Mosaic placental labyrinths containing <i>LMα5</i>−/− trophoblasts and hLMα5-expressing endothelial cells show hLMα5 deposition and normal vascularization.

<p>(A) Schematic diagram of the strategy for forcing expression of hLMα5 in endothelial cells on the <i>LMα5−/−</i> background. Cre recombinase driven by the Tie2 promoter removes a floxed STOP located between the <i>Rosa26</i> promoter and the reverse tetracycline transactivator (rtTA). rtTA binds and activates the tetracycline-inducible TetO₇ promoter in the presence of doxycycline, thereby driving transcription of the hLMα5 cDNA in endothelial cells. (B–G) <i>LMα5−/−;ROSA26TA;hLMα5;Tie2cre</i> embryos (top panels) were compared with <i>LMα5−/−</i> embryos (bottom panels). Mouse LMα5 was undetectable in kidney (B, B′) or placenta (E, E′). Human LMα5 was detected in both kidney and placental vasculatures of <i>LMα5−/−;ROSA26TA;hLMα5;Tie2cre</i> embryos (C, F) but not of <i>LMα5−/−</i> embryos (C′,F′), both of which show the typical <i>LMα5</i> null phenotype (D, D′). Expression of hLMα5 in endothelial cells was associated with a normalized placental labyrinth architecture, demonstrated by the LM-111 antibody staining pattern (compare G and G′).</p

Mosaic placental labyrinths containing wild-type trophoblasts and <i>LMα5</i>−/− endothelial cells show LMα5 deposition and normal vascularization.

<p>(A, B) Schematic diagrams of the strategy for conditional mouse <i>LMα5</i> mutation. Using the Cre/loxP system, we generated <i>LMα5^flox/ko</i>; Sox2Cre embryos. Sox2cre, when inherited from a male, is active in epiblast, but not in trophectoderm. Thus, epiblast-derived cells (A), which include the embryo proper as well as extraembryonic endothelial cells, are not able to synthesize LMα5, but trophoblasts, which derive from trophectoderm (B), can. (C–H; C′–H′) Analysis of LMα5 expression and tissue architecture in control (top rows) and <i>LMα5^flox/ko</i>; Sox2cre mutant (bottom rows) embryos. LMα5 was not expressed in the kidney of <i>LMα5^flox/ko</i>; Sox2cre embryos (C′; counterstained with anti-nidogen in D′; compare with control, C and D), which show developmental abnormalities typical of <i>LMα5</i>−/− embryos (E′; arrows indicate exencephaly and syndactyly) not seen in control (E). In contrast, LMα5 was present in the placental labyrinth of <i>LMα5^flox/ko</i>; Sox2cre embryos (F′) and of control (F), and placental LM-111 and PECAM expression and localization were similar to those observed in control <i>LMα5+/</i>− placenta (G–H, G′–H′). Cytokeratin 8 (CK8) was used to identify trophoblasts (G, G′).</p

LMα5 is expressed in both endothelial cells and trophoblasts in the normal placenta.

<p>(A) Fluorescence-activated cell sorting was performed on dissociated E18.5 wild-type labyrinth cells after staining with a phycoerythrin (PE)-conjugated CD31/PECAM antibody. CD31+ (endothelial cell) and CD31− (trophoblast; indicated as baseline) populations were collected. (B) RT-PCR using RNA prepared from the two cell types showed that LMα5 was expressed in both: Lane 1, DNA marker; 2 and 3, LMα5 in CD31(−) and (+) cells, respectively; 4, negative control; 5 and 6, GAPDH in CD31(−) and (+) cells, respectively. (C) RNA was subjected to real time RT-PCR to quantitate the levels of laminin α1 (lama1), α5 (lama5), β1 (lamb1), and β2 (lamb2) mRNAs. Error bars represent standard deviations.</p

Analysis of placental labyrinth vasculature at E14.5.

<p>Frozen sections of placenta were stained with antibodies to LM-111 to label all basement membranes, to cytokeratin 8 (CK8) to label trophoblasts (green in A–C, A′–C′), and to PECAM to label endothelial cells (green in D–F, D′–F′). The reduced vascular complexity in the <i>LMα5−/−</i> labyrinth (B, E) was rescued and made similar to normal (A, D) by hLMα5 secretion from <i>LMα5−/−;ROSA26TA;hLMα5;Tie2cre</i> endothelial cells (C, F) exposed to doxycycline.</p

<i>Scribble^Δpodocyte</i> mice develop normal podocyte foot processes and show no increased susceptibility to glomerular stress.

<p>(<b>A, B</b>) No obvious histological abnormalities can be detected in PAS staining of <i>Scribble^Δpodocyte</i> kidney sections at P1 and at 6 weeks of age compared to control littermates. (<b>C, D</b>) Transmission electron micrographs display normal podocyte architecture with foot processes without any obvious ultrastructural defect. (<b>E</b>) No difference in the number of podocytes per sectioned glomerulus could be detected (ns, not significant, n = 3 each, 30 glomeruli for each mouse were analyzed). (<b>F</b>) During a one year follow up <i>Scribble^Δpodocyte</i> mice develop no significant albuminuria. (<b>G</b>) No difference in albuminuria can be detected between <i>Scribble^Δpodocyte</i> mice and control littermates in the BSA-overload model (n = 5 each) and (<b>H</b>) the ADR model (n = 6 each). Scale bars: 20 µm in (A) and (B), 1 µm in (C) and (D).</p

Multiplexed Imaging of Nanoparticles in Tissues Using Laser Desorption/Ionization Mass Spectrometry

Imaging of nanomaterials in biological tissues provides vital information for the development of nanotherapeutics and diagnostics. Multiplexed imaging of different nanoparticles (NPs) greatly reduces costs, the need to use multiple animals, and increases the biodistribution information that can enhance diagnostic applications and accelerate the screening of potential therapeutics. Various approaches have been developed for imaging NPs; however, the readout of existing imaging techniques relies on specific properties of the core material or surface ligands, and these techniques are limited because of the relatively small number of NPs that can be simultaneously measured in a single experiment. Here, we demonstrate the use of laser desorption/ionization mass spectrometry (LDI-MS) in an imaging format to investigate surface chemistry dictated intraorgan distribution of NPs. This new LDI-MS imaging method enables multiplexed imaging of NPs with potentially unlimited readouts and without additional labeling of the NPs. It provides the capability to detect and image attomole levels of NPs with almost no interferences from biomolecules. Using this new imaging approach, we find that the intraorgan distributions of same-sized NPs are directly linked to their surface chemistry

Migration of apical and basolateral polarity proteins during podocyte differentiation.

<p>Frozen kidney sections of newborn Wistar rat (P0) were stained using antibodies against the apical membrane protein Podocalyxin, the apical polarity protein Par3 and the basolateral polarity protein Scribble and were subjected to confocal laser microscopy. Since glomerular development is asynchronous, kidneys of newborn rats display various glomerular developmental stages. Each panel displays the expression pattern of the accordant proteins during glomerular development (from left to right): Developmental stages ranging from comma-shaped body (I), s-shaped body (II), capillary loop stage (III to IV), to a maturing glomerulus (V). (<b>A</b>) Whereas Par3 is expressed during comma-shaped body stage and localizes to the apical sited cell-cell junctions, expression of Podocalyxin starts during s-shaped body stage, when Par3 and the cell-cell contacts translocate along the lateral side of immature podocytes to basal. During this translocation the apical membrane area, marked by Podocalyxin, increases while the basolateral membrane area shrinks relatively. Arrows indicate translocation of Par3 from the apical cell-cell contacts in I to the developing foot processes in V. (<b>B</b>) Scribble localizes basal of Par3 at the cell-cell junctions and at the basolateral membrane during comma-shaped body stage (I) and translocates like Par3 during podocyte differentation to the developing foot processes in V. (<b>C</b>) While Podocalyxin and Par3 as well as Par3 and Scribble display an partial overlap of their localization (yellow in A and B), no overlap of Podocalyxin and Scribble can be detected indicating a localization to completely distinct membrane areas with Podocalyxin as an apical membrane marker and Scribble as a basolateral marker protein. Scale bars: 5 µm.</p

PARK7 modulates autophagic proteolysis through binding to the N-terminally arginylated form of the molecular chaperone HSPA5

<p>Macroautophagy is induced under various stresses to remove cytotoxic materials, including misfolded proteins and their aggregates. These protein cargoes are collected by specific autophagic receptors such as SQSTM1/p62 (sequestosome 1) and delivered to phagophores for lysosomal degradation. To date, little is known about how cells sense and react to diverse stresses by inducing the activity of SQSTM1. Here, we show that the peroxiredoxin-like redox sensor PARK7/DJ-1 modulates the activity of SQSTM1 and the targeting of ubiquitin (Ub)-conjugated proteins to macroautophagy under oxidative stress caused by TNFSF10/TRAIL (tumor necrosis factor [ligand] superfamily, member 10). In this mechanism, TNFSF10 induces the N-terminal arginylation (Nt-arginylation) of the endoplasmic reticulum (ER)-residing molecular chaperone HSPA5/BiP/GRP78, leading to cytosolic accumulation of Nt-arginylated HSPA5 (R-HSPA5). In parallel, TNFSF10 induces the oxidation of PARK7. Oxidized PARK7 acts as a co-chaperone-like protein that binds the ER-derived chaperone R-HSPA5, a member of the HSPA/HSP70 family. By forming a complex with PARK7 (and possibly misfolded protein cargoes), R-HSPA5 binds SQSTM1 through its Nt-Arg, facilitating self-polymerization of SQSTM1 and the targeting of SQSTM1-cargo complexes to phagophores. The 3-way interaction among PARK7, R-HSPA5, and SQSTM1 is stabilized by the Nt-Arg residue of R-HSPA5. PARK7-deficient cells are impaired in the targeting of R-HSPA5 and SQSTM1 to phagophores and the removal of Ub-conjugated cargoes. Our results suggest that PARK7 functions as a co-chaperone for R-HSPA5 to modulate autophagic removal of misfolded protein cargoes generated by oxidative stress.</p

Development of podocyte foot processes in cultured embryonic <i>Scribble</i> knockout kidneys and in <i>circletail</i> mutant mice.

<p>(<b>A</b>) Western blot of total embryo lysates shows complete loss of Scribble protein in <i>Scribble</i> knockout embryos (<i>Scribble^−/−</i>) (ns, non specific). (<b>B</b>) Kidneys of <i>Scribble</i> knockout and wildtype littermate embryos were harvested at P12.5 and grew in DMEM medium for 6 days. (<b>C</b>) Immunofluorescence staining against WT1, which is expressed in podocytes and embryonic kidney epithelial cells, and Scribble reveals complete loss of Scribble in <i>Scribble</i> knockout kidney culture (arrows indicate glomeruli), (<b>D</b>) while expression of the podocyte marker proteins Podocin and Nephrin as well as the apical polarity protein Par3 can be detected in <i>Scribble</i> knockout kidney culture glomeruli. (<b>E</b>) Electron micrographs show development of podocyte foot processes connected by slit diaphragms in <i>Scribble</i> knockout and wildtype kidney culture glomeruli (P, podocyte; GBM, glomerular basement membrane). Arrow heads indicate localization of foot processes, arrows indicate slit diaphragms. (<b>F</b>) <i>Circletail</i> mutant mice, bearing a mutation in the <i>Scribble</i> gene, which results in a shortened protein lacking third and fourth c-terminal PDZ domains, die during late embryonic development. Kidneys of <i>Crc/Crc</i> mutant mice and control littermates where harvested for electron microscopy at E18.5. Transmission electron micrographs display podocytes with normal foot process architecture and slit-diaphragms and without any obvious abnormalities. Scale bars: 400 µm in (B), 20 µm in (C) and (D), 2 µm in (E) left and middle panel, 500 nm in (E) right panel, 2 µm in (F) upper panel, and 500 nm in (F) lower panel.</p

Dual-Mode Mass Spectrometric Imaging for Determination of <i>in Vivo</i> Stability of Nanoparticle Monolayers

Effective correlation of the <i>in vitro</i> and <i>in vivo</i> stability of nanoparticle-based platforms is a key challenge in their translation into the clinic. Here, we describe a dual imaging method that site-specifically reports the stability of monolayer-functionalized nanoparticles <i>in vivo</i>. This approach uses laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) imaging to monitor the distributions of the nanoparticle core material and laser desorption/ionization mass spectrometry (LDI-MS) imaging to report on the monolayers on the nanoparticles. Quantitative comparison of the images reveals nanoparticle stability at the organ and suborgan level. The stability of particles observed in the spleen was location-dependent and qualitatively similar to <i>in vitro</i> studies. In contrast, <i>in vivo</i> stability of the nanoparticles in the liver differed dramatically from <i>in vitro</i> studies, demonstrating the importance of <i>in vivo</i> assessment of nanoparticle stability