RESUMO
After partitioning of cytoplasmic contents by cleavage furrow ingression, animal cells remain connected by an intercellular bridge, which subsequently splits by abscission. Here, we examined intermediate stages of abscission in human cells by using live imaging, three-dimensional structured illumination microscopy, and electron tomography. We identified helices of 17-nanometer-diameter filaments, which narrowed the cortex of the intercellular bridge to a single stalk. The endosomal sorting complex required for transport (ESCRT)-III co-localized with constriction zones and was required for assembly of 17-nanometer-diameter filaments. Simultaneous spastin-mediated removal of underlying microtubules enabled full constriction at the abscission site. The identification of contractile filament helices at the intercellular bridge has broad implications for the understanding of cell division and of ESCRT-III-mediated fission of large membrane structures.
Assuntos
Divisão Celular , Complexos Endossomais de Distribuição Requeridos para Transporte/química , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Actinas/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ciclo Celular/metabolismo , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Tomografia com Microscopia Eletrônica , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Células HeLa , Humanos , Imageamento Tridimensional , Microscopia Eletrônica , Proteínas Nucleares/metabolismo , Conformação Proteica , Multimerização Proteica , Interferência de RNA , EspastinaAssuntos
Caenorhabditis elegans/química , Glicolipídeos/química , Larva/química , Animais , Caenorhabditis elegans/crescimento & desenvolvimento , Caenorhabditis elegans/metabolismo , Glicolipídeos/isolamento & purificação , Glicolipídeos/metabolismo , Larva/crescimento & desenvolvimento , Larva/metabolismo , TemperaturaRESUMO
HeLa cells are widely used as a model system to study cell division. The last step of cell division, abscission, occurs at an about 1 µm wide intercellular bridge that connects the post-mitotic sister cells. Abscission often occurs long after ingression of the cleavage furrow, and no efficient methods to synchronize cells to this stage are available. Here, we have developed a correlative fluorescence time-lapse imaging and electron microscopic approach using Aclar sheets with engraved grid patterns. This grid pattern, leaving a negative imprint on thin-layer embedded samples, allows identification of cells selected from the time-lapse imaging for serial-section electron microscopy. This method facilitates the ultrastructural analysis of specific stages of abscission.
Assuntos
Citocinese , Células HeLa/ultraestrutura , Microscopia Eletrônica/métodos , Imagem com Lapso de Tempo/métodos , Membrana Celular/ultraestrutura , Citoesqueleto/ultraestrutura , Células HeLa/fisiologia , Histocitoquímica/métodos , Humanos , Microscopia Eletrônica/instrumentação , Imagem com Lapso de Tempo/instrumentaçãoRESUMO
During mitosis, the connections of microtubules (MTs) to centrosomes and kinetochores are dynamic. From in vitro studies, it is known that the dynamic behavior of MTs is related to the structure of their ends, but we know little about the structure of MT ends in spindles. Here, we use high-voltage electron tomography to study the centrosome- and kinetochore-associated ends of spindle MTs in embryonic cells of the nematode, Caenorhabditis elegans. Centrosome-associated MT ends are either closed or open. Closed MT ends are more numerous and are uniformly distributed around the centrosome, but open ends are found preferentially on kinetochore-attached MTs. These results have structural implications for models of MT interactions with centrosomes.
