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Endothelial nitric oxide synthase (eNOS)-related vessel relaxation is a highly coordinated process that regulates blood flow and pressure and is dependent on caveolae. Here, we investigated the role of caveolar plasma membrane stabilization by the dynamin-related ATPase EHD2 on eNOS-nitric oxide (NO)-dependent vessel relaxation. Loss of EHD2 in small arteries led to increased numbers of caveolae that were detached from the plasma membrane. Concomitantly, impaired relaxation of mesenteric arteries and reduced running wheel activity were observed in EHD2 knockout mice. EHD2 deletion or knockdown led to decreased production of nitric oxide (NO) although eNOS expression levels were not changed. Super-resolution imaging revealed that eNOS was redistributed from the plasma membrane to internalized detached caveolae in EHD2-lacking tissue or cells. Following an ATP stimulus, reduced cytosolic Ca(2+) peaks were recorded in human umbilical vein endothelial cells (HUVECs) lacking EHD2. Our data suggest that EHD2-controlled caveolar dynamics orchestrates the activity and regulation of eNOS/NO and Ca(2+) channel localization at the plasma membrane.

The balance between proliferation and differentiation of muscle stem cells is tightly controlled, ensuring the maintenance of a cellular pool needed for muscle growth and repair. We demonstrate here that the transcriptional regulator Hes1 controls the balance between proliferation and differentiation of activated muscle stem cells in both developing and regenerating muscle. We observed that Hes1 is expressed in an oscillatory manner in activated stem cells where it drives the oscillatory expression of MyoD. MyoD expression oscillates in activated muscle stem cells from postnatal and adult muscle under various conditions: when the stem cells are dispersed in culture, when they remain associated with single muscle fibers, or when they reside in muscle biopsies. Unstable MyoD oscillations and long periods of sustained MyoD expression are observed in differentiating cells. Ablation of the Hes1 oscillator in stem cells interfered with stable MyoD oscillations and led to prolonged periods of sustained MyoD expression, resulting in increased differentiation propensity. This interfered with the maintenance of activated muscle stem cells, and impaired muscle growth and repair. We conclude that oscillatory MyoD expression allows the cells to remain in an undifferentiated and proliferative state and is required for amplification of the activated stem cell pool.

Das actin-related Protein 3 (Arp3) ist eine Untereinheit des Arp2/3-Komplexes. Der Arp2/3-Komplex ist für die Nukleation von verzweigten Aktinnetzen in eukaryotischen Zellen verantwortlich, die in physiologisch wichtigen Prozessen der Zelle, wie Bewegung, Stofftransport und Zellteilung, sowie an der Gestalt von Zellen beteiligt sind. Die Deletion des Arp3-Gens in der Maus führt zu einem embryonal letalen Phänotyp. Um die Untersuchung späterer zellulärer Funktionen von Arp3 und dem Arp2/3-Komplex zu ermöglichen, habe ich eine konditionelle Arp3-Mausmutante hergestellt und die Rolle des Arp3 in der Skelettmuskelentwicklung analysiert. Die Deletion von Arp3 in Muskelvorläuferzellen hat keinen offensichtlichen Einfluss auf den Beginn der Myogenese, führt aber zur Reduktion von Skelettmuskeln während der fötalen Entwicklung. Als Ursache des verminderten Muskelwachstums wurde zum einen ein Myoblastenfusionsdefekt aufgedeckt, zudem zeigte sich ein Wachstumsdefekt Arp3-defizienter Myoblasten. Auch Apoptose wurde im Muskelgewebe nach Arp3-Deletion festgestellt. Die Deletion von Arp3 in differenzierten Muskelzellen stört die embryonale und fötale Myogenese nicht. Dennoch sterben Arp3-defiziente Nachkommen kurz nach der Geburt ohne offensichtliche organische Ursachen. Arp3-defiziente Tiere unterliegen wahrscheinlich den Geschwistern im Wettbewerb um die Muttermilch. Die Akkumulation des Arp3 an der Z-Scheibe der Sarkomere deutet darauf hin, dass das Arp3-Protein möglicherweise eine Funktion in der Fibrillogenese im erwachsenem Muskel hat. Arp3-defiziente embryonale Maus-Fibroblasten weisen eine gestörte Mitose und / oder Zytokinese auf, wodurch das Zellvolumen, der DNA-Gehalt und die Anzahl der Zentrosomen zunehmen. Zudem verändern Fibroblasten nach der Deletion beider Arp3-Allele ihre Gestalt: Die Ausbildung der Lamellipodien ist weitgehend gehemmt. Als Konsequenz zeigen Arp3-defiziente Fibroblasten starke Beeinträchtigung der Migration., Arp3 is one of seven subunits of the Arp2/3 complex and essential for complex stability. The Arp2/3 complex is an actin nucleator to form branched actin filaments, which are involved in important physiological processes of eukaryotic cells like migration, cell devision, exocytosis as well as in cell morphology. Arp3 knockout mice show an early lethal phenotype. To study the role of Arp3 in later stages of skeletal muscle development in vivo and in mouse embryonic fibroblasts in vitro, a conditional Arp3 allele has been generated. The specific deletion of Arp3 in skeletal muscle precursor cells does apparently not affect embryonic skeletal muscle development but results in reduction of skeletal muscle during fetal embryogenesis. This is caused by impaired fusion and proliferation of Arp3-deficient myoblasts, as well as apoptosis. The deletion of Arp3 in differentiated skeletal muscle cells has no influence on skeletal muscle growth during embryonic and fetal development. Nevertheless, Arp3 mutants die shortly after birth without any obvious organic cause. Arp3 protein accumulates at the Z-disc of the striated muscle, suggesting a role of Arp3 in fibrillogenesis of the differentiated muscle. The deletion of Arp3 in mouse embryonic fibroblasts in vitro results in a proliferation defect, centrosome hyperamplification and increased DNA content, indicating a role of Arp3 in cytokinesis. In addition, Arp3-deficient fibroblasts show an impaired formation of lamellipodia, resulting in impaired cell migration.