Podocyte-Specific Sialylation-Deficient Mice Serve as a Model for Human FSGS.

BACKGROUND: The etiology of steroid-resistant nephrotic syndrome, which manifests as FSGS, is not completely understood. Aberrant glycosylation is an often underestimated factor for pathologic processes, and structural changes in the glomerular endothelial glycocalyx have been correlated with models of nephrotic syndrome. Glycans are frequently capped by sialic acid (Sia), and sialylation's crucial role for kidney function is well known. Human podocytes are highly sialylated; however, sialylation's role in podocyte homeostasis remains unclear. METHODS: We generated a podocyte-specific sialylation-deficient mouse model (PCmas-/- ) by targeting CMP-Sia synthetase, and used histologic and ultrastructural analysis to decipher the phenotype. We applied CRISPR/Cas9 technology to generate immortalized sialylation-deficient podocytes (asialo-podocytes) for functional studies. RESULTS: Progressive loss of sialylation in PCmas-/- mice resulted in onset of proteinuria around postnatal day 28, accompanied by foot process effacement and loss of slit diaphragms. Podocyte injury led to severe glomerular defects, including expanded capillary lumen, mesangial hypercellularity, synechiae formation, and podocyte loss. In vivo, loss of sialylation resulted in mislocalization of slit diaphragm components, whereas podocalyxin localization was preserved. In vitro, asialo-podocytes were viable, able to proliferate and differentiate, but showed impaired adhesion to collagen IV. CONCLUSIONS: Loss of cell-surface sialylation in mice resulted in disturbance of podocyte homeostasis and FSGS development. Impaired podocyte adhesion to the glomerular basement membrane most likely contributed to disease development. Our data support the notion that loss of sialylation might be part of the complex process causing FSGS. Sialylation, such as through a Sia supplementation therapy, might provide a new therapeutic strategy to cure or delay FSGS and potentially other glomerulopathies.

Iron-regulatory proteins secure iron availability in cardiomyocytes to prevent heart failure.

Aims: Iron deficiency (ID) is associated with adverse outcomes in heart failure (HF) but the underlying mechanisms are incompletely understood. Intracellular iron availability is secured by two mRNA-binding iron-regulatory proteins (IRPs), IRP1 and IRP2. We generated mice with a cardiomyocyte-targeted deletion of Irp1 and Irp2 to explore the functional implications of ID in the heart independent of systemic ID and anaemia. Methods and results: Iron content in cardiomyocytes was reduced in Irp-targeted mice. The animals were not anaemic and did not show a phenotype under baseline conditions. Irp-targeted mice, however, were unable to increase left ventricular (LV) systolic function in response to an acute dobutamine challenge. After myocardial infarction, Irp-targeted mice developed more severe LV dysfunction with increased HF mortality. Mechanistically, the activity of the iron-sulphur cluster-containing complex I of the mitochondrial electron transport chain was reduced in left ventricles from Irp-targeted mice. As demonstrated by extracellular flux analysis in vitro, mitochondrial respiration was preserved at baseline but failed to increase in response to dobutamine in Irp-targeted cardiomyocytes. As shown by 31P-magnetic resonance spectroscopy in vivo, LV phosphocreatine/ATP ratio declined during dobutamine stress in Irp-targeted mice but remained stable in control mice. Intravenous injection of ferric carboxymaltose replenished cardiac iron stores, restored mitochondrial respiratory capacity and inotropic reserve, and attenuated adverse remodelling after myocardial infarction in Irp-targeted mice but not in control mice. As shown by electrophoretic mobility shift assays, IRP activity was significantly reduced in LV tissue samples from patients with advanced HF and reduced LV tissue iron content. Conclusions: ID in cardiomyocytes impairs mitochondrial respiration and adaptation to acute and chronic increases in workload. Iron supplementation restores cardiac energy reserve and function in iron-deficient hearts.

Pulmonary transplantation of macrophage progenitors as effective and long-lasting therapy for hereditary pulmonary alveolar proteinosis.

Hereditary pulmonary alveolar proteinosis (herPAP) is a rare lung disease caused by mutations in the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor genes, resulting in disturbed alveolar macrophage differentiation, massive alveolar proteinosis, and life-threatening respiratory insufficiency. So far, the only effective treatment for herPAP is repetitive whole-lung lavage, a merely symptomatic and highly invasive procedure. We introduce pulmonary transplantation of macrophage progenitors as effective and long-lasting therapy for herPAP. In a murine disease model, intrapulmonary transplanted macrophage progenitors displayed selective, long-term pulmonary engraftment and differentiation into functional alveolar macrophages. A single transplantation ameliorated the herPAP phenotype for at least 9 months, resulting in significantly reduced alveolar proteinosis, normalized lung densities in chest computed tomography, and improved lung function. A significant and sustained disease resolution was also observed in a second, humanized herPAP model after intrapulmonary transplantation of human macrophage progenitors. The therapeutic effect was mediated by long-lived, lung-resident macrophages, which displayed functional and phenotypical characteristics of primary human alveolar macrophages. Our findings present the concept of organotopic transplantation of macrophage progenitors as an effective and long-lasting therapy of herPAP and may also serve as a proof of principle for other diseases, expanding current stem cell-based strategies toward potent concepts using the transplantation of differentiated cells.

Standardized, App-based disinfection of iPads in a clinical and nonclinical setting: comparative analysis.

BACKGROUND: With the use of highly mobile tools like tablet PCs in clinical settings, an effective disinfection method is a necessity. Since manufacturers do not allow cleaning methods that make use of anything but a dry fleece, other approaches have to be established to ensure patient safety and to minimize risks posed by microbiological contamination. OBJECTIVE: The ability of isopropanol wipes to decontaminate iPads was evaluated prospectively in a observer blinded, comparative analysis of devices used in a clinical and a nonclinical setting. METHODS: 10 new iPads were randomly deployed to members of the nursing staff of 10 clinical wards, to be used in a clinical setting over a period of 4 weeks. A pre-installed interactive disinfection application (deBac-app, PLRI MedAppLab, Germany) was used on a daily basis. Thereafter, the number and species of remaining microorganisms on the surface of the devices (13 locations; front and back) was evaluated using contact agar plates. Following this, the 10 iPads were disinfected and randomly deployed to medical informatics professionals who also used the devices for 4 weeks but were forbidden to use disinfecting agents. The quality of a single, standardized disinfection process was then determined by a final surface disinfection process of all devices in the infection control laboratory. No personal data were logged with the devices. The evaluation was performed observer blinded with respect to the clinical setting they were deployed in and personnel that used the devices. RESULTS: We discovered a 2.7-fold (Mann-Whitney U test, z=-3.402, P=.000670) lower bacterial load on the devices used in the clinical environment that underwent a standardized daily disinfection routine with isopropanol wipes following the instructions provided by "deBac-app". Under controlled conditions, an average reduction of the mainly Gram-positive normal skin microbiological load of 99.4% (Mann-Whitney U test, z=-3.1798, P=.001474) for the nonclinical group and 98.1% (Mann-Whitney U test, z=3.1808, P=.001469) for the clinical group was achieved using one complete disinfecting cycle. CONCLUSIONS: Normal use of tablet PCs leads to a remarkable amount of microbial surface contamination. Standardized surface disinfection with isopropanol wipes as guided by the application significantly reduces this microbial load. When performed regularly, the disinfection process helps with maintaining a low germ count during use. This should reduce the risk of subsequent nosocomial pathogen transmission. Unfortunately, applying a disinfection procedure such as the one we propose may lead to losing the manufacturer's warranty for the devices; this remains an unsolved issue.

Mitogen-activated protein kinase-activated protein kinases 2 and 3 regulate SERCA2a expression and fiber type composition to modulate skeletal muscle and cardiomyocyte function.

The mitogen-activated protein kinase (MAPK)-activated protein kinases 2 and 3 (MK2/3) represent protein kinases downstream of the p38 MAPK. Using MK2/3 double-knockout (MK2/3(-/-)) mice, we analyzed the role of MK2/3 in cross-striated muscle by transcriptome and proteome analyses and by histology. We demonstrated enhanced expression of the slow oxidative skeletal muscle myofiber gene program, including the peroxisome proliferator-activated receptor gamma (PPARγ) coactivator 1α (PGC-1α). Using reporter gene and electrophoretic gel mobility shift assays, we demonstrated that MK2 catalytic activity directly regulated the promoters of the fast fiber-specific myosin heavy-chain IId/x and the slow fiber-specific sarco/endoplasmic reticulum Ca(2+)-ATPase 2 (SERCA2) gene. Elevated SERCA2a gene expression caused by a decreased ratio of transcription factor Egr-1 to Sp1 was associated with accelerated relaxation and enhanced contractility in MK2/3(-/-) cardiomyocytes, concomitant with improved force parameters in MK2/3(-/-) soleus muscle. These results link MK2/3 to the regulation of calcium dynamics and identify enzymatic activity of MK2/3 as a critical factor for modulating cross-striated muscle function by generating a unique muscle phenotype exhibiting both reduced fatigability and enhanced force in MK2/3(-/-) mice. Hence, the p38-MK2/3 axis may represent a novel target for the design of therapeutic strategies for diseases related to fiber type changes or impaired SERCA2 function.

Deficits in sialylation impair podocyte maturation.

The role of sialylation in kidney biology is not fully understood. The synthesis of sialoglycoconjugates, which form the outermost structures of animal cells, requires CMP-sialic acid, which is a product of the nuclear enzyme CMAS. We used a knock-in strategy to create a mouse with point mutations in the canonical nuclear localization signal of CMAS, which relocated the enzyme to the cytoplasm of transfected cells without affecting its activity. Although insufficient to prevent nuclear entry in mice, the mutation led to a drastically reduced concentration of nuclear-expressed enzyme. Mice homozygous for the mutation died from kidney failure within 72 hours after birth. The Cmas(nls) mouse exhibited podocyte foot process effacement, absence of slit diaphragms, and massive proteinuria, recapitulating features of nephrin-knockout mice and of patients with Finnish-type congenital nephrotic syndrome. Although the Cmas(nls) mouse displayed normal sialylation in all organs including kidney, a critical shortage of CMP-sialic acid prevented sialylation of nephrin and podocalyxin in the maturing podocyte where it is required during the formation of foot processes. Accordingly, the sialylation defects progressed with time and paralleled the morphologic changes. In summary, sialylation is critical during the development of the glomerular filtration barrier and required for the proper function of nephrin. Whether altered sialylation impairs nephrin function in human disease requires further study.

A tumor-derived population (SCCOHT-1) as cellular model for a small cell ovarian carcinoma of the hypercalcemic type.

The small cell ovarian carcinoma of the hypercalcemic type (SCCOHT) represents an aggressive tumor with poor prognosis predominantly affecting young women and so far, no cell line or animal model is available to investigate this devastating disease. Biopsy material from a recurrent SCCOHT was subjected to an explant culture to obtain an adherent and continuously proliferating cell population. Morphological and functional characterization revealed a heterogeneous population (SCCOHT-1) of about 13 µm in diameter and approximately 36 h of doubling time. Flow cytometric analysis of surface markers demonstrated the expression of CD15, CD29, CD44 and CD90 paralleled by the presence of cytokeratins and vimentin. Cytogenetic analysis and high-resolution oligo-array comparative genomic hybridization (aCGH) demonstrated a stable karyotype including deletions of the PARK2, CSMD1, GRIN2B and ATF7IP genes. Following lentiviral transduction with a GFP vector, the labeled SCCOHT-derived cells were subjected to CCE to separate distinct subpopulations as evidenced by cell cycle analysis. Subcutaneous injection of these subpopulations into NOD/SCID mice exhibited hypercalcemia and a tumor development in 100% of the mice. Re-cultivation of the mouse tumors revealed an outgrowth of SCCOHT-derived phenotypes and all cell populations expressed high telomerase activity. Moreover, histopathological evaluation demonstrated close similarities between the mouse tumors and the original patient tumor. In conclusion, SCCOHT-1 cells provide a study platform to investigate this rare disease and to examine effective and sufficient therapeutic strategies for this rather unknown type of cancer.

Differential regulation of node formation, nodal ciliogenesis and cilia positioning by Noto and Foxj1.

The mouse transcription factor Noto is expressed in the node and controls node morphogenesis, formation of nodal cilia and left-right asymmetry. Noto acts upstream of Foxj1, which regulates ciliogenesis in other mouse tissues. However, the significance of Foxj1 for the formation of cilia in the mouse node is unclear; in non-amniote species Foxj1 is required for ciliogenesis in the structures equivalent to the node. Here, we analyzed nodes, nodal cilia and nodal flow in mouse embryos in which we replaced the Noto-coding sequence with that of Foxj1, or in embryos that were deficient for Foxj1. We show that Foxj1 expressed from the Noto locus is functional and restores the formation of structurally normal motile cilia in the absence of Noto. However, Foxj1 is not sufficient for the correct positioning of cilia on the cell surface within the plane of the nodal epithelium, and cannot restore normal node morphology. We also show that Foxj1 is essential for ciliogenesis upstream of Rfx3 in the node. Thus, the function of Foxj1 in vertebrate organs of asymmetry is conserved, and Noto regulates node morphogenesis and the posterior localization of cilia on node cells independently of Foxj1.

Kir4.1 channel expression is essential for parietal cell control of acid secretion.

Kir4.1 channels were found to colocalize with the H(+)/K(+)-ATPase throughout the parietal cell (PC) acid secretory cycle. This study was undertaken to explore their functional role. Acid secretory rates, electrophysiological parameters, PC ultrastructure, and gene and protein expression were determined in gastric mucosae of 7-8-day-old Kir4.1-deficient mice and WT littermates. Kir4.1(-/-) mucosa secreted significantly more acid and initiated secretion significantly faster than WT mucosa. No change in PC number but a relative up-regulation of H(+)/K(+)-ATPase gene and protein expression (but not of other PC ion transporters) was observed. Electron microscopy revealed fully fused canalicular membranes and a lack of tubulovesicles in resting state Kir4.1(-/-) PCs, suggesting that Kir4.1 ablation may also interfere with tubulovesicle endocytosis. The role of this inward rectifier in the PC apical membrane may therefore be to balance between K(+) loss via KCNQ1/KCNE2 and K(+) reabsorption by the slow turnover of the H(+)/K(+)-ATPase, with consequences for K(+) reabsorption, inhibition of acid secretion, and membrane recycling. Our results demonstrate that Kir4.1 channels are involved in the control of acid secretion and suggest that they may also affect secretory membrane recycling.

A comparison of GFP-tagged clathrin light chains with fluorochromated light chains in vivo and in vitro.

Clathrin triskelia consist of three heavy chains and three light chains (LCs). Green fluorescent protein (GFP)-tagged LCs are widely utilized to follow the dynamics of clathrin in living cells, but whether they reflect faithfully the behavior of clathrin triskelia in cells has not been investigated yet thoroughly. As an alternative approach, we labeled purified LCs either with Alexa 488 or Cy3 dye and compared them with GFP-tagged LC variants. Cy3-labeled light chains (Cy3-LCs) were microinjected into HeLa cells either directly or in association with heavy chains. Within 1-2 min the Cy3-LC heavy chain complexes entered clathrin-coated structures, whereas uncomplexed Cy3-LC did not within 2 h. These findings show that no significant exchange of LCs occurs over the time-course of an endocytic cycle. To explore whether GFP-tagged LCs behave functionally like endogenous LCs, we characterized them biochemically. Unlike wild-type LCs, recombinant LCs with a GFP attached to either end did not efficiently inhibit clathrin assembly in vitro, whereas Cy3- and Alexa 488-labeled LC behaved similar to wild-type LCs in vitro and in vivo. Thus, fluorochromated LCs are a valuable tool for investigating the complex behavior of clathrin in living cells.

Carbon dioxide modifies the morphology and function of mesothelial cells and facilitates transepithelial neuroblastoma cell migration.

BACKGROUND: The response of mesothelial cells to surgical trauma and bacterial contamination is poorly defined. We have recently shown that CO(2) pneumoperitoneum increases systemic metastasis of neuroblastoma cells in a murine model. Thus, we hypothesized that CO(2) alters the morphology and function of mesothelial cells and facilitates transmesothelial tumor cell migration. MATERIALS AND METHODS: Murine mesothelial cells were exposed to 100% CO(2) and 5% CO(2) as control. Scanning electron microscopy (SEM) investigations, as well as LPS-induced granulocyte-colony stimulating factor (G-CSF) production and mitochondrial activity (MTT assay) were measured. Transmesothelial migration of neuroblastoma cells (Neuro2a) was determined using a transwell chamber system. RESULTS: CO(2) incubation was associated with a significant destruction of the microvillar formation in SEM. Migration studies showed that the barrier function of the mesothelial monolayer decreased. A significantly increased migration of neuroblastoma cells was identified after 100% CO(2) exposure (P < 0.05). Although the conversion of MTT as an indicator of mitochondrial activity was only slightly and not significantly reduced after CO(2) incubation, the release of G-CSF induced by LPS was completely blocked during the incubation with 100% CO(2) (P < 0.05). The capacity of G-CSF release recovered after the incubation. CONCLUSION: We observed that peritoneal mesothelial cells lose their typical cell morphology by CO(2) incubation, which is accompanied by facilitated migration of neuroblastoma cells. Moreover, the synthesis of immunological factors is blocked, but this effect is not long lasting. These mechanisms may explain an increased metastasis rate of neuroblastoma cells after CO(2) pneumoperitoneum, which was recently observed in a murine model.

KCNQ1 is the luminal K+ recycling channel during stimulation of gastric acid secretion.

Parietal cell (PC) proton secretion via H(+)/K(+)-ATPase requires apical K(+) recycling. A variety of K(+) channels and transporters are expressed in the PC and the molecular nature of the apical K(+) recycling channel is under debate. This study was undertaken to delineate the exact function of KCNQ1 channels in gastric acid secretion. Acid secretory rates and electrophysiological parameters were determined in gastric mucosae of 7- to 8-day-old KCNQ1(+/+), (+/-) and (-/-) mice. Parietal cell ultrastructure, abundance and gene expression levels were quantified. Glandular structure and PC abundance, and housekeeping gene expression did not differ between the KCNQ1(-/-) and (+/+) mucosae. Microvillar secretory membranes were intact, but basal acid secretion was absent and forskolin-stimulated acid output reduced by approximately 90% in KCNQ1(-/-) gastric mucosa. Application of a high K(+) concentration to the luminal membrane restored normal acid secretory rates in the KCNQ1(-/-) mucosa. The study demonstrates that the KCNQ1 channel provides K(+) to the extracellular K(+) binding site of the H(+)/K(+)-ATPase during acid secretion, and no other gastric K(+) channel can substitute for this function.

New components of 'basal laminar deposits' in age-related macular degeneration.

Two new components of basal laminar deposit (BlamD) occurring in samples of submacular neovascular membranes surgically removed from patients with a wet (exudative) form of age-related macular degeneration are described. They are: (1) minute ribbon-like structures which occur singly and/or in a bunch and extend from the inner surface of the BlamD layer into the extracellular matrix (ECM) beneath the retinal pigment epithelium (RPE). The ribbons are composed of polarized molecules, aggregating in parallel, aligned transversally in register, morphologically similar to isolated collagen molecules of the short-chain type. Deeper in the BlamD but always close to its inner surface, aspects suggesting a transition between ribbons and (2) long-spacing collagen (LSC)-like aggregates characterized by periods bordered by a single dense band were observed. This band could arise from the globular domains of the polarized monomers, which assemble in parallel and display all their terminal extensions at the same end of each period resulting in the single dense band. The presence of ribbons and of LSC-like aggregates in the BlamD layer and the concomitant choroidal neovascularization (CNV) suggest that the events might be correlated. The newly formed vessels crossing Bruch's membrane and invading the BlamD layer could induce physicochemical changes in the ECM of the RPE, providing the required environmental conditions for the polymerization of collagen molecules into aggregates with the LSC-like pattern. With the deposition of new components, the thickness of BlamD increases and further impairs the supply of nutrients and oxygen, thus sustaining CNV.

Serum-free differentiation of murine embryonic stem cells into alveolar type II epithelial cells.

Alveolar type II (AT2) epithelial cells have important functions including the production of surfactant and regeneration of lost alveolar type I epithelial cells. The ability of in vitro production of AT2 cells would offer new therapeutic options in treating pulmonary injuries and disorders including genetically based surfactant deficiencies. Aiming at the generation of AT2-like cells, the differentiation of murine embryonic stem cells (mESCs) toward mesendodermal progenitors (MEPs) was optimized using a "Brachyury-eGFP-knock in" mESC line. eGFP expression demonstrated generation of up to 65% MEPs at day 4 after formation of embryoid bodies (EBs) under serum-free conditions. Plated EBs were further differentiated into AT2-like cells for a total of 25 days in serum-free media resulting in the expression of endodermal marker genes (FoxA2, Sox17, TTR, TTF-1) and of markers for distal lung epithelium (surfactant proteins (SP-) A, B, C, and D, CCSP, aquaporin 5). Notably, expression of SP-C as the only known AT2 cell specific marker could be detected after serum-induction as well as under serum-free conditions. Cytoplasmic localization of SP-C was demonstrated by confocal microscopy. The presence of AT2-like cells was confirmed by electron microscopy providing evidence for polarized cells with apical microvilli and lamellar body-like structures. Our results demonstrate the differentiation of AT2-like cells from mESCs after serum-induction and under serum-free conditions. The established serum-free differentiation protocol will facilitate the identification of key differentiation factors leading to a more specific and effective generation of AT2-like cells from ESCs.

Type II pneumocyte-restricted green fluorescent protein expression after lentiviral transduction of lung epithelial cells.

Type II alveolar epithelial (AT2) cell-specific reporter expression has been highly useful in the study of embryology and alveolar regeneration in transgenic mice. Technologies enabling efficient gene transfer and cell type-restricted transgene expression in AT2 cells would allow for correction of AT2 cell-based diseases such as genetic surfactant deficiencies. Moreover, such approaches are urgently required to investigate differentiation of AT2 cells from adult and embryonic stem cells of other species than mouse. Using a human surfactant protein C (SP-C) promoter fragment, we have constructed lentiviral vectors enabling AT2-restricted transgene expression and identification of stem cell-derived AT2 cells. Lung epithelial cell lines M3E3/C3, H441, RLE-6TN, A549, MLE-12, and MLE-15 were characterized at the molecular and ultrastructural levels to identify cell lines useful to assess the cell type specificity of our vector constructs. After transduction, no green fluorescent protein (GFP) expression was observed in nontarget cells including bronchial H441 cells, pulmonary A549 cells, fibroblasts, smooth muscle cells, and endothelial cells. In contrast, and in correlation with endogenous SP-C expression, lentiviral transduction resulted in stable GFP expression in MLE-12 and MLE-15 AT2 cells. In conclusion, we have constructed a lentiviral vector mediating SP-C promoter-dependent GFP expression. Transgene expression strictly corresponds with an AT2 phenotype of the transduced cells. In particular, the generated vector should facilitate local alveolar gene therapy and investigation of alveolar regeneration and stem cell differentiation.

Bending a membrane: how clathrin affects budding.

Receptor-mediated endocytosis of ligands, such as transferrin and LDL, is suppressed when clathrin synthesis is blocked by RNA interference in HeLa cells. We have found that domains containing the adapter complex 2 (AP2)-coated vesicle adapter and the endocytic accessory proteins CALM (clathrin assembly lymphoid myeloid leukemia protein), epsin, and eps15/eps15R (EGF receptor pathway substrate 15-related) nevertheless persist at the plasma membrane. They are similar in size and number to those seen in clathrin-expressing cells. Here we characterize these membrane domains by fluorescence and electron microscopy in detail. Fluorescence recovery after photobleaching measurements suggest that the exchange between membrane-bound and free cytosolic AP2 molecules is not significantly influenced by the depletion of clathrin. The AP2 membrane domains are dispersed upon interfering with protein-protein interactions that involve the alpha appendage domain of AP2. Electron microscopy of cellular cortices revealed that the AP2 membrane domains lack any curvature, suggesting that clathrin is essential for driving coated pit invagination. A model for coated vesicle formation, incorporating a mechanism commonly referred to as a "Brownian ratchet," is consistent with our observations.

Clathrin and clathrin-accessory proteins in rat kidney cortex epithelia.

Several vectorial transport routes in mammalian cells involve clathrin and associated proteins. In kidney epithelia urine production requires numerous transport processes. However, only little is known about the distribution of clathrin and its associated proteins in this organ in situ. We now report on the presence and distribution of clathrin and its accessory proteins AP1, AP2, Eps15, Epsin, CALM and Clint/EpsinR in the epithelia of the rat kidney cortex using immunoblotting, immunofluorescence and immuno-electron microscopy. Our data show that all investigated proteins are ubiquitously present in rat kidney cortex epithelia, however, with distinct distribution patterns. In the renal corpuscle, podocytes showed the most conspicuous labelling. Clathrin, AP2 and CALM were highly expressed in foot processes, while AP1 was primarily localized in the cell body. In the proximal tubule all proteins were present in dots along the plasma membrane and most conspicuous below the brush border. However, clathrin and AP2 co-localized in vesicle subtypes distinct from those containing clathrin and AP1. In the distal tubule and in the cortical collecting duct all proteins were found in the apex of the cells; however, AP1 and Clint/EpsinR showed additional staining in perinuclear dots. The occurrence and distribution of the investigated proteins in kidney epithelia are discussed with respect to their possible involvement in the functions of the specific nephron segment.

Generation and characterization of functional cardiomyocytes from rhesus monkey embryonic stem cells.

Embryonic stem cells (ESCs) from mice and humans (hESCs) have been shown to be able to efficiently differentiate toward cardiomyocytes (CMs). Because murine ESCs and hESCs do not allow for establishment of pre-clinical allogeneic transplantation models, the aim of our study was to generate functional CMs from rhesus monkey ESCs (rESCs). Although formation of ectodermal and neuronal/glial cells appears to be the default pathway of the rESC line R366.4, we were able to change this commitment and to direct generation of endodermal/mesodermal cells and further differentiation toward CMs. Differentiation of rESCs resulted in an average of 18% of spontaneously contracting embryoid bodies (EBs) from rESCs. Semiquantitative reverse transcription-polymerase chain reaction analyses demonstrated expression of marker genes typical for endoderm, mesoderm, cardiac mesoderm, and CMs, including brachyury, goosecoid, Tbx-5, Tbx-20, Mesp1, Nkx2.5, GATA-4, FOG-2, Mlc2a, MLC2v, ANF, and alpha-MHC in rESC-derived CMs. Immunohistological and ultrastructural studies showed expression of CM-typical proteins, including sarcomeric actinin, troponin T, titin, connexin 43, and cross-striated muscle fibrils. Electrophysiological studies by means of multielectrode arrays revealed evidence of functionality, electrical coupling, and beta-adrenergic signaling of the generated CMs. This is the first study demonstrating generation of functional CMs derived from rESCs. In contrast to hESCs, rESCs allow for establishment of pre-clinical allogeneic transplantation models. Moreover, rESC-derived CMs represent a cell source for the development of high-throughput assays for cardiac safety pharmacology.

Effect of clathrin assembly lymphoid myeloid leukemia protein depletion on clathrin coat formation.

The endocytic accessory clathrin assembly lymphoid myeloid leukemia protein (CALM) is the ubiquitously expressed homolog of the neuron-specific protein AP180 that has been implicated in the retrieval of synaptic vesicle. Here, we show that CALM associates with the alpha-appendage domain of the AP2 adaptor via the three peptide motifs 420DPF, 375DIF and 489FESVF and to a lesser extent with the amino-terminal domain of the clathrin heavy chain. Reducing clathrin levels by RNA interference did not significantly affect CALM localization, but depletion of AP2 weakens its association with the plasma membrane. In cells, where CALM levels were reduced by RNA interference, AP2 and clathrin remained organized in somewhat enlarged bright fluorescent puncta. Electron microscopy showed that the depletion of CALM drastically affected the clathrin lattice structure. Round-coated buds, which are the predominant features in control cells, were replaced by irregularly shaped buds and long clathrin-coated tubules. Moreover, we noted an increase in the number of very small cages that formed on flat lattices. Furthermore, we noticed a redistribution of endosomal markers and AP1 in cells that were CALM depleted. Taken together, our findings indicate a critical role for CALM in the regulation and orderly progression of coated bud formation at the plasma membrane.