Stephanie Groos

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 α‐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.

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

Macrophage progenitors are an effective and long-lasting therapy of hereditary pulmonary alveolar proteinosis. Macrophages Treat Rare Lung Disease Innate immune cell transplant into the lung could be an effective treatment for a rare lung disease. Happle et al. report that transplanting macrophage progenitors into lungs of a mouse model of hereditary pulmonary alveolar proteinosis (herPAP) improved lung function for up to 9 months after transplant. herPAP is caused by mutations in the granulocyte-macrophage colony-stimulating factor receptor genes, resulting in disturbed alveolar macrophage differentiation and life-threatening respiratory problems. A single transplantation of macrophage progenitors into a mouse model of herPAP resulted in differentiation into functional alveolar macrophages. If these data hold true in humans, this could not only provide a new treatment modality for herPAP but also serve as a proof of principle for other genetic diseases. 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.

Re-evaluation of Epoxy Resin Sections for Light and Electron Microscopic Immunostaining

Epoxy resins provide optimal tissue morphology at both the light and the electron microscopic level and therefore enable correlative studies on semithin and thin sections from the same tissue block. Here we report on an approach to retain these advantages for immunolabeling studies by adapting and combining well-known techniques, i.e., surface etching with sodium ethoxide and heat-mediated antigen retrieval. We propose a simple procedure for immunostaining semithin and thin epoxy resin sections. To check its applicability, well characterized, commercially available antibodies (against E-cadherin, α-catenin, and β-catenin) were used on sections of human small intestine. By light microscopy, the immunostaining efficiency was compared on cryo-, paraffin, and epoxy semithin sections processed in parallel. The most detailed results were obtained on semithin sections, where the labeling precisely delineated the lateral plasma membrane of the enterocytes. At the electron microscopic level the procedure did not damage the structures and allowed an efficient, reproducible immunogold labeling extending homogeneously over exceptionally wide tissue areas. The three antibodies specifically labeled the zonula adherens of the junctional complex between epithelial cells and, in agreement with light microscopic observations, the lateral plasma membrane.

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.

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 ∼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.

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 α‐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 β‐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.

Mitogen-Activated Protein Kinase-Activated Protein Kinases 2 and 3 Regulate SERCA2a Expression and Fiber Type Composition To Modulate Skeletal Muscle and Cardiomyocyte Function

ABSTRACT 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 Ca2+-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.

Changes in epithelial cell turnover and extracellular matrix in human small intestine after TPN.

BACKGROUND The atrophy and architectural remodeling of the jejunal mucosa arising in adults receiving total parenteral nutrition (TPN) has been suggested to originate from a disturbance in tissue homeostasis. The present study aims at examining (1) whether there are differences in proliferation and apoptosis of epithelial cells between enterally and parenterally nourished patients and (2) whether the distribution pattern of extracellular matrix (ECM) proteins known to influence cell turnover along the the crypt-villus axis is changed after TPN. METHODS The mitotic frequency and the proliferation index [using an antibody against Ki-67 antigen (MIB 1)] were determined on epoxy semithin and paraffin sections, respectively. Morphological techniques and the TUNEL assay were applied to detect apoptotic events. Immunolocalization of collagen IV, laminin, fibronectin, tenascin, and collagen VI was performed on cryosections. RESULTS After TPN the cell renewal was significantly enhanced, while epithelial cell death was drastically reduced. The comparison of TPN and EN patients revealed differences in the distribution patterns of the ECM proteins laminin, fibronectin, and tenascin along the crypt-villus axis. Moreover, after TPN an increased expression of collagen types IV and VI was observed. CONCLUSIONS TPN in human adults is associated with alterations in epithelial cell turnover and changes in expression and/or localization of ECM proteins. Thus, the inverted route of nutrient supply in patients might modify environmental tissue conditions, which may influence the interactions between intestinal epithelial cells and the extracellular matrix.

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.