Nop2 is expressed during proliferation of neural stem cells and in adult mouse and human brain

The nucleolar protein 2 gene encodes a protein specific for the nucleolus. It is assumed that it plays a role in the synthesis of ribosomes and regulation of the cell cycle. Due to its link to cell proliferation, higher expression of Nop2 indicates a worse tumor prognosis. In this work we used Nop2(gt1gaj) gene trap mouse strain. While lethality of homozygous animals suggested a vital role of this gene, heterozygous animals allowed the detection of expression of Nop2 in various tissues, including mouse brain. Histochemistry, immunohistochemistry and immunoelectron microscopy techniques, applied to a mature mouse brain, human brain and on mouse neural stem cells revealed expression of Nop2 in differentiating cells, including astrocytes, as well as in mature neurons. Nop2 was detected in various regions of mouse and human brain, mostly in large pyramidal neurons. In the human, Nop2 was strongly expressed in supragranular and infragranular layers of the somatosensory cortex and in layer III of the cingulate cortex. Also, Nop2 was detected in CA1 and the subiculum of the hippocampus. Subcellular analyses revealed predominant location of Nop2 within the dense fibrillar component of the nucleolus. To test if Nop2 expression correlates to cell proliferation occurring during tissue regeneration, we induced strokes in mice by middle cerebral artery occlusion. Two weeks after stroke, the number of Nop2/nestin double positive cells in the region affected by ischemia and the periventricular zone substantially increased. Our findings suggest a newly discovered role of Nop2 in both mature neurons and in cells possibly involved in the regeneration of nervous tissue

Biophysical properties of cationic lipophosphoramidates: Vesicle morphology, bilayer hydration and dynamics

International audienceCationic lipids are used to deliver genetic material to living cells. Their proper biophysical characterization is needed in order to design and control this process. In the present work we characterize some properties of recently synthetized cationic lipophosphoramidates. The studied compounds share the same structure of their hydrophobic backbone, but differ in their hydrophilic cationic headgroup, which is formed by a trimethylammonium, a trimethylarsonium or a dicationic moiety. Dynamic light scattering and cryo-transmission electron microscopy proves that the studied lipophosphoramidates create stable unilamellar vesicles. Fluorescence of polarity probe, Laurdan, analyzed using time-dependent fluorescence shift method (TDFS) and generalized polarization (GP) gives important information about the phase, hydration and dynamics of the lipophosphoramidate bilayers. While all of the compounds produced lipid bilayers that were sufficiently fluid for their potential application in gene therapy, their polarity/hydration and mobility was lower than for the standard cationic lipid – DOTAP. Mixing cationic lipophosphoramidates with DOPC helps to reduce this difference. The structure of the cationic headgroup has an important and complex influence on bilayer hydration and mobility. Both TDFS and GP methods are suitable for the characterization of cationic amphiphiles and can be used for screening of the newly synthesized compounds

Quantitative Proteomics Analysis of Macrophage-Derived Lipid Rafts Reveals Induction of Autophagy Pathway at the Early Time of Francisella tularensis LVS Infection

Francisella tularensis is a highly infectious intracellular pathogen that has evolved an efficient strategy to subvert host defense response to survive inside the host. The molecular mechanisms regulating these host–pathogen interactions and especially those that are initiated at the time of the bacterial entry via its attachment to the host plasma membrane likely predetermine the intracellular fate of pathogen. Here, we provide the evidence that infection of macrophages with F. tularensis leads to changes in protein composition of macrophage-derived lipid rafts, isolated as detergent-resistant membranes (DRMs). Using SILAC-based quantitative proteomic approach, we observed the accumulation of autophagic adaptor protein p62 at the early stages of microbe–host cell interaction. We confirmed the colocalization of the p62 with ubiquitinated and LC3-decorated intracellular F. tularensis microbes with its maximum at 1 h postinfection. Furthermore, the infection of p62-knockdown host cells led to the transient increase in the intracellular number of microbes up to 4 h after in vitro infection. Together, these data suggest that the activation of the autophagy pathway in F. tularensis infected macrophages, which impacts the early phase of microbial proliferation, is subsequently circumvented by ongoing infection