RESUMO
Although strategies for directed differentiation of human pluripotent stem cells (hPSCs) into lung and airway have been established, terminal maturation of the cells remains a vexing problem. We show here that in collagen I 3D cultures in the absence of glycogen synthase kinase 3 (GSK3) inhibition, hPSC-derived lung progenitors (LPs) undergo multilineage maturation into proximal cells, type I alveolar epithelial cells and morphologically mature type II cells. Enhanced cell cycling, one of the signaling outputs of GSK3 inhibition, plays a role in the maturation-inhibiting effect of GSK3 inhibition. Using this model, we show NOTCH signaling induced a distal cell fate at the expense of a proximal and ciliated cell fate, whereas WNT signaling promoted a proximal club cell fate, thus implicating both signaling pathways in proximodistal specification in human lung development. These findings establish an approach to achieve multilineage maturation of lung and airway cells from hPSCs, demonstrate a pivotal role of GSK3 in the maturation of lung progenitors and provide novel insight into proximodistal specification during human lung development.
Assuntos
Técnicas de Cultura de Células/métodos , Diferenciação Celular , Linhagem da Célula , Quinase 3 da Glicogênio Sintase/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Pulmão/citologia , Piridinas/farmacologia , Animais , Padronização Corporal/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem da Célula/efeitos dos fármacos , Colágeno Tipo I/metabolismo , Genoma Humano , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/ultraestrutura , Camundongos , Receptores Notch/metabolismo , Reprodutibilidade dos Testes , Via de Sinalização Wnt/efeitos dos fármacosRESUMO
Primary cilia are microtubule structures that extend from the distal end of the mature, mother centriole. CEP164 is a component of the distal appendages carried by the mother centriole that is required for primary cilium formation. Recent data have implicated CEP164 as a ciliopathy gene and suggest that CEP164 plays some roles in the DNA damage response (DDR). We used reverse genetics to test the role of CEP164 in the DDR. We found that conditional depletion of CEP164 in chicken DT40 cells using an auxin-inducible degron led to no increase in sensitivity to DNA damage induced by ionising or ultraviolet irradiation. Disruption of CEP164 in human retinal pigmented epithelial cells blocked primary cilium formation but did not affect cellular proliferation or cellular responses to ionising or ultraviolet irradiation. Furthermore, we observed no localisation of CEP164 to the nucleus using immunofluorescence microscopy and analysis of multiple tagged forms of CEP164. Our data suggest that CEP164 is not required in the DDR.
Assuntos
Núcleo Celular/metabolismo , Reparo do DNA , Proteínas dos Microtúbulos/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Animais , Núcleo Celular/patologia , Galinhas , Cílios/genética , Cílios/metabolismo , Dano ao DNA , Edição de Genes , Células HeLa , Humanos , Células Jurkat , Proteínas dos Microtúbulos/genética , Epitélio Pigmentado da Retina/patologia , Raios Ultravioleta/efeitos adversosRESUMO
Centrins are small, highly conserved members of the EF-hand superfamily of calcium-binding proteins that are found throughout eukaryotes. They play a major role in ensuring the duplication and appropriate functioning of the ciliary basal bodies in ciliated cells. They have also been localised to the centrosome, which is the major microtubule organising centre in animal somatic cells. We describe the identification, cloning and characterisation of centrins in multiple eukaryotic species. Although centrins have been implicated in centriole biogenesis, recent results have indicated that centrosome duplication can, in fact, occur in the absence of centrins. We discuss these data and the non-centrosomal functions that are emerging for the centrins. In particular, we discuss the involvement of centrins in nucleotide excision repair, a process that repairs the DNA lesions that are induced primarily by ultraviolet irradiation. We discuss how centrin may be involved in these diverse processes and contribute to nuclear and cytoplasmic events.
Assuntos
Proteínas de Ligação ao Cálcio/fisiologia , Proteínas de Ciclo Celular/fisiologia , Centríolos/metabolismo , Proteínas Cromossômicas não Histona/fisiologia , Reparo do DNA , Genoma , Animais , Proteínas de Ligação ao Cálcio/química , Proteínas de Ciclo Celular/química , Centrossomo/metabolismo , Cílios/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Motivos EF Hand , Evolução Molecular , Humanos , Conformação Proteica , Fuso Acromático/genética , Vertebrados/metabolismoRESUMO
Intraflagellar transport (IFT) trains, built around IFT-A and IFT-B complexes, are carried by opposing motors to import and export ciliary cargo. While transported by kinesin-2 on anterograde IFT trains, the dynein-2 motor adopts an autoinhibitory conformation until it needs to be activated at the ciliary tip to power retrograde IFT. Growing evidence has linked the IFT-A complex to retrograde IFT; however, its roles in this process remain unknown. Here, we use CRISPR-Cas9-mediated genome editing to disable the dynein-2 autoinhibition mechanism in Caenorhabditis elegans and assess its impact on IFT with high-resolution live imaging and photobleaching analyses. Remarkably, this dynein-2 "hot-wiring" approach reignites retrograde motility inside IFT-A-deficient cilia without triggering tug-of-war events. In addition to providing functional evidence that multiple mechanisms maintain dynein-2 inhibited during anterograde IFT, our data establish key roles for IFT-A in mediating motor-train coupling during IFT turnaround, promoting retrograde IFT initiation, and modulating dynein-2 retrograde motility.
Assuntos
Proteínas de Caenorhabditis elegans , Dineínas , Animais , Dineínas/metabolismo , Transporte Biológico , Cílios/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Flagelos/metabolismoRESUMO
The dynein-2 motor complex drives retrograde intraflagellar transport (IFT), playing a pivotal role in the assembly and functions of cilia. However, the mechanisms that regulate dynein-2 motility remain poorly understood. Here, we identify the Caenorhabditis elegans WDR60 homologue, WDR-60, and dissect the roles of this intermediate chain using genome editing and live imaging of endogenous dynein-2/IFT components. We find that loss of WDR-60 impairs dynein-2 recruitment to cilia and its incorporation onto anterograde IFT trains, reducing retrograde motor availability at the ciliary tip. Consistent with this, we show that fewer dynein-2 motors power WDR-60-deficient retrograde IFT trains, which move at reduced velocities and fail to exit cilia, accumulating on the distal side of the transition zone. Remarkably, disrupting the transition zone's NPHP module almost fully restores ciliary exit of underpowered retrograde trains in wdr-60 mutants. This work establishes WDR-60 as a major contributor to IFT, and the NPHP module as a roadblock to dynein-2 passage through the transition zone.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Cílios/metabolismo , Proteínas do Citoesqueleto/metabolismo , Dineínas/metabolismo , Flagelos/metabolismo , Animais , Proteínas de Caenorhabditis elegans/química , Proteínas do Citoesqueleto/química , Dineínas/química , Proteínas de Fluorescência Verde/metabolismo , Cinética , Mutação/genética , Domínios Proteicos , Células Receptoras Sensoriais/metabolismoRESUMO
Cilia are microtubule-based organelles that carry out a wide range of critical functions throughout the development of higher animals. Regardless of their type, all cilia rely on a motor-driven, bidirectional transport system known as intraflagellar transport (IFT). Of the many components of the IFT machinery, IFT20 is one of the smallest subunits. Nevertheless, IFT20 has been shown to play critical roles in the assembly of several types of mammalian cilia. Here we show that the IFT20 homolog in Caenorhabditis elegans, IFT-20, is also important for correct cilium assembly in sensory neurons. Strikingly, however, we find that IFT-20-deficient animals are able to assemble short, vestigial cilia. In spite of this, we show that practically all IFT-20-deficient animals fail to respond to environmental cues that are normally detected by cilia to modulate their behavior. Altogether, our results indicate that IFT-20 is critical for both the correct assembly and function of cilia in C. elegans.
RESUMO
Autophagy plays critical roles in neurodegeneration and development, but how this pathway is organized and regulated in neurons remains poorly understood. Here, we find that the dynein adaptor RILP is essential for retrograde transport of neuronal autophagosomes, and surprisingly, their biogenesis as well. We find that induction of autophagy by mTOR inhibition specifically upregulates RILP expression and its localization to autophagosomes. RILP depletion or mutations in its LC3-binding LIR motifs strongly decrease autophagosome numbers suggesting an unexpected RILP role in autophagosome biogenesis. We find that RILP also interacts with ATG5 on isolation membranes, precluding premature dynein recruitment and autophagosome transport. RILP inhibition impedes autophagic turnover and causes p62/sequestosome-1 aggregation. Together, our results identify an mTOR-responsive neuronal autophagy pathway, wherein RILP integrates the processes of autophagosome biogenesis and retrograde transport to control autophagic turnover. This pathway has important implications for understanding how autophagy contributes to neuronal function, development, and disease.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteína 5 Relacionada à Autofagia/metabolismo , Autofagia , Proteínas Associadas aos Microtúbulos/metabolismo , Neurônios/fisiologia , Serina-Treonina Quinases TOR/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Autofagossomos , Proteína 5 Relacionada à Autofagia/genética , Transporte Biológico , Dineínas/metabolismo , Células HeLa , Humanos , Masculino , Proteínas Associadas aos Microtúbulos/genética , Neurônios/citologia , Ratos , Ratos Sprague-Dawley , Serina-Treonina Quinases TOR/genética , Proteínas rab de Ligação ao GTP/genética , proteínas de unión al GTP Rab7RESUMO
Cytoplasmic dynein participates in multiple aspects of neocortical development. These include neural progenitor proliferation, morphogenesis, and neuronal migration. The cytoplasmic dynein light intermediate chains (LICs) 1 and 2 are cargo-binding subunits, though their relative roles are not well understood. Here, we used in utero electroporation of shRNAs or LIC functional domains to determine the relative contributions of the two LICs in the developing rat brain. We find that LIC1, through BicD2, is required for apical nuclear migration in neural progenitors. In newborn neurons, we observe specific roles for LIC1 in the multipolar to bipolar transition and glial-guided neuronal migration. In contrast, LIC2 contributes to a novel dynein role in the little-studied mode of migration, terminal somal translocation. Together, our results provide novel insight into the LICs' unique functions during brain development and dynein regulation overall.
Assuntos
Encéfalo/embriologia , Movimento Celular , Dineínas do Citoplasma/metabolismo , Células-Tronco Neurais/metabolismo , Neurogênese , Animais , Encéfalo/citologia , Dineínas do Citoplasma/genética , Eletroporação , Células-Tronco Neurais/citologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Ratos , Ratos Sprague-DawleyRESUMO
Over the past two decades, substantial progress has been made in visualizing and understanding neuronal cell migration and morphogenesis during brain development. Distinct mechanisms have evolved to support migration of the various cell types that compose the developing neocortex. A specific subset of molecular motors, so far consisting of cytoplasmic dynein 1, Kif1a and myosin II, are responsible for cytoskeletal and nuclear transport in these cells. This review focuses on the emerging roles for each of these motor proteins in the migratory mechanisms of neocortical cell types. We discuss how migration can be cell cycle regulated and how coordination of motor activity is required to ensure migratory direction. © 2016 Wiley Periodicals, Inc.
Assuntos
Movimento Celular/fisiologia , Neocórtex/embriologia , Células-Tronco Neurais/metabolismo , Neurônios/metabolismo , Animais , Dineínas/metabolismo , Humanos , Cinesinas/metabolismo , Miosina Tipo II/metabolismo , Neocórtex/citologia , Células-Tronco Neurais/citologia , Neurônios/citologiaRESUMO
Microcephaly is a cortical malformation disorder characterized by an abnormally small brain. Recent studies have revealed severe cases of microcephaly resulting from human mutations in the NDE1 gene, which is involved in the regulation of cytoplasmic dynein. Here using in utero electroporation of NDE1 short hairpin RNA (shRNA) in embryonic rat brains, we observe cell cycle arrest of proliferating neural progenitors at three distinct stages: during apical interkinetic nuclear migration, at the G2-to-M transition and in regulation of primary cilia at the G1-to-S transition. RNAi against the NDE1 paralogue NDEL1 has no such effects. However, NDEL1 overexpression can functionally compensate for NDE1, except at the G2-to-M transition, revealing a unique NDE1 role. In contrast, NDE1 and NDEL1 RNAi have comparable effects on postmitotic neuronal migration. These results reveal that the severity of NDE1-associated microcephaly results not from defects in mitosis, but rather the inability of neural progenitors to ever reach this stage.
Assuntos
Microcefalia/etiologia , Proteínas Associadas aos Microtúbulos/genética , Células-Tronco Neurais/patologia , Animais , Encéfalo/embriologia , Encéfalo/patologia , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/genética , Pontos de Checagem do Ciclo Celular/genética , Movimento Celular/genética , Cílios/patologia , Modelos Animais de Doenças , Feminino , Técnicas de Silenciamento de Genes , Humanos , Microcefalia/genética , Microcefalia/patologia , Proteínas Associadas aos Microtúbulos/antagonistas & inibidores , Modelos Neurológicos , Mutação , Neuroglia/patologia , Gravidez , RNA Interferente Pequeno/genética , RatosRESUMO
Development of the cerebral cortex is a very dynamic process, involving a series of complex morphogenetic events. Following division of progenitor cells in the ventricular zone, neurons undergo a series of morphological changes and migrate outward toward the cortical plate, where they differentiate and integrate into functional circuits. Errors at several of stages during neurogenesis and migration cause a variety of severe cortical malformations. A number of disease genes encode factors associated with the cytoskeleton, which plays a crucial role throughout cortical development. Methods for regulating gene expression coupled with imaging of subcellular structures have provided important insight into the mechanisms governing normal and abnormal brain development. We describe here a series of protocols for imaging motor protein-dependent processes in real time in the developing rat brain.
Assuntos
Córtex Cerebral/metabolismo , Proteínas Motores Moleculares/genética , Células-Tronco Neurais/metabolismo , Animais , Movimento Celular/fisiologia , Córtex Cerebral/citologia , Eletroporação/métodos , Embrião de Mamíferos/inervação , Células Ependimogliais/citologia , Regulação da Expressão Gênica/genética , Proteínas de Fluorescência Verde/genética , Proteínas Luminescentes/genética , Microtúbulos/metabolismo , Células-Tronco Neurais/citologia , Transporte Proteico/fisiologia , Interferência de RNA , RNA Interferente Pequeno/genética , Ratos , Ratos Sprague-Dawley , Proteína Vermelha FluorescenteRESUMO
A wide range of subcellular organelles, pathogens, and macromolecular complexes are actively transported within neuronal and nonneuronal cells by microtubule motors. Transport speeds range up to 2-3 µm/s, which requires millisecond- and nanometer-scale resolution for proper imaging and analysis. Dissecting the contributions of multiple motor types has been challenging because of their functional interdependence and the complexity of individual motor behavior. In this chapter, we describe several methods for motor inhibition coupled with high-resolution particle tracking of vesicular and virus cargoes to provide a detailed and quantitative understanding of motor behavior and regulation. We discuss long-term inhibition, as well as short-term inhibition methods when needed to minimize complications from motor protein interactions.
Assuntos
Transporte Axonal/fisiologia , Hipocampo/metabolismo , Organelas/metabolismo , Animais , Células COS , Linhagem Celular Tumoral , Chlorocebus aethiops , Dineínas do Citoplasma/metabolismo , Endossomos/metabolismo , Células HeLa , Hipocampo/citologia , Humanos , Cinesinas/metabolismo , Lisossomos/metabolismo , Microtúbulos/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética , Ratos , Vírus/metabolismoRESUMO
The short rib polydactyly syndromes (SRPSs) are a heterogeneous group of autosomal recessive, perinatal lethal skeletal disorders characterized primarily by short, horizontal ribs, short limbs and polydactyly. Mutations in several genes affecting intraflagellar transport (IFT) cause SRPS but they do not account for all cases. Here we identify an additional SRPS gene and further unravel the functional basis for IFT. We perform whole-exome sequencing and identify mutations in a new disease-producing gene, cytoplasmic dynein-2 light intermediate chain 1, DYNC2LI1, segregating with disease in three families. Using primary fibroblasts, we show that DYNC2LI1 is essential for dynein-2 complex stability and that mutations in DYNC2LI1 result in variable length, including hyperelongated, cilia, Hedgehog pathway impairment and ciliary IFT accumulations. The findings in this study expand our understanding of SRPS locus heterogeneity and demonstrate the importance of DYNC2LI1 in dynein-2 complex stability, cilium function, Hedgehog regulation and skeletogenesis.
Assuntos
Cílios/metabolismo , Dineínas do Citoplasma/genética , Citoesqueleto/genética , Fibroblastos/metabolismo , Síndrome de Costela Curta e Polidactilia/genética , Transporte Biológico/genética , Feminino , Flagelos/metabolismo , Proteínas Hedgehog , Humanos , Masculino , Mutação , LinhagemRESUMO
Centrosomes, the principal microtubule-organizing centers of animal somatic cells, consist of two centrioles embedded in the pericentriolar material (PCM). Pericentrin is a large PCM protein that is required for normal PCM assembly. Mutations in PCNT cause primordial dwarfism. Pericentrin has also been implicated in the control of DNA damage responses. To test how pericentrin is involved in cell cycle control after genotoxic stress, we disrupted the Pcnt locus in chicken DT40 cells. Pericentrin-deficient cells proceeded through mitosis more slowly, with a high level of monopolar spindles, and were more sensitive to spindle poisons than controls. Centriole structures appeared normal by light and electron microscopy, but the PCM did not recruit γ-tubulin efficiently. Cell cycle delays after ionizing radiation (IR) treatment were normal in pericentrin-deficient cells. However, pericentrin disruption in Mcph1-/- cells abrogated centrosome hyperamplification after IR. We conclude that pericentrin controls genomic stability by both ensuring appropriate mitotic spindle activity and centrosome regulation.
Assuntos
Antígenos/genética , Proteínas Aviárias/genética , Proteínas de Ciclo Celular/genética , Centríolos/efeitos da radiação , Reparo do DNA , Mitose/efeitos da radiação , Animais , Antígenos/metabolismo , Proteínas Aviárias/metabolismo , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Centríolos/genética , Centríolos/metabolismo , Galinhas , Dano ao DNA , Deleção de Genes , Loci Gênicos , Instabilidade Genômica/efeitos da radiação , Regiões Promotoras Genéticas , Radiação Ionizante , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismoRESUMO
Centrosomes, the principal microtubule-organising centres in animal cells, contain centrins, small, conserved calcium-binding proteins unique to eukaryotes. Centrin2 binds to xeroderma pigmentosum group C protein (XPC), stabilising it, and its presence slightly increases nucleotide excision repair (NER) activity in vitro. In previous work, we deleted all three centrin isoforms present in chicken DT40 cells and observed delayed repair of UV-induced DNA lesions, but no centrosome abnormalities. Here, we explore how centrin2 controls NER. In the centrin null cells, we expressed centrin2 mutants that cannot bind calcium or that lack sites for phosphorylation by regulatory kinases. Expression of any of these mutants restored the UV sensitivity of centrin null cells to normal as effectively as expression of wild-type centrin. However, calcium-binding-deficient and T118A mutants showed greatly compromised localisation to centrosomes. XPC recruitment to laser-induced UV-like lesions was only slightly slower in centrin-deficient cells than in controls, and levels of XPC and its partner HRAD23B were unaffected by centrin deficiency. Interestingly, we found that overexpression of the centrin interactor POC5 leads to the assembly of linear, centrin-dependent structures that recruit other centrosomal proteins such as PCM-1 and NEDD1. Together, these observations suggest that assembly of centrins into complex structures requires calcium binding capacity, but that such assembly is not required for centrin activity in NER.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Cálcio/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Reparo do DNA , Animais , Autoantígenos/genética , Autoantígenos/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteínas de Transporte/genética , Proteínas de Ciclo Celular/genética , Linhagem Celular , Linhagem Celular Tumoral , Centrossomo/metabolismo , Centrossomo/ultraestrutura , Galinhas , Dano ao DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Immunoblotting , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Mutação Puntual , Ligação ProteicaAssuntos
Ciclo Celular , Microcefalia/patologia , Animais , Núcleo Celular/metabolismo , Humanos , Modelos Biológicos , Mutação/genética , RatosRESUMO
Cilia are found on most human cells and exist as motile cilia or non-motile primary cilia. Primary cilia play sensory roles in transducing various extracellular signals, and defective ciliary functions are involved in a wide range of human diseases. Centrosomes are the principal microtubule-organizing centers of animal cells and contain two centrioles. We observed that DNA damage causes centriole splitting in non-transformed human cells, with isolated centrioles carrying the mother centriole markers CEP170 and ninein but not kizuna or cenexin. Loss of centriole cohesion through siRNA depletion of C-NAP1 or rootletin increased radiation-induced centriole splitting, with C-NAP1-depleted isolated centrioles losing mother markers. As the mother centriole forms the basal body in primary cilia, we tested whether centriole splitting affected ciliogenesis. While irradiated cells formed apparently normal primary cilia, most cilia arose from centriolar clusters, not from isolated centrioles. Furthermore, C-NAP1 or rootletin knockdown reduced primary cilium formation. Therefore, the centriole cohesion apparatus at the proximal end of centrioles may provide a target that can affect primary cilium formation as part of the DNA damage response.