM sections to analyze the host cell membrane alterations induced by HCV. Three different types of membrane alteration were observed: vesicles in clusters (ViCs), contiguous vesicles (CVs), and double-membrane vesicles (DMVs). The main ultrastructural change observed early in infection was the formation of a network of CVs surrounding the lipid droplets. Later stages in the infectious cycle were characterized by a large increase in the number of DMVs, which may be derived from the CVs. These DMVs are thought to constitute the membranous structures harboring the viral replication complexes in which viral replication is firmly and permanently established and to protect the virus against double-stranded RNA-triggered host antiviral responses

Oviductal Extracellular Vesicles Enhance Porcine In Vitro Embryo Development by Modulating the Embryonic Transcriptome

Oviductal extracellular vesicles (oEVs) have been identified as important components of the oviductal fluid (OF) and have been pointed to as key modulators of gamete/embryo-maternal interactions. Here, we determined the functional impact of oEVs on embryo development and the embryonic transcriptome in porcine. Experiment 1 examined the effect of oEVs and OF on embryo development. In vitro-produced embryos were cultured with oEVs or OF for 2 or 7 days using an in vitro sequential system or without supplementation (control). Experiment 2 analyzed transcriptomic alterations of EV-treated embryos versus control and the oEVs RNA cargo by RNA-sequencing. Two days of EV treatment enhanced embryo development over time when compared to other treatments. Different RNA expression profiles between embryos treated with EVs for two or seven days and untreated controls were obtained, with 54 and 59 differentially expressed (DE) genes and six and seven DE miRNAs, respectively. In oEV RNA cargo, 12,998 RNAs and 163 miRNAs were identified. Integrative analyses pointed to specific oEV components that might act as modulators of the embryonic transcriptome, such as S100A11, ANXA2 or miR-21-5p. Overall, the findings suggested that oEVs could be a potential strategy to improve porcine IVP outcomes, particularly by using two days of EV treatment

Correlative Scanning-Transmission Electron Microscopy Reveals that a Chimeric Flavivirus Is Released as Individual Particles in Secretory Vesicles.

International audienceThe intracellular morphogenesis of flaviviruses has been well described, but flavivirus release from the host cell remains poorly documented. We took advantage of the optimized production of an attenuated chimeric yellow fever/dengue virus for vaccine purposes to study this phenomenon by microscopic approaches. Scanning electron microscopy (SEM) showed the release of numerous viral particles at the cell surface through a short-lived process. For transmission electron microscopy (TEM) studies of the intracellular ultrastructure of the small number of cells releasing viral particles at a given time, we developed a new correlative microscopy method: CSEMTEM (for correlative scanning electron microscopy - transmission electron microscopy). CSEMTEM analysis suggested that chimeric flavivirus particles were released as individual particles, in small exocytosis vesicles, via a regulated secretory pathway. Our morphological findings provide new insight into interactions between flaviviruses and cells and demonstrate that CSEMTEM is a useful new method, complementary to SEM observations of biological events by intracellular TEM investigations

Spatio-temporal expression patterns of aurora kinases a, B, and C and cytoplasmic polyadenylation-element-binding protein in bovine oocytes during meiotic maturation.

International audienceMaturation of immature bovine oocytes requires cytoplasmic polyadenylation and synthesis of a number of proteins involved in meiotic progression and metaphase-II arrest. Aurora serine-threonine kinases--localized in centrosomes, chromosomes, and midbody--regulate chromosome segregation and cytokinesis in somatic cells. In frog and mouse oocytes, Aurora A regulates polyadenylation-dependent translation of several mRNAs such as MOS and CCNB1, presumably by phosphorylating CPEB, and Aurora B phosphorylates histone H3 during meiosis. We analyzed the expression of three Aurora kinase genes--AURKA, AURKB, and AURKC--in bovine oocytes during meiosis by reverse transcription followed by quantitative real-time PCR and immunodetection. Aurora A was the most abundant form in oocytes, both at mRNA and protein levels. AURKA protein progressively accumulated in the oocyte cytoplasm during antral follicle growth and in vitro maturation. AURKB associated with metaphase chromosomes. AURKB, AURKC, and Thr-phosphorylated AURKA were detected at a contractile ring/midbody during the first polar body extrusion. CPEB, localized in oocyte cytoplasm, was hyperphosphorylated during prophase/metaphase-I transition. Most CPEB degraded in metaphase-II oocytes and remnants remained localized in a contractile ring. Roscovitine, U0126, and metformin inhibited meiotic divisions; they all induced a decrease of CCNB1 and phospho-MAPK3/1 levels and prevented CPEB degradation. However, only metformin depleted AURKA. The Aurora kinase inhibitor VX680 at 100 nmol/L did not inhibit meiosis but led to multinuclear oocytes due to the failure of the polar body extrusion. Thus, in bovine oocyte meiosis, massive destruction of CPEB accompanies metaphase-I/II transition, and Aurora kinases participate in regulating segregation of the chromosomes, maintenance of metaphase-II, and formation of the first polar body

Solid state synthesis of carbon-encapsulated iron carbide nanoparticles and their interaction with living cells †

Superparamagnetic carbon-encapsulated iron carbide nanoparticles (NPs), Fe 7 C 3 @C, with unique properties, were produced from pure ferrocene by high pressure-high temperature synthesis. These NPs combine the merits of nanodiamonds and SPIONs but lack their shortcomings which limit their use for biomedical applications. Investigation of these NPs by X-ray diffraction, electron microscopy techniques, X-ray spectroscopic and magnetic measurement methods has demonstrated that this method of synthesis yields NPs with perfectly controllable physical properties. Using magnetic and subsequent fractional separation of magnetic NPs from residual carbon, the aqueous suspensions of Fe 7 C 3 @C NPs with an average particle size of $25 nm were prepared. The suspensions were used for in vitro studies of the interaction of Fe 7 C 3 @C NPs with cultured mammalian cells. The dynamics of interaction of the living cells with Fe 7 C 3 @C was studied by optical microscopy using time-lapse video recording and also by transmission electron microscopy. Using novel highly sensitive cytotoxicity tests based on the cell proliferation assay and long-term live cell observations it was shown that the internalization of Fe 7 C 3 @C NPs has no cytotoxic effect on cultured cells and does not interfere with the process of their mitotic division, a fundamental property that ensures the existence of living organisms. The influence of NPs on the proliferative activity of cultured cells was not detected as well. These results indicate that the carbon capsules of Fe 7 C 3 @C NPs are air-tight which could offer great opportunities for future use of these superparamagnetic NPs in biology and medicine

A Journey through Time on the Discovery of Cell Cycle Regulation

All living organisms on Earth are made up of cells, which are the functional unit of life. Eukaryotic organisms can consist of a single cell (unicellular) or a group of either identical or different cells (multicellular). Biologists have always been fascinated by how a single cell, such as an egg, can give rise to an entire organism, such as the human body, composed of billions of cells, including hundreds of different cell types. This is made possible by cell division, whereby a single cell divides to form two cells. During a symmetric cell division, a mother cell produces two daughter cells, while an asymmetric cell division results in a mother and a daughter cell that have different fates (different morphologies, cellular compositions, replicative potentials, and/or capacities to differentiate). In biology, the cell cycle refers to the sequence of events that a cell must go through in order to divide. These events, which always occur in the same order, define the different stages of the cell cycle: G1, S, G2, and M. What is fascinating about the cell cycle is its universality, and the main reason for this is that the genetic information of the cell is encoded by exactly the same molecular entity with exactly the same structure: the DNA double helix. Since both daughter cells always inherit their genetic information from their parent cell, the underlying fundamentals of the cell cycle—DNA replication and chromosome segregation—are shared by all organisms. This review goes back in time to provide a historical summary of the main discoveries that led to the current understanding of how cells divide and how cell division is regulated to remain highly reproducible