Die Rattenfänger von Balingen : wie die Bundeswehr mit Musik neuen Nachwuchs sucht

<div><p>Nuclear lamins are important structural and functional proteins in mammalian cells, but little is known about the mechanisms and cofactors that regulate their traffic into the nucleus. Here, we demonstrate that trafficking of lamin A, but not lamin B1, and its assembly into the nuclear envelope are regulated by sorting nexin 6 (SNX6), a major component of the retromer that targets proteins and other molecules to specific subcellular locations. SNX6 interacts with lamin A <i>in vitro</i> and <i>in vivo</i> and links it to the outer surface of the endoplasmic reticulum in human and mouse cells. SNX6 transports its lamin A cargo to the nuclear envelope in a process that takes several hours. Lamin A protein levels in the nucleus augment or decrease, respectively, upon gain or loss of SNX6 function. We further show that SNX6-dependent lamin A nuclear import occurs across the nuclear pore complex via a RAN-GTP-dependent mechanism. These results identify SNX6 as a key regulator of lamin A synthesis and incorporation into the nuclear envelope.</p></div

Study of viability and expression of stem-cell markers of cells treated with MG132 and RA.

<p>(<b>A</b>) Quantitative data of CD34-expressing cells (+) or non-expressing cells (-) that are dead or remain alive after the treatment at completion of a 3 day treatment. (<b>B</b>) Quantification of Nanog-expressing (+) or non-expressing cells (-) at completion of a 3 day treatment. (<b>C</b>) Confocal microscopy analysis of Oct4 stained cells at completion of a 3 day treatment. Scale bar = 15μm.</p

Effects of the combined RA/MG132 treatment on the expression stem cell- related markers.

<p>(<b>A</b>) Western blot analysis at completion of a 3 day treatment with (+) or without (-) RA and/or MG132, as indicated, and (<b>B</b>) at completion of a 3 day treatment plus 5 additional days in the absence of compounds.</p

Pre- and posttreatment levels of biomarkers in patients and in controls.

<p>A: Circulating endothelial cells (CECs), B: Microparticles (MPs), C: Endogen thrombin generation (TG); D: Procoagulant phospholipid-dependent clotting time (PPLCT). Marker levels and their standard deviations are shown for pre-t: pretreatment; post-t: posttreatment; c: controls. Logarithmic transformation of data was made to normalize the distributions. NS: no significant.</p

Effects of the combined RA/MG132 treatment on the expression of Nestin, Oct4 and IR-red viability at completion of a 3 day treatment plus 5 additional days of recovery without compounds.

<p>(<b>A</b>) Flow cytometry dot plots and graphical representation of viable Nestin labelled (+) or not labelled (-) cells. The percentage of cells is indicated in each quadrant. (<b>B</b>) Flow cytometry dot plots and graphical representation of viable Oct4 labelled (+) or not labelled (-) cells. The percentage of cells is indicated in each quadrant.</p

Phospho-specific antibodies (pAIB1) confirm that AIB1 is phosphorylated on Ser728 exclusively during mitosis.

<p>(A) Confocal microscopy analysis of an asynchonous culture of MCF-7 cells stained with DAPI (blue), anti-pAIB1(S728) (Alexa Fluor 488) and anti-β-tubulin (Alexa Fluor 633) to reveal the mitotic spindle. Scale bar: 10 µm. Only mitotic cells stain positively with pAIB1(S728) antibodies and suggest that pAIB1(S728) is excluded from chromatin. (B) Western blot analysis of MCF-7 cells arrested at mitosis with nocodazole. Cell lysates were probed with the indicated antibodies, revealing that anti-pAIB1(S728) antibodies detect exclusively the slower electrophoretic mobility band.</p

Combination of MG132 and RA induces p53-independent apoptosis and RelA inhibition.

<p>(A, B) The apoptosis induced by MG132 and MG132/RA is p53 independent. (<b>A</b>) Luciferase assay performed on SK-N-BE(2) cells and (<b>B</b>) SH-SY5Y cells to assess transcriptional activity of p53 upon treatment. SK-N-BE(2) cells express mutant p53, SH-SY5Y neuroblastoma cells are wildtype for p53. LRU= Luciferase reference unit. (<b>C</b>) Representative western blot showing the accumulation of p53 protein in response to MG132 treatment. MG132 treatment does not induce up-regulation of the p53 responsive gene p21/Waf1 in SK-N-BE(2) cells, (<b>D</b>) in contrast to SH-SY5Y cells. (<b>E</b>, <b>F</b>) Effects of combined RA/MG132 treatment on NF-κB signalling. Both MG132 as well as RA cause significant decrease of nuclear expression of RelA (p65) protein. (<b>E</b>) Immunofluorescent detection of sub-cellular localization of RelA (p65) protein under the different treatment conditions. Arrows indicate RelA (p65) protein in the nucleus. Scale bar = 25μm. (<b>F</b>) Quantitative data. The reduction of the cell fraction with nuclear RelA expression in response to combined treatment is statistically significant (*) as compared to control (p< 0.037), to RA treated cells (<i>p</i>=0.037), but also to MG132 treated cells (<i>p</i>=0.016).</p

Post-translational modification of AIB1 occurs at the entry to mitosis.

<p>(A) Western blot analysis of HeLa cells arrested at the different stages of the cell-cycle as indicated in the scheme. Anti-AIB1 antibodies reveal an electrophoretic mobility shift in mitotic cells (lane 6). Antibodies against indicated cyclins were used to confirm cell-cycle progression through the different stages. β-actin was used as loading control. Chemically-induced cell cycle arrest was produced using the following drugs at the doses and times described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028602#s4" target="_blank">materials and methods</a>: lane 1: cyclosporin A; lane 2: wortmannin; lane 3: L-mimosine; lane 4: hydroxyurea; lane 5: etoposide, and lane 6: nocodazole. (B) Morphology of asynchronous culture of HeLa cells and cells treated with nocodazole to arrest them at the beginning of mitosis. Rounded up cells (light refracting) are cells that have entered mitosis. The increased number of rounded uping cells under nocodazole treatment visually confirms the enriched mitotic population. (C) Western blot analysis of asynchronous growing cells (A), nocodazole-treated cells and mitotic cells (M) isolated from an asynchronous culture by shake-off. Nocodazole-treated cells were divided into rounded up cells (R.up) and those that still remained attached (Adh.) to the surface of the culture dish. (D) Flow cytometry analysis was used to confirm the DNA content of cells treated as indicated in (C).</p

Desphosphorylation of AIB1 occurs at mitosis exit.

<p>(A) Floating MCF7 cells arrested at mitosis with nocodazole were washed and liberated to progress through mitosis for the times indicated, in the presence or absence of 20 µM MG132 and 10 µg/ml cycloheximide (CHX). Western blot analysis was performed for AIB1 to monitor its phosphorylation status. Cyclin B1, protein phosphatase 1 (PP1), and the inhibitory phosphorylation of PP1 (P-PP1) were also assessed. β-actin was used as a loading control. (B) Flow cytometry analysis of MCF-7 cells treated as above was used to confirm cell-cycle progression as determined by DNA content.</p

Clinical characteristics of patients.

<p>KPS: Karnofsky performance scale; S. Biopsy: stereotactic biopsy.</p