Cytoplasme | EPFL Graph Search

Le cytoplasme désigne le contenu d'une cellule vivante et la région comprise entre la membrane plasmique et le noyau d'une cellule eucaryote ou le nucléoïde d'une cellule procaryote. En d'autres termes, le protoplasme délimité par la membrane plasmique est constitué du cytoplasme et du noyau chez les cellules eucaryotes, du cytoplasme et du nucléoïde pour les cellules procaryotes. Il est constitué d'environ 80 % d'eau et se définit comme une émulsion colloïdale à l'aspect granuleux constituée de cytosol, solution aqueuse de sels minéraux et de divers composés organiques qui, avec le cytosquelette, forme le hyaloplasme. Chez les eucaryotes, le cytoplasme comprend plusieurs organites comme les réticulum endoplasmiques, des vacuoles, des mitochondries ou des chloroplastes formant le morphoplasme, mais ne comprend pas le noyau (dont le contenu est appelé nucléoplasme). Il comprend également des ribosomes, qui ne sont pas considérés comme des organites. Chez les procaryotes, le cytoplasme e

How adeno-associated virus Rep78 relocalises Cdc25B to arrest the cell cycle

Florence Magnin

Adeno-associated virus (AAV) is a small DNA virus belonging to the family of the parvoviridae. AAV infects the human population, however no disease has been associated with this virus. On the contrary, AAV was reported to have oncosuppressive activities. The AAV genome contains two genes: the Rep gene encoding four non-structural proteins (Rep78, 68, 52 and 40) and the Cap gene encoding the three capsid proteins. Rep78 is implicated in transcription, replication and site-specific integration of the viral DNA. It has been shown to block the cell cycle in G1, G2 and S phase with a complete inhibition of DNA synthesis. To be able to induce such a strong cell cycle arrest, Rep78 affects several proteins implicated in the regulation of the cell cycle, such as the family of the cell division cycle 25 (Cdc25) phosphatases. Rep78 has been shown to inactivate Cdc25A by binding to it and preventing it from accessing its substrates. Cdc25B shares conserved domains with Cdc25A and moreover has an important role in cell cycle regulation, especially in giving the signal for entry into mitosis. Thus, in this work, the interaction between Cdc25B and Rep78 was examined in more detail. Rep78 was found to bind to Cdc25B and to alter its intracellular localisation. Cdc25B is nuclear, but has to migrate into the cytoplasm at the end of the G2 phase to activate proteins needed for the entry into mitosis. The expression of Rep78, which is nuclear, leads to the sequestration of Cdc25B in the nucleus. Moreover, Rep78 by interacting with Cdc25B not only changes its localisation, but also inhibits its phosphatase activity. Our model is that Rep78 interacts with Cdc25B and thus retains it in the nucleus. This sequestration of Cdc25B in the nucleus prevents it from activating the proteins in the cytoplasm needed to give the entry signal into mitosis, thus contributing to the G2/M arrest induced by Rep78. Moreover, the inhibition of Cdc25B phosphatase activity by Rep78 may also contribute to the cell cycle block. Rep78 also interacts with a number of cellular proteins implicated in DNA replication, DNA repair or recombination. Interactions between Rep78 and mini-chromosome maintenance 3 (MCM3), DNA polymerases α and δ, proliferating cell nuclear antigen (PCNA), Rad50 and Rad51 were identified in this work as well as possible interactions between Rep78 and Ataxia telangiectasia mutated (ATM) or Ataxia telangiectasia and Rad3 related (ATR) proteins. These interactions may contribute to AAV genome replication as well as its integration into the host DNA.

EPFL2009

Characterizing the localization and role of lin-5 and era-1 mRNAs in early C. elegans embryos

Zoltán Péter Spiró

Asymmetric cell division is essential for the generation of diversity during development and the function of stem cell lineages. The Caenorhabditis elegans zygote is an attractive model to investigate the mechanisms of spindle positioning during asymmetric cell division. In this polarized cell, the asymmetric distribution of cortical force generators along the antero-posterior axis and pulling on astral microtubules leads to the unequal cleavage of the one-cell embryo. The mechanisms underlying such cortical force generation are thought to act strictly at the protein level. In this thesis work we report that the mRNA encoding the cortical force generator component LIN-5 is enriched around centrosomes in early embryos, in a manner that depends on microtubules and dynein. We established that the lin-5 coding sequence is necessary and sufficient for mRNA enrichment around centrosomes in C. elegans. In addition, we found that lin-5 mRNA is mislocalized in lin-5(ev571) mutant embryos, which harbor a 9 nucleotide insertion in the coding sequence. Moreover, an intragenic revertant of lin-5(ev571), lin-5(ev571he63), also exhibits mislocalized lin-5 mRNA distribution. We demonstrated that this is accompanied by diminished pulling forces on the posterior spindle pole, suggesting that centrosomal localization of lin-5 mRNA is important for robust pulling forces. We found also that lin-5 mRNA centrosomal enrichment is slightly asymmetric during anaphase, with more transcripts present on the anterior side. We developed a novel FRAP-based assay, which revealed that lin-5 is translated/folded preferentially in the cytoplasm compared to centrosomes. Furthermore, we found that morpholino-mediated inhibition of lin-5 translation diminishes pulling forces on the posterior side during anaphase. Together, these findings lead us to propose that preferential translation/folding of lin-5 in the posterior cytoplasm following release of the mRNA from the posterior centrosome contributes to asymmetric cortical distribution of force generators, and thus to proper spindle positioning. Moreover, we found that the mRNA of an uncharacterized gene, era-1 is enriched on the anterior side of the zygote and is inherited by the anterior blastomeres. Similar to era-1 mRNA, a YFP fusion of ERA-1 protein is also asymmetrically distributed. Moreover, asymmetric distribution of both era-1 mRNA and YFP-ERA-1 protein requires the era-1 3'UTR. Furthermore, the RNA-binding protein MEX-5 is needed for both asymmetric era-1 mRNA localization and for its translational activation. Furthermore, we report that the clathrin heavy chain CHC-1 negatively regulates pulling forces acting on centrosomes during interphase and on spindle poles during mitosis in one-cell C. elegans embryos. We establish a similar role for the cytokinesis/apoptosis/RNA-binding protein CAR-1 and uncover that CAR-1 is needed to maintain normal levels of CHC-1. We demonstrate that CHC-1 is necessary for proper organization of the cortical acto-myosin network and for full cortical tension. Furthermore, we establish that the centrosome positioning phenotype of embryos depleted of CHC-1 is alleviated by stabilizing the acto-myosin network. Conversely, we demonstrate that slight perturbations of the acto-myosin network results in excess centrosome movements. Overall, our findings lead us to propose that clathrin plays a critical role in centrosome positioning by promoting acto-myosin cortical tension.

EPFL2014

3D correlative electron microscopy reveals continuity of Brucella-containing vacuoles with the endoplasmic reticulum

Shyan Huey Low, Henning Paul-Julius Stahlberg

Entry of the facultative intracellular pathogen Brucella into host cells results in the formation of endosomal Brucella-containing vacuoles (eBCVs) that initially traffic along the endocytic pathway. eBCV acidification triggers the expression of a type IV secretion system that translocates bacterial effector proteins into host cells. This interferes with lysosomal fusion of eBCVs and supports their maturation to replicative Brucella-containing vacuoles (rBCVs). Bacteria replicate in rBCVs to large numbers, eventually occupying most of the cytoplasmic volume. As rBCV membranes tightly wrap each individual bacterium, they are constantly being expanded and remodeled during exponential bacterial growth. rBCVs are known to carry endoplasmic reticulum (ER) markers; however, the relationship of the vacuole to the genuine ER has remained elusive. Here, we have reconstructed the 3-dimensional ultrastructure of rBCVs and associated ER by correlative structured illumination microscopy (SIM) and focused ion beam/scanning electron microscopic tomography (FIB/SEM). Studying B. abortus-infected HeLa cells and trophoblasts derived from B. melitensis-infected mice, we demonstrate that rBCVs are complex and interconnected compartments that are continuous with neighboring ER cisternae, thus supporting a model that rBCVs are extensions of genuine ER.

The Company of Biologists2018