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1.
Elife ; 102021 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-33871354

RESUMO

During the first days of mammalian development, the embryo forms the blastocyst, the structure responsible for implanting the mammalian embryo. Consisting of an epithelium enveloping the pluripotent inner cell mass and a fluid-filled lumen, the blastocyst results from a series of cleavage divisions, morphogenetic movements, and lineage specification. Recent studies have identified the essential role of actomyosin contractility in driving cytokinesis, morphogenesis, and fate specification, leading to the formation of the blastocyst. However, the preimplantation development of contractility mutants has not been characterized. Here, we generated single and double maternal-zygotic mutants of non-muscle myosin II heavy chains (NMHCs) to characterize them with multiscale imaging. We found that Myh9 (NMHC II-A) is the major NMHC during preimplantation development as its maternal-zygotic loss causes failed cytokinesis, increased duration of the cell cycle, weaker embryo compaction, and reduced differentiation, whereas Myh10 (NMHC II-B) maternal-zygotic loss is much less severe. Double maternal-zygotic mutants for Myh9 and Myh10 show a much stronger phenotype, failing most of the attempts of cytokinesis. We found that morphogenesis and fate specification are affected but nevertheless carry on in a timely fashion, regardless of the impact of the mutations on cell number. Strikingly, even when all cell divisions fail, the resulting single-celled embryo can initiate trophectoderm differentiation and lumen formation by accumulating fluid in increasingly large vacuoles. Therefore, contractility mutants reveal that fluid accumulation is a cell-autonomous process and that the preimplantation program carries on independently of successful cell division.


Assuntos
Blastocisto/metabolismo , Divisão Celular , Mutação , Cadeias Pesadas de Miosina/genética , Miosina não Muscular Tipo IIB/genética , Animais , Ciclo Celular , Diferenciação Celular , Citocinese , Bases de Dados Genéticas , Técnicas de Cultura Embrionária , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia de Vídeo , Morfogênese , Cadeias Pesadas de Miosina/metabolismo , Miosina não Muscular Tipo IIB/metabolismo , Fatores de Tempo , Imagem com Lapso de Tempo
2.
J Mol Biol ; 433(24): 167305, 2021 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-34655654

RESUMO

Numerous genetic methods facilitate the detection of binary protein-protein interactions (PPIs) by exogenous overexpression, which can lead to false results. Here, we describe CellFIE, a CRISPR- and cell fusion-based PPI detection method, which enables the mapping of interactions between endogenously tagged two-hybrid proteins. We demonstrate the specificity and reproducibility of CellFIE in a matrix mapping approach, validating the interactions of VCP with ASPL and UBXD1, and the self-interaction of TDP-43 under endogenous conditions. Furthermore, we show that CellFIE can be used to quantify changes of endogenous PPIs upon stress induction or drug treatment. For the first time, CellFIE facilitates systematic mapping of interactions between endogenously tagged proteins and represents a novel tool to characterize PPIs in live cells under dynamic conditions.


Assuntos
Fusão Celular , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Mapeamento de Interação de Proteínas/métodos , Humanos , Técnicas do Sistema de Duplo-Híbrido
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