Estudo da Função Mitótica da Proteína Cep57 Humana na Regulação das Interacções dos Microtúbulos com os Centrossomas e Cinetocoros
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2009
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A distribuição equitativa dos cromossomas durante a divisão celular é essencial à viabilidade do organismo. Erros na segregação dos cromossomas estão na origem da aneuploidia associada a doenças do recém-nascido e tumorigénese. Deste modo, é fundamental a compreensão dos mecanismos moleculares envolvidos na fidelidade da segregação cromossómica.
A separação dos cromossomas durante a mitose requer a formação de uma estrutura simétrica bipolar, designada fuso mitótico, que é formada por microtúbulos nucleados a partir dos centrossomas em células animais. Os cromossomas são ligados ao fuso através da interacção dos cinetocoros com os microtúbulos. Existe um elevado número de proteínas associadas ao fuso, centrossomas e cinetocoros que regulam a bipolaridade, os movimentos cromossómicos e a fidelidade da segregação.
A proteína xCep57 foi previamente caracterizada em Xenopus como uma proteína adaptadora das ligações dos microtúbulos aos centrossomas e cinetocoros. A imunodepleção desta proteína num sistema in vitro de extractos de oócitos compromete a estrutura do fuso, o alinhamento dos cromossomas e a estabilidade das ligações cinetocoro-microtúbulo e centrosoma-microtúbulo. Consistentemente, esta proteína interactua com proteínas cinetocorianas reguladoras das ligações ao fuso bem como a γ-tubulina que faz parte dos anéis de nucleação de microtúbulos nos centrossomas.
Tem sido questionada a possibilidade da xCep57 ser o homólogo funcional da proteína Dam1 de levedura, para a qual foi demonstrado um papel fulcral na acoplagem da ligação do cinetocoro aos microtúbulos dinâmicos. Existe assim grande expectativa quanto à identificação de um homólogo em eucariotas superiores de função determinante na regulação dos movimentos cromossómicos e fidelidade de segregação.
Neste trabalho, partiu-se para um estudo funcional da proteína Cep57 humana com recurso à metodologia de RNA de interferência (RNAi) e microscopia de fluorescência confocal, com o objectivo de se caracterizar a função desta proteína num sistema in vivo através da sua repressão específica por RNAi. Verificou-se por imunofluorescência que a proteína Cep57 se localiza nos centrossomas em interfase e mitose, mas não se detectou nos cinetocoros. Também se demonstrou que a proteína Cep57 se liga directamente a microtúbulos in vitro. A análise do fenótipo mitótico de células depletadas de Cep57 por RNAi revelou a existência de problemas no alinhamento dos cromossomas na placa metafásica e na organização do fuso mitótico. As ligações cinetocoro-microtúbulos estão significativamente comprometidas e a distância interpolar está diminuída. No entanto, ao contrário dos resultados anteriormente descritos que apontam para a interacção de xCep57 com as proteínas cinetocorianas Zwint e Mis12, neste estudo não se observou a diminuição destas proteínas no cinetocoro de células RNAi-depletadas. Deste modo, o fenótipo mitótico observado parece resultar de uma função da proteína Cep57 no centrossoma e não no cinetocoro, possivelmente, e em particular, na ancoragem dos microtúbulos aos centrossomas. Curiosamente, através de um estudo in silico, descobriu-se a existência de um segundo homólogo de xCep57 em humano e em ratinho. Deixa-se assim em aberto a questão de, em humano, poderem existir duas proteínas homólogas de xCep57 com função acopladora aos microtúbulos, actuando uma nos centrossomas (provavelmente a Cep57 aqui descrita) e outra nos cinetocoros. Estudos futuros centrar-se-ão no esclarecimento desta questão.
The equal distribution of chromosomes during cell division is essential to the viability of the organism. Errors in chromosome segregation are the cause of aneuploidy associated with diseases of the newborn and tumorigenesis. Thus, it is essential the understanding of the molecular mechanisms involved in the fidelity of chromosome segregation. The separation of chromosomes during mitosis requires the formation of a symmetric bipolar structure, called spindle, which is formed by microtubules nucleated from centrosomes in animal cells. Chromosomes are attached to the spindle through interaction of the kinetochores with microtubules. There are a large number of proteins associated with the spindle, centrosomes and kinetochores governing spindle bipolarity, chromosome movement and the fidelity of chromosome segregation. xCep57 protein was previously characterized in Xenopus as an adapter of the connections of microtubules to the centrosomes and kinetochores. Immunodepletion of this protein in an in vitro system of oocyte extracts compromises the structure of the spindle, the alignment of chromosomes and stability of kinetochore-microtubule as well as centrosome-microtuble connections. Consistently, this protein interacts with regulatory kinetochore proteins linked to the spindle and γ-tubulin which is a main component of the nucleation rings of microtubules at centrosomes. It has been called into question the possibility of xCep57 be the functional homolog of the Dam1 yeast protein, for which a central role in coupling the attachment of kinetochore to dynamic microtubules has been shown. Thus, there are great expectations to the identification of a homolog with decisive role in regulating the movement and fidelity of chromosome segregation in higher eukaryotes. In this work, a functional study of the human protein Cep57 has been carried out in an in vivo system using the RNA interference (RNAi) methodology and confocal microscopy analysis. It was found by immunofluorescence that the Cep57 protein is located in centrosomes during both interphase and mitosis, but could not be detected in kinetochores. It was also demonstrated that the protein Cep57 binds directly to microtubules in vitro. Phenotypic analysis of Cep57 RNAi-depleted mitotic cells revealed the existence of chromosome alignment and spindle defects. The kinetochore-microtubule connections are significantly compromised and the interpolar distance is decreased. Futhermore, the kinetochore localization of known interactors of xCep57 is not altered in Cep57 RNAi-depleted cells consistently to the fact that human Cep57 could not be detected at kinetochores. Thus, overall, the mitotic phenotype observed seems to result from a centrosomal function of Cep57, possibly in microtubule anchorage to centrosomes. Interestingly, through an in silico study, we found the existence of a second homolog of xCep57 in human and mouse. Therefore, an open question remains whether both xCep57 protein homologues in human and mouse have a function in microtubule attachment, with Cep57 described here acting at the centrosome level and the other Cep57-related protein perhaps acting at the kinetochore level. Future studies will be focused on addressing this question.
The equal distribution of chromosomes during cell division is essential to the viability of the organism. Errors in chromosome segregation are the cause of aneuploidy associated with diseases of the newborn and tumorigenesis. Thus, it is essential the understanding of the molecular mechanisms involved in the fidelity of chromosome segregation. The separation of chromosomes during mitosis requires the formation of a symmetric bipolar structure, called spindle, which is formed by microtubules nucleated from centrosomes in animal cells. Chromosomes are attached to the spindle through interaction of the kinetochores with microtubules. There are a large number of proteins associated with the spindle, centrosomes and kinetochores governing spindle bipolarity, chromosome movement and the fidelity of chromosome segregation. xCep57 protein was previously characterized in Xenopus as an adapter of the connections of microtubules to the centrosomes and kinetochores. Immunodepletion of this protein in an in vitro system of oocyte extracts compromises the structure of the spindle, the alignment of chromosomes and stability of kinetochore-microtubule as well as centrosome-microtuble connections. Consistently, this protein interacts with regulatory kinetochore proteins linked to the spindle and γ-tubulin which is a main component of the nucleation rings of microtubules at centrosomes. It has been called into question the possibility of xCep57 be the functional homolog of the Dam1 yeast protein, for which a central role in coupling the attachment of kinetochore to dynamic microtubules has been shown. Thus, there are great expectations to the identification of a homolog with decisive role in regulating the movement and fidelity of chromosome segregation in higher eukaryotes. In this work, a functional study of the human protein Cep57 has been carried out in an in vivo system using the RNA interference (RNAi) methodology and confocal microscopy analysis. It was found by immunofluorescence that the Cep57 protein is located in centrosomes during both interphase and mitosis, but could not be detected in kinetochores. It was also demonstrated that the protein Cep57 binds directly to microtubules in vitro. Phenotypic analysis of Cep57 RNAi-depleted mitotic cells revealed the existence of chromosome alignment and spindle defects. The kinetochore-microtubule connections are significantly compromised and the interpolar distance is decreased. Futhermore, the kinetochore localization of known interactors of xCep57 is not altered in Cep57 RNAi-depleted cells consistently to the fact that human Cep57 could not be detected at kinetochores. Thus, overall, the mitotic phenotype observed seems to result from a centrosomal function of Cep57, possibly in microtubule anchorage to centrosomes. Interestingly, through an in silico study, we found the existence of a second homolog of xCep57 in human and mouse. Therefore, an open question remains whether both xCep57 protein homologues in human and mouse have a function in microtubule attachment, with Cep57 described here acting at the centrosome level and the other Cep57-related protein perhaps acting at the kinetochore level. Future studies will be focused on addressing this question.
Descrição
Dissertação de Mestrado em Genética Molecular, Comparativa e Tecnológica
Palavras-chave
fuso mitótico , Cep57 , microtúbulos , centrossoma , cinetocoro