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Beta-propeller protein-associated neurodegeneration is a subtype of monogenic neurodegeneration with brain iron accumulation caused by de novo mutations in WDR45. The WDR45 protein functions as a beta-propeller scaffold and plays a putative role in autophagy through its interaction with phospholipids and autophagy-related proteins. Loss of WDR45 function due to disease-causing mutations has been linked to defects in autophagic flux in patient and animal cells. However, the role of WDR45 in iron homeostasis remains elusive. Here we studied patient-specific WDR45 mutant fibroblasts and induced pluripotent stem cell-derived midbrain neurons. Our data demonstrated that loss of WDR45 increased cellular iron levels and oxidative stress, accompanied by mitochondrial abnormalities, autophagic defects, and diminished lysosomal function. Restoring WDR45 levels partially rescued oxidative stress and the susceptibility to iron treatment, and activation of autophagy reduced the observed iron overload in WDR45 mutant cells. Our data suggest that iron-containing macromolecules and organelles cannot effectively be degraded through the lysosomal pathway due to loss of WDR45 function.

Mutations in the Parkin gene (PARK2) have been linked to a recessive form of Parkinson’s disease (PD) characterized by the loss of dopaminergic neurons in the substantia nigra. Deficiencies of mitochondrial respiratory chain complex I activity have been observed in the substantia nigra of PD patients, and loss of Parkin results in the reduction of complex I activity shown in various cell and animal models. Using co-immunoprecipitation and proximity ligation assays on endogenous proteins, we demonstrate that Parkin interacts with mitochondrial Stomatin-like protein 2 (SLP-2), which also binds the mitochondrial lipid cardiolipin and functions in the assembly of respiratory chain proteins. SH-SY5Y cells with a stable knockdown of Parkin or SLP-2, as well as induced pluripotent stem cell-derived neurons from Parkin mutation carriers, showed decreased complex I activity and altered mitochondrial network morphology. Importantly, induced expression of SLP-2 corrected for these mitochondrial alterations caused by reduced Parkin function in these cells. In-vivo Drosophila studies showed a genetic interaction of Parkin and SLP-2, and further, tissue-specific or global overexpression of SLP-2 transgenes rescued parkin mutant phenotypes, in particular loss of dopaminergic neurons, mitochondrial network structure, reduced ATP production, and flight and motor dysfunction. The physical and genetic interaction between Parkin and SLP-2 and the compensatory potential of SLP-2 suggest a functional epistatic relationship to Parkin and a protective role of SLP-2 in neurons. This finding places further emphasis on the significance of Parkin for the maintenance of mitochondrial function in neurons and provides a novel target for therapeutic strategies.

Beta-propeller protein-associated neurodegeneration is a subtype of monogenic neurodegeneration with brain iron accumulation caused by de novo mutations in WDR45. The WDR45 protein functions as a beta-propeller scaffold and plays a putative role in autophagy through its interaction with phospholipids and autophagy-related proteins. Loss of WDR45 function due to disease-causing mutations has been linked to defects in autophagic flux in patient and animal cells. However, the role of WDR45 in iron homeostasis remains elusive. Here we studied patient-specific WDR45 mutant fibroblasts and induced pluripotent stem cell-derived midbrain neurons. Our data demonstrated that loss of WDR45 increased cellular iron levels and oxidative stress, accompanied by mitochondrial abnormalities, autophagic defects, and diminished lysosomal function. Restoring WDR45 levels partially rescued oxidative stress and the susceptibility to iron treatment, and activation of autophagy reduced the observed iron overload in WDR45 mutant cells. Our data suggest that iron-containing macromolecules and organelles cannot effectively be degraded through the lysosomal pathway due to loss of WDR45 function.

Mechanotransduction pathways are activated in response to biophysical stimuli during the development or homeostasis of organs and tissues. In zebrafish, the blood-flow-sensitive transcription factor Klf2a promotes VEGF-dependent angiogenesis. However, the means by which the Klf2a mechanotransduction pathway is regulated to prevent continuous angiogenesis remain unknown. Here we report that the upregulation of klf2 mRNA causes enhanced egfl7 expression and angiogenesis signaling, which underlies cardiovascular defects associated with the loss of cerebral cavernous malformation (CCM) proteins in the zebrafish embryo. Using CCM-protein-depleted human umbilical vein endothelial cells, we show that the misexpression of KLF2 mRNA requires the extracellular matrix-binding receptor beta 1 integrin and occurs in the absence of blood flow. Downregulation of beta 1 integrin rescues ccm mutant cardiovascular malformations in zebrafish. Our work reveals a beta 1 integrin-Klf2-Egfl7-signaling pathway that is tightly regulated by CCM proteins. This regulation prevents angiogenic overgrowth and ensures the quiescence of endothelial cells.

SummarySignaling by Nodal and Bmp is essential for cardiac laterality. How activities of these pathways translate into left-right asymmetric organ morphogenesis is largely unknown. We show that, in zebrafish, Nodal locally reduces Bmp activity on the left side of the cardiac field. This effect is mediated by the extracellular matrix enzyme Hyaluronan synthase 2, expression of which is induced by Nodal. Unilateral reduction of Bmp signaling results in lower expression of nonmuscle myosin II and higher cell motility on the left, driving asymmetric displacement of the entire cardiac field. In silico modeling shows that left-right differences in cell motility are sufficient to induce a robust, directional migration of cardiac tissue. Thus, the mechanism underlying the formation of cardiac left-right asymmetry involves Nodal modulating an antimotogenic Bmp activity.

In model teleost fishes like the medaka and the zebrafish many genes which have been identified in genome sequencing projects await their functional characterization. Techniques for the effective generation of transgenic animals are a prerequisite for this challenging task, and, due to their transparency, fish offer the possibility to combine the use of fluorescent proteins and developmental analysis in vivo. Here we describe the application of the Ac/Ds transposon system to generate transgenic medaka reporter and gene trap lines. We determined a germline transmission rate of 30% in our experiments using constructs ranging in size from 1.8 to 6 kilobase pairs. The genomic integration site of the Ds-elements can be easily identified which is an important feature for gene trap mutagenesis experiments and similar approaches. We constructed gene trap vectors with functional elements of medaka sequences that produce in frame fusions of the endogenous sequence to EGFP. These vectors mimic endogenous expression of the trapped allele in transgenic animals and are capable to interfere with the expression of the wild type allele in the homozygous individuals.

Argumentationen begegnen Schulerinnen und Schulern im Rahmen vieler wissenschaftlicher Disziplinen. Die Mathematik mit ihren spezifischen Regeln stellt sie allerdings vor grose Probleme. Internationale und nationale Studien zeigen, dass viele Lernende bis zum Ende ihrer Schulzeit kaum in der Lage sind, selbstandig mathematische Beweise zu formulieren. Eine mogliche Erklarung dafur ist, dass die spezifischen Regeln fur akzeptable Argumentationen in der Mathematik fur die meisten Schulerinnen und Schuler nicht transparent sind. Nach einer konzeptuellen Fassung dieses Methodenwissens stellen wir eine Reanalyse zweier quantitativer empirischer Studien mit speziellem Fokus auf diesem Wissen und dem Zusammenhang mit geometriebezogener Beweis- und Argumentationskompetenz vor.

Der folgende Beitrag beschaftigt sich mit der Untersuchung von Geschlechtereffekten in dem sehr speziellen und spezifisch mathematischen Bereich des Beweisens und Begrundens. Die vorgestellten Ergebnisse basieren auf einer Reanalyse von vier quantitativ-empirischen Studien, die in den letzten sechs Jahren im Rahmen eines Forschungsvorhabens zum geometrischen Beweisen und Begrunden durchgefuhrt wurden1. Die Stichproben umfassten dabei insgesamt knapp 2800 Gymnasiastinnen und Gymnasiasten im 7. bzw. 8. Schuljahr. Die Studien bestatigen Ergebnisse einiger weniger entsprechender Untersuchungen auf diesem Gebiet und zeigen, dass nennenswerte Unterschiede zwischen den Geschlechtern nicht nachgewiesen werden konnen.

The tight junctions-associated MAGUK protein nagie oko is closely related to Drosophila Stardust, mouse protein associated with lin-seven 1 (Pals1), and human MAGUK p55 subfamily member 5 (Mpp5). As a component of the evolutionarily conserved Crumbs protein complex, nagie oko is essential for the maintenance of epithelial cell polarity. Here, we show that nagie oko contains a predicted nuclear export and two conserved nuclear localization signals. We find that loss of the predicted nuclear export signal results in nuclear protein accumulation. We show that nagie oko nuclear import is redundantly controlled by the two nuclear localization signals and the evolutionarily conserved region 1 (ECR1), which links nagie oko with Par6-aPKC. Finally, deletion forms of nagie oko that lack nuclear import and export signals complement several nagie oko mutant defects in cell polarity and epithelial integrity. This finding provides an entry point to potentially novel and unknown roles of this important cell polarity regulator. Developmental Dynamics 237:83–90, 2008. © 2007 Wiley-Liss, Inc.

The giant sarcomeric protein titin (up to 3.7 MDa) spans the half-sarcomere and has been regarded as blueprint for sarcomere assembly. Only little is known about the kinetics of titin's integration into the myofilament and its mechanistic role in sarcomere assembly. We study sarcomere dynamics by recombining fluorophores into the titin filament. In cardiomyocytes derived from knock-in mice expressing a titin fusion protein with eGFP attached to M-Band exon 6, FRAP experiments show an unexpected level of mobility of M-band titin that is calcium dependent. To investigate titin's role in sarcomere assembly and disassembly we inserted dsRed into titin's Z-disc region. The insertion of the fluorescent proteins at the N- and C-terminus of titin does not interfere with normal cellular function and double heterozygous knock-in animals have no obvious phenotype. The animals survive, are fertile, and have normal body and heart weights. Their myocytes allow us to simultaneously visualize titin's N- and the C-terminus using live cell imaging. Towards elucidating titin's role in myofilament dynamics we follow titin expression as well as sarcomere assembly and disassembly in differentiated mouse ES-cells, embryonic and neonatal myocytes, and satellite cells derived from titin-eGFP and -dsRed double heterozygous mice. During myofibrillogenesis in cardiomyocytes and skeletal muscle cells, titin integrates simultaneously into the Z-disc and M-band of the sarcomere. Finally, we use our animal models to follow titin synthesis, transport and degradation in developing and mature myocytes. The combination of fluorescent proteins, mouse genetics, live imaging, and super resolution microscopy provides a deeper understanding of sarcomere assembly and dynamics.

The zebrafish MAGUK protein Nagie oko is a member of the evolutionarily conserved Crumbs protein complex and functions as a scaffolding protein involved in the stabilization of multi-protein assemblies at the tight junction. During zebrafish embryogenesis, mutations in nagie oko cause defects in both epithelial polarity and cardiac morphogenesis. We used deletion constructs of Nagie oko in functional rescue experiments to define domains essential for cell polarity, maintenance of epithelial integrity and cardiac morphogenesis. Inability of Nagie oko to interact with Crumbs proteins upon deletion of the PDZ domain recreates all aspects of the nagie oko mutant phenotype. Consistent with this observation, apical localization of Nagie oko within the myocardium and neural tube is dependent on Oko meduzy/Crumbs2a. Disruption of direct interactions with Patj or Lin-7, two other members of the Crumbs protein complex, via the bipartite L27 domains produces only partial nagie oko mutant phenotypes and does not impair correct junctional localization of the truncated Nagie oko deletion protein within myocardial cells. Similarly, loss of the evolutionarily conserved region 1 domain, which mediates binding to Par6, causes only a subset of the nagie oko mutant epithelial phenotypes. Finally, deletion of the C-terminus, including the entire guanylate kinase and the SH3 domains, renders the truncated Nagie oko protein inactive and recreates all features of the nagie oko mutant phenotype when tested in functional complementation assays. Our observations reveal a previously unknown diversity of alternative multi-protein assembly compositions of the Crumbs–Nagie-oko and Par6-aPKC protein complexes that are highly dependent on the developmental context.

Megalin/LRP2 is an endocytic receptor in the proximal tubules of the mammalian kidney that plays a central role in the clearance of metabolites from the glomerular filtrate. To establish a genetic model system for elucidation of molecular components of this retrieval pathway, we characterized orthologous transport processes in the zebrafish. We show that expression of megalin/LRP2 and its co-receptor cubilin is conserved in the larval zebrafish pronephros and demarcates a segment of the pronephric duct that is active in clearance of tracer from the ultrafiltrate. Knock-down of megalin/LRP2 causes lack of Rab4-positive endosomes in the proximal pronephric duct epithelium and abrogates apical endocytosis. Similarly, knock-down of the megalin/LRP2 adaptor Disabled 2 also blocks renal clearance processes. These results demonstrate the conservation of the megalin/LRP2 retrieval pathway between the larval zebrafish pronephros and the mammalian kidney and set the stage for dissection of the renal endocytic machinery in a simple model organism. Using this model system, we provide first genetic evidence that renal tubular endocytosis and formation of endosomes is a ligand-induced process that crucially depends on megalin/LRP2 activity.