RESUMO
Actin dynamics is essential for T-cell development. We show here that cofilin1 is the key molecule for controlling actin filament turnover in this process. Mice with specific depletion of cofilin1 in thymocytes showed increased steady-state levels of actin filaments, and associated alterations in the pattern of thymocyte migration and adhesion. Our data suggest that cofilin1 is controlling oscillatory F-actin changes, a parameter that influences the migration pattern in a 3-D environment. In a collagen matrix, cofilin1 controls the speed and resting intervals of migrating thymocytes. Cofilin1 was not involved in thymocyte proliferation, cell survival, apoptosis or surface receptor trafficking. However, in cofilin1 mutant mice, impaired adhesion and migration resulted in a specific block of thymocyte differentiation from CD4/CD8 double-positive thymocytes towards CD4 and CD8 single-positive cells. Our data suggest that tuning of the dwelling time of thymocytes in the thymic niches is tightly controlled by cofilin1 and essential for positive selection during T-cell differentiation. We describe a novel role of cofilin1 in the physiological context of migration-dependent cell differentiation.
Assuntos
Actinas , Timócitos , Actinas/genética , Animais , Linfócitos T CD8-Positivos , Diferenciação Celular , Movimento Celular , Cofilina 1 , CamundongosRESUMO
BACKGROUNDS & AIMS: Increased permeability is implicated in the pathogenesis of intestinal disease. In vitro and in vivo studies have linked down-regulation of the scaffolding protein ZO-1, encoded by the TJP1 gene, to increased tight junction permeability. This has not, however, been tested in vivo. Here, we assessed the contributions of ZO-1 to in vivo epithelial barrier function and mucosal homeostasis. METHODS: Public Gene Expression Omnibus data sets and biopsy specimens from patients with inflammatory bowel disease (IBD) and healthy control individuals were analyzed. Tjp1f/f;vil-CreTg mice with intestinal epithelial-specific ZO-1 knockout (ZO-1KO.IEC) mice and Tjp1f/f mice littermates without Cre expression were studied using chemical and immune-mediated models of disease as well as colonic stem cell cultures. RESULTS: ZO-1 transcript and protein expression were reduced in biopsy specimens from patients with IBD. Despite mildly increased intestinal permeability, ZO-1KO.IEC mice were healthy and did not develop spontaneous disease. ZO-1KO.IEC mice were, however, hypersensitive to mucosal insults and displayed defective repair. Furthermore, ZO-1-deficient colonic epithelia failed to up-regulate proliferation in response to damage in vivo or Wnt signaling in vitro. ZO-1 was associated with centrioles in interphase cells and mitotic spindle poles during division. In the absence of ZO-1, mitotic spindles failed to correctly orient, resulting in mitotic catastrophe and abortive proliferation. ZO-1 is, therefore, critical for up-regulation of epithelial proliferation and successful completion of mitosis. CONCLUSIONS: ZO-1 makes critical, tight junction-independent contributions to Wnt signaling and mitotic spindle orientation. As a result, ZO-1 is essential for mucosal repair. We speculate that ZO-1 down-regulation may be one cause of ineffective mucosal healing in patients with IBD.
Assuntos
Proliferação de Células , Colo/metabolismo , Células Epiteliais/metabolismo , Doenças Inflamatórias Intestinais/metabolismo , Mucosa Intestinal/metabolismo , Mitose , Proteína da Zônula de Oclusão-1/metabolismo , Animais , Células Cultivadas , Colo/patologia , Bases de Dados Genéticas , Modelos Animais de Doenças , Células Epiteliais/patologia , Humanos , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/patologia , Mucosa Intestinal/patologia , Camundongos Knockout , Permeabilidade , Fuso Acromático/genética , Fuso Acromático/metabolismo , Fuso Acromático/patologia , Via de Sinalização Wnt , Cicatrização , Proteína da Zônula de Oclusão-1/genéticaRESUMO
The role of the actin cytoskeleton in the sequence of physiological epithelial repair in the intact epithelium has yet to be elucidated. Here, we explore the role of actin in gastric repair in vivo and in vitro gastric organoids (gastroids). In response to two-photon-induced cellular damage of either an in vivo gastric or in vitro gastroid epithelium, actin redistribution specifically occurred in the lateral membranes of cells neighboring the damaged cell. This was followed by their migration inward to close the gap at the basal pole of the dead cell, in parallel with exfoliation of the dead cell into the lumen. The repair and focal increase of actin was significantly blocked by treatment with EDTA or the inhibition of actin polymerization. Treatment with inhibitors of myosin light chain kinase, myosin II, trefoil factor 2 signaling or phospholipase C slowed both the initial actin redistribution and the repair. While Rac1 inhibition facilitated repair, inhibition of RhoA/Rho-associated protein kinase inhibited it. Inhibitors of focal adhesion kinase and Cdc42 had negligible effects. Hence, initial actin polymerization occurs in the lateral membrane, and is primarily important to initiate dead cell exfoliation and cell migration to close the gap.
Assuntos
Actinas/metabolismo , Mucosa Gástrica/lesões , Organoides/lesões , Multimerização Proteica/fisiologia , Reepitelização/fisiologia , Estômago/citologia , Animais , Movimento Celular , Células Cultivadas , Células Epiteliais/fisiologia , Feminino , Mucosa Gástrica/metabolismo , Mucosa Gástrica/fisiologia , Masculino , Camundongos , Camundongos Transgênicos , Organoides/citologia , Organoides/fisiologia , Polimerização , Regeneração/fisiologia , Estômago/lesõesRESUMO
The cytoskeleton is widely considered essential for neurulation, yet the mouse spinal neural tube can close despite genetic and non-genetic disruption of the cytoskeleton. To investigate this apparent contradiction, we applied cytoskeletal inhibitors to mouse embryos in culture. Preventing actomyosin cross-linking, F-actin assembly or myosin II contractile activity did not disrupt spinal closure. In contrast, inhibiting Rho kinase (ROCK, for which there are two isoforms ROCK1 and ROCK2) or blocking F-actin disassembly prevented closure, with apical F-actin accumulation and adherens junction disturbance in the neuroepithelium. Cofilin-1-null embryos yielded a similar phenotype, supporting the hypothesis that there is a key role for actin turnover. Co-exposure to Blebbistatin rescued the neurulation defects caused by RhoA inhibition, whereas an inhibitor of myosin light chain kinase, ML-7, had no such effect. We conclude that regulation of RhoA, Rho kinase, LIM kinase and cofilin signalling is necessary for spinal neural tube closure through precise control of neuroepithelial actin turnover and actomyosin disassembly. In contrast, actomyosin assembly and myosin ATPase activity are not limiting for closure.
Assuntos
Actinas/metabolismo , Actomiosina/metabolismo , Tubo Neural/embriologia , Quinases Associadas a rho/metabolismo , Actinas/genética , Actomiosina/genética , Animais , Cofilina 1/genética , Cofilina 1/metabolismo , Quinases Lim/genética , Quinases Lim/metabolismo , Camundongos , Camundongos Mutantes , Proteínas rho de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/metabolismo , Quinases Associadas a rho/genética , Proteína rhoA de Ligação ao GTPRESUMO
Actin is a regulator of synaptic vesicle mobilization and exocytosis, but little is known about the mechanisms that regulate actin at presynaptic terminals. Genetic data on LIMK1, a negative regulator of actin-depolymerizing proteins of the ADF/cofilin family, suggest a role for ADF/cofilin in presynaptic function. However, synapse physiology is fully preserved upon genetic ablation of ADF in mice, and n-cofilin mutant mice display defects in postsynaptic plasticity, but not in presynaptic function. One explanation for this phenomenon is overlapping functions of ADF and n-cofilin in presynaptic physiology. Here, we tested this hypothesis and genetically removed ADF together with n-cofilin from synapses. In double mutants for ADF and n-cofilin, synaptic actin dynamics was impaired and more severely affected than in single mutants. The resulting cytoskeletal defects heavily affected the organization, mobilization, and exocytosis of synaptic vesicles in hippocampal CA3-CA1 synapses. Our data for the first time identify overlapping functions for ADF and n-cofilin in presynaptic physiology and vesicle trafficking. We conclude that n-cofilin is a limiting factor in postsynaptic plasticity, a function which cannot be substituted by ADF. On the presynaptic side, the presence of either ADF or n-cofilin is sufficient to control actin remodeling during vesicle release.
Assuntos
Actinas/metabolismo , Cofilina 1/metabolismo , Destrina/metabolismo , Exocitose/fisiologia , Transporte Proteico/fisiologia , Sinapses/fisiologia , Vesículas Sinápticas/metabolismo , Animais , Cofilina 1/genética , Destrina/genética , Estimulação Elétrica , Potenciais Pós-Sinápticos Excitadores/genética , Potenciais Pós-Sinápticos Excitadores/fisiologia , Exocitose/efeitos dos fármacos , Exocitose/genética , Ácido Glutâmico/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Camundongos , Camundongos Transgênicos , Mutação/genética , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Neurônios/ultraestrutura , Fosforilação , Cloreto de Potássio/farmacologia , Prosencéfalo/citologia , Transporte Proteico/genética , Proteínas SNARE/metabolismo , Sinapses/efeitos dos fármacos , Sinapses/ultraestruturaRESUMO
Neuronal plasticity is an important process for learning, memory and complex behaviour. Rapid remodelling of the actin cytoskeleton in the postsynaptic compartment is thought to have an important function for synaptic plasticity. However, the actin-binding proteins involved and the molecular mechanisms that in vivo link actin dynamics to postsynaptic physiology are not well understood. Here, we show that the actin filament depolymerizing protein n-cofilin is controlling dendritic spine morphology and postsynaptic parameters such as late long-term potentiation and long-term depression. Loss of n-cofilin-mediated synaptic actin dynamics in the forebrain specifically leads to impairment of all types of associative learning, whereas exploratory learning is not affected. We provide evidence for a novel function of n-cofilin function in synaptic plasticity and in the control of extrasynaptic excitatory AMPA receptors diffusion. These results suggest a critical function of actin dynamics in associative learning and postsynaptic receptor availability.
Assuntos
Actinas/fisiologia , Cofilina 1/metabolismo , Aprendizagem , Plasticidade Neuronal/fisiologia , Receptores de AMPA/metabolismo , Fatores de Despolimerização de Actina/metabolismo , Actinas/metabolismo , Animais , Proteínas de Transporte/metabolismo , Membrana Celular/metabolismo , Citoesqueleto/metabolismo , Espinhas Dendríticas/metabolismo , Espinhas Dendríticas/fisiologia , Potenciação de Longa Duração/fisiologia , Memória , Camundongos , Camundongos Transgênicos , Proteínas dos Microfilamentos/metabolismoRESUMO
As signalling organelles, cilia regulate their G protein-coupled receptor content by ectocytosis, a process requiring localised actin dynamics to alter membrane shape. Photoreceptor outer segments comprise an expanse of folded membranes (discs) at the tip of highly-specialised connecting cilia, into which photosensitive GPCRs are concentrated. Discs are shed and remade daily. Defects in this process, due to mutations, cause retinitis pigmentosa (RP). Whilst fundamental for vision, the mechanism of photoreceptor disc generation is poorly understood. Here, we show membrane deformation required for disc genesis is driven by dynamic actin changes in a process akin to ectocytosis. We show RPGR, a leading RP gene, regulates actin-binding protein activity central to this process. Actin dynamics, required for disc formation, are perturbed in Rpgr mouse models, leading to aborted membrane shedding as ectosome-like vesicles, photoreceptor death and visual loss. Actin manipulation partially rescues this, suggesting the pathway could be targeted therapeutically. These findings help define how actin-mediated dynamics control outer segment turnover.
Assuntos
Actinas , Proteínas do Olho , Retinose Pigmentar , Animais , Actinas/metabolismo , Camundongos , Retinose Pigmentar/metabolismo , Retinose Pigmentar/genética , Proteínas do Olho/metabolismo , Proteínas do Olho/genética , Cílios/metabolismo , Humanos , Segmento Externo das Células Fotorreceptoras da Retina/metabolismo , Camundongos Knockout , Camundongos Endogâmicos C57BL , Membrana Celular/metabolismoRESUMO
Cerebellar granule neurons (CGNs) exploit Bergmann glia (BG) fibres for radial migration, and cell-cell contacts have a pivotal role in this process. Nevertheless, little is known about the mechanisms that control CGN-BG interaction. Here we demonstrate that the actin-binding protein profilin1 is essential for CGN-glial cell adhesion and radial migration. Profilin1 ablation from mouse brains leads to a cerebellar hypoplasia, aberrant organization of cerebellar cortex layers and ectopic CGNs. Conversely, neuronal progenitor proliferation, tangential migration of neurons and BG morphology appear to be independent of profilin1. Our mouse data and the mapping of developmental neuropathies to the chromosomal region of PFN1 suggest a similar function for profilin1 in humans.
Assuntos
Movimento Celular , Cerebelo/metabolismo , Neuroglia/metabolismo , Neurônios/metabolismo , Profilinas/metabolismo , Animais , Axônios/metabolismo , Adesão Celular/genética , Diferenciação Celular , Movimento Celular/genética , Cerebelo/patologia , Hiperplasia/genética , Camundongos , Camundongos Transgênicos , Mutação , Neurônios/citologia , Profilinas/genéticaRESUMO
The actin depolymerizing factors (ADFs) play important roles in several cellular processes that require cytoskeletal rearrangements, such as cell migration, but little is known about the in vivo functions of ADFs in developmental events like branching morphogenesis. While the molecular control of ureteric bud (UB) branching during kidney development has been extensively studied, the detailed cellular events underlying this process remain poorly understood. To gain insight into the role of actin cytoskeletal dynamics during renal branching morphogenesis, we studied the functional requirements for the closely related ADFs cofilin1 (Cfl1) and destrin (Dstn) during mouse development. Either deletion of Cfl1 in UB epithelium or an inactivating mutation in Dstn has no effect on renal morphogenesis, but simultaneous lack of both genes arrests branching morphogenesis at an early stage, revealing considerable functional overlap between cofilin1 and destrin. Lack of Cfl1 and Dstn in the UB causes accumulation of filamentous actin, disruption of normal epithelial organization, and defects in cell migration. Animals with less severe combinations of mutant Cfl1 and Dstn alleles, which retain one wild-type Cfl1 or Dstn allele, display abnormalities including ureter duplication, renal hypoplasia, and abnormal kidney shape. The results indicate that ADF activity, provided by either cofilin1 or destrin, is essential in UB epithelial cells for normal growth and branching.
Assuntos
Cofilina 1/metabolismo , Destrina/metabolismo , Morfogênese , Ureter/metabolismo , Actinas/metabolismo , Animais , Movimento Celular , Cofilina 1/genética , Destrina/genética , Células Epiteliais/metabolismo , Epitélio/embriologia , Epitélio/metabolismo , Feminino , Imunofluorescência , Genótipo , Fator Neurotrófico Derivado de Linhagem de Célula Glial/farmacologia , Hibridização In Situ , Rim/efeitos dos fármacos , Rim/embriologia , Rim/metabolismo , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Técnicas de Cultura de Órgãos , Ureter/efeitos dos fármacos , Ureter/embriologiaRESUMO
The cellular and molecular mechanisms orchestrating the complex process by which bone marrow megakaryocytes form and release platelets remain poorly understood. Mature megakaryocytes generate long cytoplasmic extensions, proplatelets, which have the capacity to generate platelets. Although microtubules are the main structural component of proplatelets and microtubule sliding is known to drive proplatelet elongation, the role of actin dynamics in the process of platelet formation has remained elusive. Here, we tailored a mouse model lacking all ADF/n-cofilin-mediated actin dynamics in megakaryocytes to specifically elucidate the role of actin filament turnover in platelet formation. We demonstrate, for the first time, that in vivo actin filament turnover plays a critical role in the late stages of platelet formation from megakaryocytes and the proper sizing of platelets in the periphery. Our results provide the genetic proof that platelet production from megakaryocytes strictly requires dynamic changes in the actin cytoskeleton.
Assuntos
Actinas/metabolismo , Plaquetas/metabolismo , Cofilina 1/metabolismo , Destrina/metabolismo , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Animais , Plaquetas/citologia , Plaquetas/ultraestrutura , Western Blotting , Forma Celular , Tamanho Celular , Sobrevivência Celular , Cofilina 1/genética , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Destrina/genética , Fibrinogênio/metabolismo , Megacariócitos/citologia , Megacariócitos/metabolismo , Megacariócitos/ultraestrutura , Camundongos , Camundongos Knockout , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Contagem de Plaquetas , Esplenomegalia/genética , Esplenomegalia/metabolismo , Esplenomegalia/patologia , Trombina/farmacologia , Fatores de TempoRESUMO
Actin dynamics determines podocyte morphology during development and in response to podocyte injury and might be necessary for maintaining normal podocyte morphology. Because podocyte intercellular junction receptor Nephrin plays a role in regulating actin dynamics, and given the described role of cofilin in actin filament polymerization and severing, we hypothesized that cofilin-1 activity is regulated by Nephrin and is necessary in normal podocyte actin dynamics. Nephrin activation induced cofilin dephosphorylation via intermediaries that include phosphatidylinositol 3-kinase, SSH1, 14-3-3, and LIMK in a cell culture model. This Nephrin-induced cofilin activation required a direct interaction between Nephrin and the p85 subunit of phosphatidylinositol 3-kinase. In a similar fashion, cofilin-1 dephosphorylation was observed in a rat model of podocyte injury at a time when foot process spreading is initially observed. To investigate the necessity of cofilin-1 in the glomerulus, podocyte-specific Cfl1 null mice were generated. Cfl1 null podocytes developed normally. However, these mice developed persistent proteinuria by 3 months of age, although they did not exhibit foot process spreading until 8 months, when the rate of urinary protein excretion became more exaggerated. In a mouse model of podocyte injury, protamine sulfate perfusion of the Cfl1 mutant mouse induced a broadened and flattened foot process morphology that was distinct from that observed following perfusion of control kidneys, and mutant podocytes did not recover normal structure following additional perfusion with heparin sulfate. We conclude that cofilin-1 is necessary for maintenance of normal podocyte architecture and for actin structural changes that occur during induction and recovery from podocyte injury.
Assuntos
Cofilina 1/metabolismo , Podócitos/metabolismo , Actinas/metabolismo , Albuminúria/metabolismo , Animais , Linhagem Celular , Feminino , Deleção de Genes , Técnicas de Silenciamento de Genes , Humanos , Quinases Lim/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Especificidade de Órgãos , Fosfatidilinositol 3-Quinases/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Fosforilação , Podócitos/patologia , Podócitos/ultraestrutura , Protaminas , Ligação Proteica , Pseudópodes/metabolismo , Pseudópodes/ultraestrutura , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Injured axons fail to regenerate in the adult CNS, which contrasts with their vigorous growth during embryonic development. We explored the potential of re-initiating axon extension after injury by reactivating the molecular mechanisms that drive morphogenetic transformation of neurons during development. Genetic loss- and gain-of-function experiments followed by time-lapse microscopy, in vivo imaging, and whole-mount analysis show that axon regeneration is fueled by elevated actin turnover. Actin depolymerizing factor (ADF)/cofilin controls actin turnover to sustain axon regeneration after spinal cord injury through its actin-severing activity. This pinpoints ADF/cofilin as a key regulator of axon growth competence, irrespective of developmental stage. These findings reveal the central role of actin dynamics regulation in this process and elucidate a core mechanism underlying axon growth after CNS trauma. Thereby, neurons maintain the capacity to stimulate developmental programs during adult life, expanding their potential for plasticity. Thus, actin turnover is a key process for future regenerative interventions.
Assuntos
Actinas/metabolismo , Axônios/metabolismo , Cofilina 1/genética , Cofilina 2/genética , Destrina/genética , Cones de Crescimento/patologia , Regeneração Nervosa/genética , Traumatismos da Medula Espinal/genética , Animais , Axônios/patologia , Cofilina 1/metabolismo , Cofilina 2/metabolismo , Destrina/metabolismo , Cones de Crescimento/metabolismo , Microscopia Intravital , Camundongos , Microscopia Confocal , Neurônios/metabolismo , Neurônios/patologia , Ratos , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/patologia , Imagem com Lapso de TempoRESUMO
Analysis of actin remodeling in live cells and tissues has become an increasingly important tool to study actin-dependent cellular processes. To facilitate these experiments in the mouse we have generated a GFP-actin-expressing line (huGE) by knock-in of the GFP-actin gene into the profilin 1 locus. Here we show that GFP-actin is expressed throughout embryonic development and in all tissues except skeletal muscle, in a pattern similar to profilin 1. Particularly high expression of GFP-actin was observed in bone marrow and all blood cells. The GFP-actin fusion protein is functional as shown by its co-localization with endogenous actin in F-actin-rich structures. Therefore, the huGE mouse line provides a novel tool to monitor actin dynamics in mouse embryos and a wide range of organs.
Assuntos
Actinas/genética , Linhagem Celular , Citoesqueleto/genética , Camundongos Knockout/genética , Profilinas/genética , Actinas/metabolismo , Animais , Células Sanguíneas/citologia , Células Sanguíneas/metabolismo , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Citoesqueleto/metabolismo , Desenvolvimento Embrionário/genética , Desenvolvimento Embrionário/fisiologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Proteínas de Fluorescência Verde , Linfócitos/citologia , Linfócitos/metabolismo , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos , Células Mieloides/citologia , Células Mieloides/metabolismo , Profilinas/metabolismoRESUMO
Mitochondria form highly dynamic networks in which organelles constantly fuse and divide. The relevance of mitochondrial dynamics is evident from its implication in various human pathologies, including cancer or neurodegenerative, endocrine and cardiovascular diseases. Dynamin-related protein 1 (DRP1) is a key regulator of mitochondrial fission that oligomerizes at the mitochondrial outer membrane and hydrolyzes GTP to drive mitochondrial fragmentation. Previous studies demonstrated that DRP1 recruitment and mitochondrial fission is promoted by actin polymerization at the mitochondrial surface, controlled by the actin regulatory proteins inverted formin 2 (INF2) and Spire1C. These studies suggested the requirement of additional actin regulatory activities to control DRP1-mediated mitochondrial fission. Here we show that the actin-depolymerizing protein cofilin1, but not its close homolog actin-depolymerizing factor (ADF), is required to maintain mitochondrial morphology. Deletion of cofilin1 caused mitochondrial DRP1 accumulation and fragmentation, without altering mitochondrial function or other organelles' morphology. Mitochondrial morphology in cofilin1-deficient cells was restored upon (i) re-expression of wild-type cofilin1 or a constitutively active mutant, but not of an actin-binding-deficient mutant, (ii) pharmacological destabilization of actin filaments and (iii) genetic depletion of DRP1. Our work unraveled a novel function for cofilin1-dependent actin dynamics in mitochondrial fission, and identified cofilin1 as a negative regulator of mitochondrial DRP1 activity. We conclude that cofilin1 is required for local actin dynamics at mitochondria, where it may balance INF2/Spire1C-induced actin polymerization.
Assuntos
Actinas/genética , Cofilina 1/genética , Proteínas Quinases Associadas com Morte Celular/genética , Dinâmica Mitocondrial/genética , Citoesqueleto de Actina/genética , Actinas/metabolismo , Animais , Células Cultivadas , Destrina/genética , Fibroblastos , Forminas , Humanos , Camundongos , Proteínas dos Microfilamentos/genética , Mitocôndrias/genética , Proteínas do Tecido Nervoso/genética , Ligação Proteica , Multimerização Proteica/genéticaRESUMO
Mutations in the Retinitis Pigmentosa GTPase Regulator (RPGR) cause X-linked RP (XLRP), an untreatable, inherited retinal dystrophy that leads to premature blindness. RPGR localises to the photoreceptor connecting cilium where its function remains unknown. Here we show, using murine and human induced pluripotent stem cell models, that RPGR interacts with and activates the actin-severing protein gelsolin, and that gelsolin regulates actin disassembly in the connecting cilium, thus facilitating rhodopsin transport to photoreceptor outer segments. Disease-causing RPGR mutations perturb this RPGR-gelsolin interaction, compromising gelsolin activation. Both RPGR and Gelsolin knockout mice show abnormalities of actin polymerisation and mislocalisation of rhodopsin in photoreceptors. These findings reveal a clinically-significant role for RPGR in the activation of gelsolin, without which abnormalities in actin polymerisation in the photoreceptor connecting cilia cause rhodopsin mislocalisation and eventual retinal degeneration in XLRP.Mutations in the Retinitis Pigmentosa GTPase Regulator (RPGR) cause retinal dystrophy, but how this arises at a molecular level is unclear. Here, the authors show in induced pluripotent stem cells and mouse knockouts that RPGR mediates actin dynamics in photoreceptors via the actin-severing protein, gelsolin.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas do Olho/metabolismo , Gelsolina/metabolismo , Retinose Pigmentar/metabolismo , Actinas/metabolismo , Animais , Proteínas de Transporte/genética , Cílios/metabolismo , Modelos Animais de Doenças , Proteínas do Olho/genética , Gelsolina/genética , Humanos , Células-Tronco Pluripotentes Induzidas , Camundongos , Camundongos Knockout , Células Fotorreceptoras de Vertebrados/metabolismo , Transporte Proteico , Retinose Pigmentar/genética , Rodopsina/metabolismoRESUMO
BACKGROUND: Actin depolymerizing proteins of the actin depolymerizing factor (ADF)/cofilin family are essential for actin dynamics, which is critical for synaptic function. Two ADF/cofilin family members, ADF and n-cofilin, are highly abundant in the brain, where they are present in excitatory synapses. Previous studies demonstrated the relevance of n-cofilin for postsynaptic plasticity, associative learning, and anxiety. These studies also suggested overlapping functions for ADF and n-cofilin. METHODS: We performed pharmacobehavioral, electrophysiologic, and electron microscopic studies on ADF and n-cofilin single mutants and double mutants (named ACC mice) to characterize the importance of ADF/cofilin activity for synapse physiology and mouse behavior. RESULTS: The ACC mice, but not single mutants, exhibited hyperlocomotion, impulsivity, and impaired working memory. Hyperlocomotion and impulsive behavior were reversed by methylphenidate, a psychostimulant commonly used for the treatment of attention-deficit/hyperactivity disorder (ADHD). Also, ACC mice displayed a disturbed morphology of striatal excitatory synapses, accompanied by strongly increased glutamate release. Blockade of dopamine or glutamate transmission resulted in normal locomotion. CONCLUSIONS: Our study reveals that ADHD can result from a disturbed balance between excitation and inhibition in striatal circuits, providing novel insights into the mechanisms underlying this neurobehavioral disorder. Our results link actin dynamics to ADHD, suggesting that mutations in actin regulatory proteins may contribute to the etiology of ADHD in humans.
Assuntos
Transtorno do Deficit de Atenção com Hiperatividade/fisiopatologia , Transtorno do Deficit de Atenção com Hiperatividade/psicologia , Cofilina 1/fisiologia , Corpo Estriado/ultraestrutura , Destrina/fisiologia , Animais , Transtorno do Deficit de Atenção com Hiperatividade/genética , Estimulantes do Sistema Nervoso Central/farmacologia , Cofilina 1/genética , Cofilina 1/metabolismo , Destrina/genética , Modelos Animais de Doenças , Antagonistas de Dopamina , Fosfoproteína 32 Regulada por cAMP e Dopamina/metabolismo , Potenciais Pós-Sinápticos Excitadores , Glutamatos/metabolismo , Comportamento Impulsivo/efeitos dos fármacos , Comportamento Impulsivo/fisiologia , Masculino , Memória de Curto Prazo/fisiologia , Metilfenidato/farmacologia , Camundongos , Camundongos Knockout , Atividade Motora/genética , Comportamento de Nidação , Neurônios/metabolismo , Neurônios/ultraestrutura , Fenótipo , Receptores Dopaminérgicos/fisiologia , Substância Negra/metabolismo , Sinapses/ultraestruturaRESUMO
Genetic co-depletion of the actin-severing proteins ADF and CFL1 triggers catastrophic loss of adult homeostasis in multiple tissues. There is impaired cell-cell adhesion in skin keratinocytes with dysregulation of E-cadherin, hyperproliferation of differentiated cells, and ultimately apoptosis. Mechanistically, the primary consequence of depleting both ADF and CFL1 is uncontrolled accumulation of contractile actin stress fibers associated with enlarged focal adhesions at the plasma membrane, as well as reduced rates of membrane protrusions. This generates increased intracellular acto-myosin tension that promotes nuclear deformation and physical disruption of the nuclear lamina via the LINC complex that normally connects regulated actin filaments to the nuclear envelope. We therefore describe a pathway involving the actin-severing proteins ADF and CFL1 in regulating the dynamic turnover of contractile actin stress fibers, and this is vital to prevent the nucleus from being damaged by actin contractility, in turn preserving cell survival and tissue homeostasis.
Assuntos
Citoesqueleto de Actina/metabolismo , Cofilina 1/metabolismo , Destrina/metabolismo , Proteína 3 Relacionada a Actina/antagonistas & inibidores , Proteína 3 Relacionada a Actina/genética , Proteína 3 Relacionada a Actina/metabolismo , Animais , Caderinas/metabolismo , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Sobrevivência Celular , Células Cultivadas , Cofilina 1/antagonistas & inibidores , Cofilina 1/genética , Destrina/deficiência , Destrina/genética , Adesões Focais/metabolismo , Forminas , Queratinócitos/citologia , Queratinócitos/metabolismo , Camundongos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/antagonistas & inibidores , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , NADPH Desidrogenase/antagonistas & inibidores , NADPH Desidrogenase/genética , NADPH Desidrogenase/metabolismo , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Membrana Nuclear/metabolismo , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Pele/metabolismo , Pele/patologiaRESUMO
Mutations in the human actin depolymerizing factor cofilin2 result in an autosomal dominant form of nemaline myopathy. Here, we report on the targeted ablation of murine cofilin2, which leads to a severe skeletal muscle specific phenotype within the first two weeks after birth. Apart from skeletal muscle, cofilin2 is also expressed in heart and CNS, however the pathology was restricted to skeletal muscle. The two close family members of cofilin2 - ADF and cofilin1 - were co-expressed in muscle, but unable to compensate for the loss of cofilin2. While primary myofibril assembly and muscle development were unaffected in cofilin2 mutant mice, progressive muscle degeneration was observed between postnatal days 3 and 7. Muscle pathology was characterized by sarcoplasmic protein aggregates, fiber size disproportion, mitochondrial abnormalities and internal nuclei. The observed muscle pathology differed from nemaline myopathy, but showed combined features of actin-associated myopathy and myofibrillar myopathy. In cofilin2 mutant mice, the postnatal expression pattern and turnover of sarcomeric α-actin isoforms were altered. Levels of smooth muscle α-actin were increased and remained high in developing muscles, suggesting that cofilin2 plays a crucial role during the exchange of α-actin isoforms during the early postnatal remodeling of the sarcomere.
Assuntos
Actinas/metabolismo , Cofilina 2/metabolismo , Músculo Esquelético/metabolismo , Doenças Musculares/genética , Agregados Proteicos/genética , Sarcômeros/metabolismo , Animais , Encéfalo/metabolismo , Cofilina 2/genética , Citoesqueleto/genética , Citoesqueleto/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Desenvolvimento Muscular , Músculo Esquelético/crescimento & desenvolvimento , Músculo Esquelético/patologia , Músculo Liso/crescimento & desenvolvimento , Músculo Liso/metabolismo , Doenças Musculares/patologia , Miocárdio/metabolismo , Especificidade de ÓrgãosRESUMO
Dynamic changes in the actin cytoskeleton are essential for immune cell function and a number of immune deficiencies have been linked to mutations, which disturb the actin cytoskeleton. In macrophages and dendritic cells, actin remodelling is critical for motility, phagocytosis and antigen presentation, however the actin binding proteins, which control antigen presentation have been poorly characterized. Here we dissect the specific roles of the family of ADF/cofilin F-actin depolymerizing factors in macrophages and in local immune responses. Macrophage migration, cell polarization and antigen presentation to T-cells require n-cofilin mediated F-actin remodelling. Using a conditional mouse model, we show that n-cofilin also controls MHC class II-dependent antigen presentation. Other cellular processes such as phagocytosis and antigen processing were found to be independent of n-cofilin. Our data identify n-cofilin as a novel regulator of antigen presentation, while ADF on the other hand is dispensable for macrophage motility and antigen presentation.
Assuntos
Actinas/metabolismo , Cofilina 1/metabolismo , Macrófagos/imunologia , Fatores de Despolimerização de Actina/metabolismo , Animais , Movimento Celular , Proliferação de Células , Forma Celular , Citocinese , Células Dendríticas/citologia , Células Dendríticas/imunologia , Deleção de Genes , Macrófagos/citologia , Macrófagos/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL , Mutação/genética , Isoformas de Proteínas/metabolismoRESUMO
Neurites are the characteristic structural element of neurons that will initiate brain connectivity and elaborate information. Early in development, neurons are spherical cells but this symmetry is broken through the initial formation of neurites. This fundamental step is thought to rely on actin and microtubule dynamics. However, it is unclear which aspects of the complex actin behavior control neuritogenesis and which molecular mechanisms are involved. Here, we demonstrate that augmented actin retrograde flow and protrusion dynamics facilitate neurite formation. Our data indicate that a single family of actin regulatory proteins, ADF/Cofilin, provides the required control of actin retrograde flow and dynamics to form neurites. In particular, the F-actin severing activity of ADF/Cofilin organizes space for the protrusion and bundling of microtubules, the backbone of neurites. Our data reveal how ADF/Cofilin organizes the cytoskeleton to drive actin retrograde flow and thus break the spherical shape of neurons.