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1.
Am J Hum Genet ; 110(7): 1086-1097, 2023 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-37339631

RESUMO

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by the degeneration of motor neurons. Although repeat expansion in C9orf72 is its most common cause, the pathogenesis of ALS isn't fully clear. In this study, we show that repeat expansion in LRP12, a causative variant of oculopharyngodistal myopathy type 1 (OPDM1), is a cause of ALS. We identify CGG repeat expansion in LRP12 in five families and two simplex individuals. These ALS individuals (LRP12-ALS) have 61-100 repeats, which contrasts with most OPDM individuals with repeat expansion in LRP12 (LRP12-OPDM), who have 100-200 repeats. Phosphorylated TDP-43 is present in the cytoplasm of iPS cell-derived motor neurons (iPSMNs) in LRP12-ALS, a finding that reproduces the pathological hallmark of ALS. RNA foci are more prominent in muscle and iPSMNs in LRP12-ALS than in LRP12-OPDM. Muscleblind-like 1 aggregates are observed only in OPDM muscle. In conclusion, CGG repeat expansions in LRP12 cause ALS and OPDM, depending on the length of the repeat. Our findings provide insight into the repeat length-dependent switching of phenotypes.


Assuntos
Esclerose Lateral Amiotrófica , Distrofias Musculares , Doenças Neurodegenerativas , Humanos , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Neurônios Motores/patologia , Distrofias Musculares/genética , Doenças Neurodegenerativas/genética , Proteína C9orf72/genética , Expansão das Repetições de DNA , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética
2.
PLoS One ; 17(3): e0264965, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35271616

RESUMO

Trisomy 21, 18, and 13 are the major autosomal aneuploidy disorders in humans. They are mostly derived from chromosome non-disjunction in maternal meiosis, and the extra trisomic chromosome can cause several congenital malformations. Various genes on the trisomic chromosomes are intricately involved in the development of disease, and fundamental treatments have not yet been established. However, chromosome therapy has been developed to correct the extra chromosome in cultured patient cells, and it was recently reported that during reprogramming into iPSCs, fibroblasts from a Down syndrome patient lost the extra chromosome 21 due to a phenomenon called trisomy-biased chromosome loss. To gain preliminary insights into the underlying mechanism of trisomy rescue during the early stages of reprogramming, we reprogrammed skin fibroblasts from patients with trisomy syndromes 21, 18, 13, and 9 to iPSC, and evaluated the genomes of the individual iPSC colonies by molecular cytogenetic techniques. We report the spontaneous correction from trisomy to disomy upon cell reprogramming in at least one cell line examined from each of the trisomy syndromes, and three possible combinations of chromosomes were selected in the isogenic trisomy-rescued iPSC clones. Single nucleotide polymorphism analysis showed that the trisomy-rescued clones exhibited either heterodisomy or segmental uniparental isodisomy, ruling out the possibility that two trisomic chromosomes were lost simultaneously and the remaining one was duplicated, suggesting instead that one trisomic chromosome was lost to generate disomic cells. These results demonstrated that trisomy rescue may be a phenomenon with random loss of the extra chromosome and subsequent selection for disomic iPSCs, which is analogous to the karyotype correction in early preimplantation embryos. Our finding is relevant for elucidating the mechanisms of autonomous karyotype correction and future application in basic and clinical research on aneuploidy disorders.


Assuntos
Síndrome de Down , Células-Tronco Pluripotentes Induzidas , Aneuploidia , Cromossomos , Síndrome de Down/genética , Humanos , Mosaicismo , Trissomia/genética , Dissomia Uniparental
3.
Methods Mol Biol ; 2374: 49-57, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34562242

RESUMO

Primary cilia are antenna-like structures that develop on the surface of quiescent G0-phase cells and receive extracellular signals including sonic hedgehog (Shh) for embryogenesis and adult tissue homeostasis. In mammalian cells, cholesterol activates the seven-transmembrane protein Smoothened to transduce the Shh signal. Germline mutations of the DHCR7 gene encoding the cholesterol biogenesis enzyme 7-dehydrocholesterol reductase cause Smith-Lemli-Opitz syndrome with ciliopathy-related symptoms such as polycystic kidney and polydactyly, implying that cholesterol is indeed involved in ciliary functions. Notably, it has been reported that the cholesterol in ciliary membranes is significantly more abundant than that in the rest of the plasma membrane. However, several studies have failed to image the enriched ciliary cholesterol. Here, we propose a set of protocols for the sensitive imaging of ciliary cholesterol using the fluorescent small compound Filipin III and the green fluorescent protein tagged Domain 4 of the exotoxin Perfringolysin O derived from the anaerobic bacterium Clostridium perfringens. These cholesterol probes should be powerful tools for understanding the physiological and pathological roles of ciliary cholesterol in the context of Shh signaling in mammalian cells.


Assuntos
Cílios , Animais , Composição de Bases , Colesterol , Proteínas Hedgehog , Filogenia , RNA Ribossômico 16S , Análise de Sequência de DNA , Transdução de Sinais
4.
Sci Rep ; 11(1): 19661, 2021 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-34608183

RESUMO

Genetic information is protected against a variety of genotoxins including ionizing radiation (IR) through the DNA double-strand break (DSB) repair machinery. Genome-wide association studies and clinical sequencing of cancer patients have suggested that a number of variants in the DNA DSB repair genes might underlie individual differences in chromosomal radiosensitivity within human populations. However, the number of established variants that directly affect radiosensitivity is still limited. In this study, we performed whole-exome sequencing of 29 Japanese ovarian cancer patients and detected the NBS1 I171V variant, which is estimated to exist at a rate of approximately 0.15% in healthy human populations, in one patient. To clarify whether this variant indeed contributes to chromosomal radiosensitivity, we generated NBS1 I171V variant homozygous knock-in HCT116 cells and mice using the CRISPR/Cas9 system. Radiation-induced micronucleus formation and chromosomal aberration frequency were significantly increased in both HCT116 cells and mouse embryonic fibroblasts (MEFs) with knock-in of the NBS1 I171V variant compared with the levels in wild-type cells. These results suggested that the NBS1 I171V variant might be a genetic factor underlying individual differences in chromosomal radiosensitivity.


Assuntos
Alelos , Substituição de Aminoácidos , Variação Biológica da População/genética , Proteínas de Ciclo Celular/genética , Instabilidade Cromossômica/efeitos da radiação , Mutação , Proteínas Nucleares/genética , Tolerância a Radiação/genética , Sítios de Ligação , Biomarcadores Tumorais , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Variações do Número de Cópias de DNA , Feminino , Edição de Genes , Técnicas de Introdução de Genes , Predisposição Genética para Doença , Humanos , Proteínas Nucleares/metabolismo , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/radioterapia , Ligação Proteica , Radiação Ionizante
5.
Aging Cell ; 19(11): e13251, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-33094908

RESUMO

Damage to the genome can accelerate aging. The percentage of aneuploid cells, that is, cells with an abnormal number of chromosomes, increases during aging; however, it is not clear whether increased aneuploidy accelerates aging. Here, we report an individual showing premature aging phenotypes of various organs including early hair loss, atrophic skin, and loss of hematopoietic stem cells; instability of chromosome numbers known as mosaic variegated aneuploidy (MVA); and spindle assembly checkpoint (SAC) failure. Exome sequencing identified a de novo heterozygous germline missense mutation of c.856C>A (p.R286S) in the mitotic activator CDC20. The mutant CDC20 showed lower binding affinity to BUBR1 during the formation of the mitotic checkpoint complex (MCC), but not during the interaction between MCC and the anaphase-promoting complex/cyclosome (APC/C)-CDC20 complex. While heterozygous knockout of CDC20 did not induce SAC failure, knock-in of the mutant CDC20 induced SAC failure and random aneuploidy in cultured cells, indicating that the particular missense mutation is pathogenic probably via the resultant imbalance between MCC and APC/C-CDC20 complex. We postulate that accelerated chromosome number instability induces premature aging in humans, which may be associated with early loss of stem cells. These findings could form the basis of a novel disease model of the aging of the body and organs.


Assuntos
Proteínas Cdc20/genética , Senilidade Prematura , Feminino , Humanos , Pessoa de Meia-Idade , Mutação
6.
EMBO J ; 39(12): e103499, 2020 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-32368833

RESUMO

Primary cilia are antenna-like organelles on the surface of most mammalian cells that receive sonic hedgehog (Shh) signaling in embryogenesis and carcinogenesis. Cellular cholesterol functions as a direct activator of a seven-transmembrane oncoprotein called Smoothened (Smo) and thereby induces Smo accumulation on the ciliary membrane where it transduces the Shh signal. However, how cholesterol is supplied to the ciliary membrane remains unclear. Here, we report that peroxisomes are essential for the transport of cholesterol into the ciliary membrane. Zellweger syndrome (ZS) is a peroxisome-deficient hereditary disorder with several ciliopathy-related features and cells from these patients showed a reduced cholesterol level in the ciliary membrane. Reverse genetics approaches revealed that the GTP exchange factor Rabin8, the Rab GTPase Rab10, and the microtubule minus-end-directed kinesin KIFC3 form a peroxisome-associated complex to control the movement of peroxisomes along microtubules, enabling communication between peroxisomes and ciliary pocket membranes. Our findings suggest that insufficient ciliary cholesterol levels may underlie ciliopathies.


Assuntos
Colesterol/metabolismo , Cílios/metabolismo , Síndrome de Zellweger/metabolismo , Células Cultivadas , Colesterol/genética , Cílios/genética , Cílios/patologia , Quinases do Centro Germinativo/genética , Quinases do Centro Germinativo/metabolismo , Humanos , Cinesinas/genética , Cinesinas/metabolismo , Microtúbulos/genética , Microtúbulos/metabolismo , Microtúbulos/patologia , Receptor Smoothened/genética , Receptor Smoothened/metabolismo , Síndrome de Zellweger/genética , Síndrome de Zellweger/patologia , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo
7.
Biochem Biophys Res Commun ; 527(3): 716-722, 2020 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-32423824

RESUMO

p27Kip1, a member of the Cip/Kip family of cyclin-dependent kinase (CDK) inhibitors, is now known as a multifunctional protein that plays crucial roles in cell architecture and migration by regulating rearrangements of the actin cytoskeleton and microtubules. The intracellular level of p27Kip1 is increased by anti-proliferative stimuli, such as mitogen deprivation and contact inhibition, which also induce formation of primary cilia, microtubule-based membranous organelles that protrude from the cell surface. However, it remains unknown whether p27Kip1 is associated with ciliogenesis. Here, we have generated p27Kip1-knockout hTERT-immortalized human retinal pigment epithelial cells, and found that ciliogenesis is almost completely disrupted in p27Kip1-knockout cells. The defect of ciliogenesis is rescued by the exogenous expression of wild-type p27Kip1 and, surprisingly, its 86-140 amino acid region, which is neither responsible for CDK inhibition nor remodeling of the actin cytoskeleton and microtubules. Moreover, transmission electron microscopy and immunofluorescence analyses reveal that p27Kip1 abrogation impairs one of the earliest events of ciliogenesis, docking of the Ehd1-associated preciliary vesicles to the distal appendages of the basal body. Our findings identify a novel CDK-independent function of p27Kip1 in primary cilia formation.


Assuntos
Cílios/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Epitélio Pigmentado da Retina/citologia , Linhagem Celular , Cílios/ultraestrutura , Inibidor de Quinase Dependente de Ciclina p27/genética , Técnicas de Inativação de Genes , Humanos , Epitélio Pigmentado da Retina/metabolismo
8.
Cells ; 9(1)2020 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-31963583

RESUMO

Chromosomal segregation errors in germ cells and early embryonic development underlie aneuploidies, which are numerical chromosomal abnormalities causing fetal absorption, developmental anomalies, and carcinogenesis. It has been considered that human aneuploidy disorders cannot be resolved by radical treatment. However, recent studies have demonstrated that aneuploidies can be rescued to a normal diploid state using genetic engineering in cultured cells. Here, we summarize a series of studies mainly applying genome editing to eliminate an extra copy of human chromosome 21, the cause of the most common constitutional aneuploidy disorder Down syndrome. We also present findings on induced pluripotent stem cell reprogramming, which has been shown to be one of the most promising technologies for converting aneuploidies into normal diploidy without the risk of genetic alterations such as genome editing-mediated off-target effects.


Assuntos
Técnicas de Reprogramação Celular/métodos , Transtornos Cromossômicos/genética , Transtornos Cromossômicos/terapia , Síndrome de Down/genética , Edição de Genes/métodos , Células-Tronco Pluripotentes Induzidas/metabolismo , Cromossomos Sexuais/genética , Trissomia/genética , Aneuploidia , Sistemas CRISPR-Cas , Transtornos Cromossômicos/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Síndrome de Down/terapia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Mosaicismo , Cromossomos Sexuais/patologia
9.
Genes Cells ; 23(12): 1023-1042, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30318703

RESUMO

The centrosome is a small but important organelle that participates in centriole duplication, spindle formation, and ciliogenesis. Each event is regulated by key enzymatic reactions, but how these processes are integrated remains unknown. Recent studies have reported that ciliogenesis is controlled by distal appendage proteins such as FBF1, also known as Albatross. However, the precise role of Albatross in the centrosome cycle, including centriole duplication and centrosome separation, remains to be determined. Here, we report a novel function for Albatross at the proximal ends of centrioles. Using Albatross monospecific antibodies, full-length constructs, and siRNAs for rescue experiments, we found that Albatross mediates centriole duplication by recruiting HsSAS-6, a cartwheel protein of centrioles. Moreover, Albatross participates in centrosome separation during mitosis by recruiting Plk1 to residue S348 of Albatross after its phosphorylation. Taken together, our results show that Albatross is a novel protein that spatiotemporally integrates different aspects of centrosome function, namely ciliogenesis, centriole duplication, and centrosome separation.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Centríolos/metabolismo , Centrossomo/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Animais , Proteínas de Ciclo Celular/metabolismo , Células HEK293 , Células HeLa/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Células NIH 3T3 , Fosforilação , Fosfosserina/metabolismo , Ligação Proteica , Domínios Proteicos , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Serina/metabolismo , Quinase 1 Polo-Like
10.
J Radiat Res ; 59(suppl_2): ii75-ii82, 2018 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-29528422

RESUMO

DNA double-strand breaks (DSBs) induced by ionizing radiation (IR) are the initial and critical step in major alteration of genetic information and cell death. To prevent deleterious effects, DNA repair systems recognize and re-join DNA DSBs in human cells. It has been suggested that there are individual differences in radiosensitivity within human populations, and that variations in DNA repair genes might contribute to this heterogeneity. Because confounding factors, including age, gender, smoking, and diverse genetic backgrounds within human populations, also influence the cellular radiosensitivity, to accurately measure the effect of candidate genetic variations on radiosensitivity, it is necessary to use human cultured cells with a uniform genetic background. However, a reverse genetics approach in human cultured cells is difficult because of their low level of homologous recombination. Engineered endonucleases used in genome editing technology, however, can enable the local activation of DNA repair pathways at the human genome target site to efficiently introduce genetic variations of interest into human cultured cells. Recently, we used this technology to demonstrate that heterozygous mutations of the ATM gene, which is responsible for a hyper-radiosensitive genetic disorder, ataxia-telangiectasia, increased the number of chromosomal aberrations after IR. Thus, understanding the heterozygous mutations of radiosensitive disorders should shed light on the genetic basis underlying individual differences in radiosensitivity within human populations.


Assuntos
Edição de Genes/métodos , Genética Populacional , Tolerância a Radiação/genética , Reparo do DNA/genética , Predisposição Genética para Doença , Humanos , Mutação/genética
11.
J Radiat Res ; 59(suppl_2): ii11-ii17, 2018 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-29053826

RESUMO

It is difficult to distinguish radiation-induced events from spontaneous events during induction of stochastic effects, especially in the case of low-dose or low-dose-rate exposures. By using a hypersensitive system for detecting somatic mutations at the HPRT1 locus, we investigated the frequency and spectrum of mutations induced by low-dose X-rays. The mutant frequencies induced by doses of >0.15 Gy were statistically significant when compared with the spontaneous frequency, and a clear dose dependency was also observed for mutant frequencies at doses of >0.15 Gy. In contrast, mutant frequencies at doses of <0.1 Gy occurred at non-significant levels. The mutation spectrum in HPRT-deficient mutants revealed that the type of mutations induced by low-dose exposures was similar to that seen in spontaneous mutants. An apparent change in mutation type was observed for mutants induced by doses of >0.2 Gy. Our observations suggest that there could be a critical dose for mutation induction at between 0.1 Gy and 0.2 Gy, where mutagenic events are induced by multiple DNA double-strand breaks (DSBs). These observations also suggest that low-dose radiation delivered at doses of <0.1 Gy may not result in DSB-induced mutations but may enhance spontaneous mutagenesis events.


Assuntos
Mutação/genética , Radiação , Animais , Linhagem Celular , Cromossomos Humanos X/genética , Cricetinae , Relação Dose-Resposta à Radiação , Loci Gênicos , Humanos , Hipoxantina Fosforribosiltransferase/deficiência , Hipoxantina Fosforribosiltransferase/genética , Modelos Genéticos , Mutagênese , Taxa de Mutação , Raios X
12.
J Hum Genet ; 63(2): 133-143, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29167553

RESUMO

Current deep-sequencing technology provides a mass of nucleotide variations associated with human genetic disorders to accelerate the identification of causative mutations. To understand the etiology of genetic disorders, reverse genetics in human cultured cells is a useful approach for modeling a disease in vitro. However, gene targeting in human cultured cells is difficult because of their low activity of homologous recombination. Engineered endonucleases enable enhancement of the local activation of DNA repair pathways at the human genome target site to rewrite the desired sequence, thereby efficiently generating disease-modeling cultured cell clones. These edited cells can be used to explore the molecular functions of a causative gene product to uncover the etiological mechanisms. The correction of mutations in patient cells using genome editing technology could contribute to the development of unique gene therapies. This technology can also be applied to screening causative mutations. Rare genetic disorders and non-exonic mutation-caused diseases remain frontier in the field of human genetics as it is difficult to validate whether the extracted nucleotide variants are mutation or polymorphism. When isogenic human cultured cells with a candidate variant reproduce the pathogenic phenotypes, it is confirmed that the variant is a causative mutation.


Assuntos
Edição de Genes/métodos , Genética Humana/métodos , Modelos Genéticos , Células Cultivadas , Humanos
13.
Hum Mol Genet ; 26(22): 4429-4440, 2017 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-28973348

RESUMO

Primary microcephaly (MCPH) is an autosomal recessive disorder characterized by congenital reduction of head circumference. Here, we identified compound heterozygous mutations c.731 C > T (p.Ser 244 Leu) and c.2413 G > T (p.Glu 805 X) in the WDR62/MCPH2 gene, which encodes the mitotic centrosomal protein WDR62, in two siblings in a Japanese family with microcephaly using whole-exome sequencing. However, the molecular and cellular pathology of microcephaly caused by WDR62/MCPH2 mutation remains unclear. To clarify the physiological role of WDR62, we used the CRISPR/Cas9 system and single-stranded oligonucleotides as a point-mutation-targeting donor to generate human cell lines with knock-in of WDR62/MCPH2 c.731 C > T (p.Ser 244 Leu) missense mutation. In normal metaphase, the mitotic spindle forms parallel to the substratum to ensure symmetric cell division, while WDR62/MCPH2-mutated cells exhibited a randomized spindle orientation caused by the impaired astral microtubule assembly. It was shown that a mitotic kinase, Polo-like kinase 1 (PLK1), is required for the maintenance of spindle orientation through astral microtubule development. In this study, we demonstrated that WDR62 is a PLK1 substrate that is phosphorylated at Ser 897, and that this phosphorylation at the spindle poles promotes astral microtubule assembly to stabilize spindle orientation. Our findings provide insights into the role of the PLK1-WDR62 pathway in the maintenance of proper spindle orientation.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fuso Acromático/fisiologia , Sequência de Bases , Proteínas de Ciclo Celular/genética , Divisão Celular/genética , Linhagem Celular , Centrossomo/metabolismo , Feminino , Técnicas de Introdução de Genes , Células HCT116 , Humanos , Recém-Nascido , Masculino , Microcefalia/genética , Microcefalia/metabolismo , Microtúbulos/genética , Microtúbulos/metabolismo , Mitose/genética , Mitose/fisiologia , Mutação de Sentido Incorreto , Proteínas do Tecido Nervoso/genética , Fosforilação , Gravidez , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Fuso Acromático/genética , Fuso Acromático/metabolismo , Quinase 1 Polo-Like
14.
Sci Rep ; 7(1): 5996, 2017 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-28729543

RESUMO

Ionizing radiation (IR) induces DNA double-strand breaks (DSBs), which are an initial step towards chromosomal aberrations and cell death. It has been suggested that there are individual differences in radiosensitivity within human populations, and that the variations in DNA repair genes might determine this heterogeneity. However, it is difficult to quantify the effect of genetic variants on the individual differences in radiosensitivity, since confounding factors such as smoking and the diverse genetic backgrounds within human populations affect radiosensitivity. To precisely quantify the effect of a genetic variation on radiosensitivity, we here used the CRISPR-ObLiGaRe (Obligate Ligation-Gated Recombination) method combined with the CRISPR/Cas9 system and a nonhomologous end joining (NHEJ)-mediated knock-in technique in human cultured cells with a uniform genetic background. We generated ATM heterozygous knock-out (ATM +/-) cell clones as a carrier model of a radiation-hypersensitive autosomal-recessive disorder, ataxia-telangiectasia (A-T). Cytokinesis-blocked micronucleus assay and chromosome aberration assay showed that the radiosensitivity of ATM +/- cell clones was significantly higher than that of ATM +/+ cells, suggesting that ATM gene variants are indeed involved in determining individual radiosensitivity. Importantly, the differences in radiosensitivity among the same genotype clones were small, unlike the individual differences in fibroblasts derived from A-T-affected family members.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/genética , Edição de Genes , Individualidade , Mutação/genética , Tolerância a Radiação/genética , Automação , Sistemas CRISPR-Cas/genética , Células Cultivadas , Células Clonais , Citocinese , Fibroblastos/metabolismo , Fibroblastos/patologia , Heterozigoto , Humanos , Testes para Micronúcleos , Modelos Biológicos , Recombinação Genética/genética
18.
Nature ; 521(7551): 217-221, 2015 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-25778702

RESUMO

Vertebrates have a unique 3D body shape in which correct tissue and organ shape and alignment are essential for function. For example, vision requires the lens to be centred in the eye cup which must in turn be correctly positioned in the head. Tissue morphogenesis depends on force generation, force transmission through the tissue, and response of tissues and extracellular matrix to force. Although a century ago D'Arcy Thompson postulated that terrestrial animal body shapes are conditioned by gravity, there has been no animal model directly demonstrating how the aforementioned mechano-morphogenetic processes are coordinated to generate a body shape that withstands gravity. Here we report a unique medaka fish (Oryzias latipes) mutant, hirame (hir), which is sensitive to deformation by gravity. hir embryos display a markedly flattened body caused by mutation of YAP, a nuclear executor of Hippo signalling that regulates organ size. We show that actomyosin-mediated tissue tension is reduced in hir embryos, leading to tissue flattening and tissue misalignment, both of which contribute to body flattening. By analysing YAP function in 3D spheroids of human cells, we identify the Rho GTPase activating protein ARHGAP18 as an effector of YAP in controlling tissue tension. Together, these findings reveal a previously unrecognised function of YAP in regulating tissue shape and alignment required for proper 3D body shape. Understanding this morphogenetic function of YAP could facilitate the use of embryonic stem cells to generate complex organs requiring correct alignment of multiple tissues.


Assuntos
Tamanho Corporal/genética , Proteínas de Peixes/metabolismo , Morfogênese/genética , Oryzias/anatomia & histologia , Oryzias/embriologia , Actomiosina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Embrião não Mamífero/anatomia & histologia , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Proteínas de Peixes/genética , Proteínas Ativadoras de GTPase/metabolismo , Genes Essenciais/genética , Gravitação , Humanos , Mutação/genética , Tamanho do Órgão/genética , Oryzias/genética , Fenótipo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Esferoides Celulares/citologia , Esferoides Celulares/metabolismo
19.
Cell Rep ; 10(5): 664-673, 2015 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-25660017

RESUMO

The primary cilium is an antenna-like, microtubule-based organelle on the surface of most vertebrate cells for receiving extracellular information. Although primary cilia form in the quiescent phase, ciliary disassembly occurs when quiescent cells re-enter the proliferative phase. It was shown that a mitotic kinase, Polo-like kinase 1 (PLK1), is required for cell-proliferation-coupled primary cilia disassembly. Here, we report that kinesin superfamily protein 2A (KIF2A), phosphorylated at T554 by PLK1, exhibits microtubule-depolymerizing activity at the mother centriole to disassemble the primary cilium in a growth-signal-dependent manner. KIF2A-deficient hTERT-RPE1 cells showed the impairment of primary cilia disassembly following growth stimulation. It was also found that the PLK1-KIF2A pathway is constitutively active in cells from patients with premature chromatid separation (PCS) syndrome and is responsible for defective ciliogenesis in this syndrome. These findings provide insights into the roles of the PLK1-KIF2A pathway in physiological cilia disassembly and cilia-associated disorders.

20.
J Neurol Sci ; 337(1-2): 219-23, 2014 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-24332946

RESUMO

Progressive myoclonic ataxia (PMA) is a clinical syndrome defined as progressive ataxia and myoclonus and infrequent seizures in the absence of progressive dementia. Due to the extremely heterogeneous nature of PMA, a large proportion of PMA cases remain molecularly undiagnosed. The aim of this study was to clarify the molecular etiology of PMA. The patient was a 52-year-old female from consanguineous parents. She developed a jerking neck movement at age 9, which gradually expanded to her entire body. On physical examination at age 47, she exhibited generalized, spontaneous myoclonus that occurred continuously. She also presented with mild limb and truncal ataxia. An electroencephalogram revealed no abnormalities. A brain MRI displayed no atrophy of the cerebellum. Electrophysiological studies suggested myoclonus of a subcortical origin. For further evaluation, we performed exome sequencing, and we identified a novel homozygous missense mutation in the MRE11 gene (NM_005590:c.140C>T:p.A47V). Subsequently, we analyzed the expression of MRE11 and related proteins (RAD50 and NBS1) via Western blot, and they were markedly decreased compared to a healthy control. Mutations in the MRE11 gene have been known to cause an ataxia-telangiectasia-like (ATLD) disorder. Accumulating evidence has indicated that its wide phenotypic variations in ATLD correspond to genotypic differences. Interestingly, our case exhibited a relatively mild decrease in NBS1 compared to previously reported cases of a homozygous missense mutation, which may account for the milder phenotype in this patient. Moreover, together with a recently reported case of an MRE11 mutation, it is suggested that MRE11 mutations can present as PMA.


Assuntos
Proteínas de Ligação a DNA/genética , Mutação/genética , Dissinergia Cerebelar Mioclônica/genética , Encéfalo/patologia , Análise Mutacional de DNA , Exoma/genética , Feminino , Humanos , Proteína Homóloga a MRE11 , Imageamento por Ressonância Magnética , Pessoa de Meia-Idade , Dissinergia Cerebelar Mioclônica/patologia
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