Induced Arp2/3 Complex Depletion Increases FMNL2/3 Formin Expression and Filopodia Formation.

The Arp2/3 complex generates branched actin filament networks operating in cell edge protrusion and vesicle trafficking. Here we employ a conditional knockout mouse model permitting tissue- or cell-type specific deletion of the murine Actr3 gene (encoding Arp3). A functional Actr3 gene appeared essential for fibroblast viability and growth. Thus, we developed cell lines for exploring the consequences of acute, tamoxifen-induced Actr3 deletion causing near-complete loss of functional Arp2/3 complex expression as well as abolished lamellipodia formation and membrane ruffling, as expected. Interestingly, Arp3-depleted cells displayed enhanced rather than reduced cell spreading, employing numerous filopodia, and showed little defects in the rates of random cell migration. However, both exploration of new space by individual cells and collective migration were clearly compromised by the incapability to efficiently maintain directionality of migration, while the principal ability to chemotax was only moderately affected. Examination of actin remodeling at the cell periphery revealed reduced actin turnover rates in Arp2/3-deficient cells, clearly deviating from previous sequestration approaches. Most surprisingly, induced removal of Arp2/3 complexes reproducibly increased FMNL formin expression, which correlated with the explosive induction of filopodia formation. Our results thus highlight both direct and indirect effects of acute Arp2/3 complex removal on actin cytoskeleton regulation

The Arp2/3 complex is crucial for colonisation of the mouse skin by melanoblasts

The Arp2/3 complex is essential for the assembly of branched filamentous actin, but its role in physiology and development is surprisingly little understood. Melanoblasts deriving from the neural crest migrate along the developing embryo and traverse the dermis to reach the epidermis, colonising the skin and eventually homing within the hair follicles. We have previously established that Rac1 and Cdc42 direct melanoblast migration in vivo. We hypothesised that the Arp2/3 complex might be the main downstream effector of these small GTPases. Arp3 depletion in the melanocyte lineage results in severe pigmentation defects in dorsal and ventral regions of the mouse skin. Arp3 null melanoblasts demonstrate proliferation and migration defects and fail to elongate as their wild-type counterparts. Conditional deletion of Arp3 in primary melanocytes causes improper proliferation, spreading, migration and adhesion to extracellular matrix. Collectively, our results suggest that the Arp2/3 complex is absolutely indispensable in the melanocyte lineage in mouse development, and indicate a significant role in developmental processes that require tight regulation of actin-mediated motility

Generation of a conditional Arp3 knockout mouse to analyze functions in skeletal muscle development in vivo and in mouse embryonic fibroblasts in vitro

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

Induced Arp2/3 Complex Depletion Increases FMNL2/3 Formin Expression and Filopodia Formation.

The Arp2/3 complex generates branched actin filament networks operating in cell edge protrusion and vesicle trafficking. Here we employ a conditional knockout mouse model permitting tissue- or cell-type specific deletion of the murine Actr3 gene (encoding Arp3). A functional Actr3 gene appeared essential for fibroblast viability and growth. Thus, we developed cell lines for exploring the consequences of acute, tamoxifen-induced Actr3 deletion causing near-complete loss of functional Arp2/3 complex expression as well as abolished lamellipodia formation and membrane ruffling, as expected. Interestingly, Arp3-depleted cells displayed enhanced rather than reduced cell spreading, employing numerous filopodia, and showed little defects in the rates of random cell migration. However, both exploration of new space by individual cells and collective migration were clearly compromised by the incapability to efficiently maintain directionality of migration, while the principal ability to chemotax was only moderately affected. Examination of actin remodeling at the cell periphery revealed reduced actin turnover rates in Arp2/3-deficient cells, clearly deviating from previous sequestration approaches. Most surprisingly, induced removal of Arp2/3 complexes reproducibly increased FMNL formin expression, which correlated with the explosive induction of filopodia formation. Our results thus highlight both direct and indirect effects of acute Arp2/3 complex removal on actin cytoskeleton regulation

Oscillations of Delta-like1 regulate the balance between differentiation and maintenance of muscle stem cells

International audienceCell-cell interactions mediated by Notch are critical for the maintenance of skeletal muscle stem cells. However, dynamics, cellular source and identity of functional Notch ligands during expansion of the stem cell pool in muscle growth and regeneration remain poorly characterized. Here we demonstrate that oscillating Delta-like 1 (Dll1) produced by myogenic cells is an indispensable Notch ligand for self-renewal of muscle stem cells in mice. Dll1 expression is controlled by the Notch target Hes1 and the muscle regulatory factor MyoD. Consistent with our mathematical model, our experimental analyses show that Hes1 acts as the oscillatory pacemaker, whereas MyoD regulates robust Dll1 expression. Interfering with Dll1 oscillations without changing its overall expression level impairs self-renewal, resulting in premature differentiation of muscle stem cells during muscle growth and regeneration. We conclude that the oscillatory Dll1 input into Notch signaling ensures the equilibrium between self-renewal and differentiation in myogenic cell communities

Canonical and non-canonical integrin-based adhesions dynamically interconvert

Abstract Adhesions are critical for anchoring cells in their environment, as signaling platforms and for cell migration. In line with these diverse functions different types of cell-matrix adhesions have been described. Best-studied are the canonical integrin-based focal adhesions. In addition, non-canonical integrin adhesions lacking focal adhesion proteins have been discovered. These include reticular adhesions also known as clathrin plaques or flat clathrin lattices, that are enriched in clathrin and other endocytic proteins, as well as extensive adhesion networks and retraction fibers. How these different adhesion types that share a common integrin backbone are related and whether they can interconvert is unknown. Here, we identify the protein stonin1 as a marker for non-canonical αVβ5 integrin-based adhesions and demonstrate by live cell imaging that canonical and non-canonical adhesions can reciprocally interconvert by the selective exchange of components on a stable αVβ5 integrin scaffold. Hence, non-canonical adhesions can serve as points of origin for the generation of canonical focal adhesions

The Arp2/3 complex is critical for colonisation of the mouse skin by melanoblasts.

The Arp2/3 complex is essential for the assembly of branched filamentous actin but its role in physiology and development is surprisingly little understood. Melanoblasts deriving from the neural crest migrate along the developing embryo and traverse the dermis to reach the epidermis colonising the skin and eventually homing within the hair follicles. We have previously established that Rac1 and Cdc42 direct melanoblast migration in vivo We hypothesised that the Arp2/3 complex might be the main downstream effector of these small GTPases. Arp3 depletion in the melanocyte lineage results in severe pigmentation defects in dorsal and ventral regions of the mouse skin. Arp3 null melanoblasts demonstrate proliferation and migration defects and fail to elongate as their wild-type counterparts. Conditional deletion of Arp3 in primary melanocytes causes improper proliferation, spreading, migration and adhesion to extracellular matrix. Collectively, our results suggest that the Arp2/3 complex is absolutely indispensable in the melanocyte lineage in mouse development, and indicate a significant role in developmental processes that require tight regulation of actin-mediated motility

Sepsis induces interleukin 6, gp130/JAK2/STAT3, and muscle wasting

Abstract Background Sepsis and inflammation can cause intensive care unit‐acquired weakness (ICUAW). Increased interleukin‐6 (IL‐6) plasma levels are a risk factor for ICUAW. IL‐6 signalling involves the glycoprotein 130 (gp130) receptor and the JAK/STAT‐pathway, but its role in sepsis‐induced muscle wasting is uncertain. In a clinical observational study, we found that the IL‐6 target gene, SOCS3, was increased in skeletal muscle of ICUAW patients indicative for JAK/STAT‐pathway activation. We tested the hypothesis that the IL‐6/gp130‐pathway mediates ICUAW muscle atrophy. Methods We sequenced RNA (RNAseq) from tibialis anterior (TA) muscle of cecal ligation and puncture‐operated (CLP) and sham‐operated wildtype (WT) mice. The effects of the IL‐6/gp130/JAK2/STAT3‐pathway were investigated by analysing the atrophy phenotype, gene expression, and protein contents of C2C12 myotubes. Mice lacking Il6st, encoding gp130, in myocytes (cKO) and WT controls, as well as mice treated with the JAK2 inhibitor AG490 or vehicle were exposed to CLP or sham surgery for 24 or 96 h. Results Analyses of differentially expressed genes in RNAseq (≥2‐log2‐fold change, P < 0.01) revealed an activation of IL‐6‐signalling and JAK/STAT‐signalling pathways in muscle of septic mice, which occurred after 24 h and lasted at least for 96 h during sepsis. IL‐6 treatment of C2C12 myotubes induced STAT3 phosphorylation (three‐fold, P < 0.01) and Socs3 mRNA expression (3.1‐fold, P < 0.01) and caused myotube atrophy. Knockdown of Il6st diminished IL‐6‐induced STAT3 phosphorylation (−30.0%; P < 0.01), Socs3 mRNA expression, and myotube atrophy. JAK2 (− 29.0%; P < 0.01) or STAT3 inhibition (−38.7%; P < 0.05) decreased IL‐6‐induced Socs3 mRNA expression. Treatment with either inhibitor attenuated myotube atrophy in response to IL‐6. CLP‐operated septic mice showed an increased STAT3 phosphorylation and Socs3 mRNA expression in TA muscle, which was reduced in septic Il6st‐cKO mice by 67.8% (P < 0.05) and 85.6% (P < 0.001), respectively. CLP caused a loss of TA muscle weight, which was attenuated in Il6st‐cKO mice (WT: −22.3%, P < 0.001, cKO: −13.5%, P < 0.001; WT vs. cKO P < 0.001). While loss of Il6st resulted in a reduction of MuRF1 protein contents, Atrogin‐1 remained unchanged between septic WT and cKO mice. mRNA expression of Trim63/MuRF1 and Fbxo32/Atrogin‐1 were unaltered between CLP‐treated WT and cKO mice. AG490 treatment reduced STAT3 phosphorylation (−22.2%, P < 0.05) and attenuated TA muscle atrophy in septic mice (29.6% relative reduction of muscle weight loss, P < 0.05). The reduction in muscle atrophy was accompanied by a reduction in Fbxo32/Atrogin‐1‐mRNA (−81.3%, P < 0.05) and Trim63/MuRF1‐mRNA expression (−77.6%, P < 0.05) and protein content. Conclusions IL‐6 via the gp130/JAK2/STAT3‐pathway mediates sepsis‐induced muscle atrophy possibly contributing to ICUAW

Efficient generation of a self-organizing neuromuscular junction model from human pluripotent stem cells

Abstract The complex neuromuscular network that controls body movements is the target of severe diseases that result in paralysis and death. Here, we report the development of a robust and efficient self-organizing neuromuscular junction (soNMJ) model from human pluripotent stem cells that can be maintained long-term in simple adherent conditions. The timely application of specific patterning signals instructs the simultaneous development and differentiation of position-specific brachial spinal neurons, skeletal muscles, and terminal Schwann cells. High-content imaging reveals self-organized bundles of aligned muscle fibers surrounded by innervating motor neurons that form functional neuromuscular junctions. Optogenetic activation and pharmacological interventions show that the spinal neurons actively instruct the synchronous skeletal muscle contraction. The generation of a soNMJ model from spinal muscular atrophy patient-specific iPSCs reveals that the number of NMJs and muscle contraction is severely affected, resembling the patient’s pathology. In the future, the soNMJ model could be used for high-throughput studies in disease modeling and drug development. Thus, this model will allow us to address unmet needs in the neuromuscular disease field

Oscillations of MyoD and Hes1 proteins regulate the maintenance of activated muscle stem cells

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