RESUMO
Entosis, or cell death by invading another cell, is typical for tumor epithelial cells. The formation of cell-in-cell structures is extensively studied in suspension cultures, but remains poorly understood in substrate-dependent cells. Here, we used electron, confocal and time-lapse microscopy in combination with pharmacological inhibition of intracellular components to study the kinetics of entosis using two human substrate-dependent tumor cultures, A431 and MCF7. In total, we identified and characterized five consecutive stages of entosis, which were common for both examined cell lines. We further demonstrated that actin filaments in the entotic as well as invading cells were crucial for entosis. Microtubules and the Golgi apparatus of entotic cells provided membrane expansion required for internalization of the invading cell. Depolymerization of microfilaments and microtubules, and disintegration of the Golgi complex inhibited entosis. We confirmed the presence of adhesive junctions and discovered the formation of desmosomes between the invading and entotic cells. The internalized cell was shown to be degraded due to the lysosomal activation in both cells whereas the disintegration of the Golgi apparatus did not affect the process. Thus, in the substrate-dependent cultures, entosis requires microfilaments, microtubules and the Golgi complex for cell invasion, but not for internalized cell degradation.
Assuntos
Citoesqueleto de Actina/ultraestrutura , Entose/genética , Complexo de Golgi/ultraestrutura , Lisossomos/ultraestrutura , Citoesqueleto de Actina/genética , Células Epiteliais , Complexo de Golgi/genética , Humanos , Cinética , Lisossomos/genética , Células MCF-7 , Microscopia Confocal , Microscopia Eletrônica , Microtúbulos/genética , Microtúbulos/ultraestrutura , Neoplasias/genética , Neoplasias/patologia , Imagem com Lapso de TempoRESUMO
Accurate segregation of chromosomes, essential for the stability of the genome, depends on 'bi-orientation'-simultaneous attachment of each individual chromosome to both poles of the mitotic spindle. On bi-oriented chromosomes, kinetochores (macromolecular complexes that attach the chromosome to the spindle) reside on the opposite sides of the chromosome's centromere. In contrast, sister kinetochores shift towards one side of the centromere on 'syntelic' chromosomes that erroneously attach to one spindle pole with both sister kinetochores. Syntelic attachments often arise during spindle assembly and must be corrected to prevent chromosome loss. It is assumed that restoration of proper centromere architecture occurs automatically owing to elastic properties of the centromere. Here we test this assumption by combining laser microsurgery and chemical biology assays in cultured mammalian cells. We find that kinetochores of syntelic chromosomes remain juxtaposed on detachment from spindle microtubules. These findings reveal that correction of syntelic attachments involves an extra step that has previously been overlooked: external forces must be applied to move sister kinetochores to the opposite sides of the centromere. Furthermore, we demonstrate that the shape of the centromere is important for spindle assembly, because bipolar spindles do not form in cells lacking centrosomes when multiple chromosomes with juxtaposed kinetochores are present. Thus, proper architecture of the centromere makes an important contribution to achieving high fidelity of chromosome segregation.
Assuntos
Centrômero/metabolismo , Segregação de Cromossomos , Mitose , Fuso Acromático/metabolismo , Animais , Linhagem Celular , Cromátides/efeitos dos fármacos , Cromátides/metabolismo , Segregação de Cromossomos/efeitos dos fármacos , Feminino , Cinetocoros/metabolismo , Macropodidae , Microtúbulos/fisiologia , Pirimidinas/farmacologia , Sintenia , Tionas/farmacologiaRESUMO
Forces in the spindle that align and segregate chromosomes produce a steady poleward flux of kinetochore microtubules (MTs [kMTs]) in higher eukaryotes. In several nonmammalian systems, flux is driven by the tetrameric kinesin Eg5 (kinesin 5), which slides antiparallel MTs toward their minus ends. However, we find that the inhibition of kinesin 5 in mammalian cultured cells (PtK1) results in only minor reduction in the rate of kMT flux from approximately 0.7 to approximately 0.5 microm/min, the same rate measured in monopolar spindles that lack antiparallel MTs. These data reveal that the majority of poleward flux of kMTs in these cells is not driven by Eg5. Instead, we favor a polar "pulling-in" mechanism in which a depolymerase localized at kinetochore fiber minus ends makes a major contribution to poleward flux. One candidate, Kif2a (kinesin 13), was detected at minus ends of fluxing kinetochore fibers. Kif2a remains associated with the ends of K fibers upon disruption of the spindle by dynein/dynactin inhibition, and these K fibers flux.
Assuntos
Cinesinas/fisiologia , Cinetocoros/fisiologia , Microtúbulos/fisiologia , Animais , Linhagem Celular , Células Cultivadas , Proteínas de Fluorescência Verde/genética , Humanos , Cinesinas/antagonistas & inibidores , Microinjeções , Modelos Biológicos , Pirimidinas/farmacologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Tionas/farmacologia , Tubulina (Proteína)/genéticaRESUMO
The large coiled-coil protein NuMA plays an essential role in organizing microtubule minus ends at spindle poles in vertebrate cells. Here, we use both in vivo and in vitro methods to examine NuMA dynamics at mitotic spindle poles. Using fluorescence recovery after photobleaching, we show that an exogenously expressed green-fluorescent-protein/NuMA fusion undergoes continuous exchange between soluble and spindle-associated pools in living cells. These dynamics require cellular energy and display an average half-time for fluorescence recovery of approximately 3 minutes. To explore how NuMA dynamics at spindle poles is regulated, we exploited the association of NuMA with microtubule asters formed in mammalian mitotic extracts. Using a monoclonal antibody specific for human NuMA, we followed the fate of human NuMA associated with microtubule asters upon dilution with a hamster mitotic extract. Consistent with in vivo data, this assay shows that NuMA can be displaced from the core of pre-assembled asters into the soluble pool. The half-time of NuMA displacement from asters under these conditions is approximately 5 minutes. Using this assay, we show that protein kinase activity and the NuMA-binding protein LGN regulate the dynamic exchange of NuMA on microtubule asters. Thus, the dynamic properties of NuMA are regulated by multiple mechanisms including protein phosphorylation and binding to the LGN protein, and the rate of exchange between soluble and microtubule-associated pools suggests that NuMA associates with an insoluble matrix at spindle poles.