Arf6 controls retromer traffic and intracellular cholesterol distribution via a phosphoinositide-based mechanism

Small GTPases play a critical role in membrane traffic. Among them, Arf6 mediates transport to and from the plasma membrane, as well as phosphoinositide signalling and cholesterol homeostasis. Here we delineate the molecular basis for the link between Arf6 and cholesterol homeostasis using an inducible knockout (KO) model of mouse embryonic fibroblasts (MEFs). We find that accumulation of free cholesterol in the late endosomes/lysosomes of Arf6 KO MEFs results from mistrafficking of Niemann–Pick type C protein NPC2, a cargo of the cation-independent mannose-6-phosphate receptor (CI-M6PR). This is caused by a selective increase in an endosomal pool of phosphatidylinositol-4-phosphate (PI4P) and a perturbation of retromer, which controls the retrograde transport of CI-M6PR via sorting nexins, including the PI4P effector SNX6. Finally, reducing PI4P levels in KO MEFs through independent mechanisms rescues aberrant retromer tubulation and cholesterol mistrafficking. Our study highlights a phosphoinositide-based mechanism for control of cholesterol distribution via retromer

Deficiencies of the Lipid-Signaling Enzymes Phospholipase D1 and D2 Alter Cytoskeletal Organization, Macrophage Phagocytosis, and Cytokine-Stimulated Neutrophil Recruitment

Cell migration and phagocytosis ensue from extracellular-initiated signaling cascades that orchestrate dynamic reorganization of the actin cytoskeleton. The reorganization is mediated by effector proteins recruited to the site of activity by locally-generated lipid second messengers. Phosphatidic acid (PA), a membrane phospholipid generated by multiple enzyme families including Phospholipase D (PLD), has been proposed to function in this role. Here, we show that macrophages prepared from mice lacking either of the classical PLD isoforms PLD1 or PLD2, or wild-type macrophages whose PLD activity has been pharmacologically inhibited, display isoform-specific actin cytoskeleton abnormalities that likely underlie decreases observed in phagocytic capacity. Unexpectedly, PA continued to be detected on the phagosome in the absence of either isoform and even when all PLD activity was eliminated. However, a disorganized phagocytic cup was observed as visualized by imaging PA, F-actin, Rac1, an organizer of the F-actin network, and DOCK2, a Rac1 activator, suggesting that PLD-mediated PA production during phagocytosis is specifically critical for the integrity of the process. The abnormal F-actin reorganization additionally impacted neutrophil migration and extravasation from the vasculature into interstitial tissues. Although both PLD1 and PLD2 were important in these processes, we also observed isoform-specific functions. PLD1-driven processes in particular were observed to be critical in transmigration of macrophages exiting the vasculature during immune responses such as those seen in acute pancreatitis or irritant-induced skin vascularization

Accounting entities in culture

The bachelor thesis on Accounting entities in culture deals with the accounting entities in the field of culture, especially with theatres. Its intention is to describe the historical development of these entities and their effective legislation in 2014, to determine their main resources of financing and to answer the question: What is the situation of private theatres in the Czech Republic? The theoretical part is focused on defining entities (theatres) in the field of culture and their historical development. It further describes legal conditions for doing business in the field of culture under commercial law and defines the types of legal forms of business in the Czech Republic. The following section analyses the specifics of the companies operating in the field of culture in terms of sales for tickets and financing of these entities in the form of grants. The practical part is focused on analysis of financial statements of 10 theatres in the Czech Republic in terms of quality reporting. In particular financial statements is analysed financial position and financial management of private theatres. In conclusion there is a brief assessment of the fulfilment of the objectives of the thesis

Bacterial growth in citrus tissue.

<p>Leaf planchets were cut from each infiltration area at 2-d intervals post inoculation to analyze bacterial growth. The values shown are means of three technical repeats with standard deviations.</p

Expression of cellulase genes in <i>X</i>. <i>citri</i> subsp. <i>citri</i>.

<p>(A) RT-PCR analysis of gene transcription in <i>X</i>. <i>citri</i> subsp. <i>citri</i> culturing in nutrient broth medium (NB) and growing in citrus. M, DL2000 marker. (B) Detection of BglC3 and EngXCA secretion using immunoblotting. Protein samples were analyzed by SDS-PAGE and immunoblotted using anti-c-Myc antibodies. TE, Total protein extract; SE, Culture supernatant.</p

Ectopic expression of nine cellulases in <i>Escherichia coli</i> BL21(DE3).

<p>(A) Degradation halos on Luria-Bertani plates supplemented with 0.5% carboxymethyl cellulose and 1.0 mM isopropyl-β-D-thiogalactopyranoside. (B) Diameters of the degradation halos. The tests were repeated three times; the data in the figure are the mean values. Two asterisks in the data column indicated significant differences at <i>P</i> = 0.01 by Student’s <i>t</i> test.</p

Transcriptome analysis of Sinorhizobium meliloti nodule bacteria in nifA mutant background

Tian Z, Zou H, Li J, et al. Transcriptome analysis of Sinorhizobium meliloti nodule bacteria in nifA mutant background. CHINESE SCIENCE BULLETIN. 2006;51(17):2079-2086.Gene expression profiles of a Sinorhizobium meliloti 1021 nifA mutant and wild type nodule bacteria were compared using whole genome microarrays. The results revealed a large scale alteration of gene expression (601 genes) in the nifA minus background. The loss of NifA altered the expression of many functional groups of genes (macromolecular metabolism, TCA cycle and respiration, nodulation and nitrogen fixation) and may lead to quite different life stages of the nodule bacteria. Upregulation of fixK and its associated genes was observed in the nifA mutant nodule bacteria. Additional quantitative real-time PCR experiments revealed that the transcript levels of fixLJ were significantly upshifted in the nifA mutant nodule bacteria. Putative NifA binding sites were predicted by a statistical method in the upstream sequences of 13 differentially regulated genes from the nifA transcriptome