RESUMO
Chromosome segregation relies on the correct assembly of a bipolar spindle. Spindle pole self-organization requires dynein-dependent microtubule (MT) transport along other MTs. However, during M-phase RanGTP triggers MT nucleation and branching generating polarized arrays with nonastral organization in which MT minus ends are linked to the sides of other MTs. This raises the question of how branched-MT nucleation and dynein-mediated transport cooperate to organize the spindle poles. Here, we used RanGTP-dependent MT aster formation in Xenopus laevis (X. laevis) egg extract to study the interplay between these two seemingly conflicting organizing principles. Using temporally controlled perturbations of MT nucleation and dynein activity, we found that branched MTs are not static but instead dynamically redistribute over time as poles self-organize. Our experimental data together with computer simulations suggest a model where dynein together with dynactin and NuMA directly pulls and move branched MT minus ends toward other MT minus ends.
Assuntos
Dineínas , Fuso Acromático , Animais , Dineínas/metabolismo , Xenopus laevis/metabolismo , Fuso Acromático/metabolismo , Microtúbulos/metabolismo , Complexo Dinactina , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas de Xenopus/metabolismoRESUMO
Bipolar spindle organization is essential for the faithful segregation of chromosomes during cell division. This organization relies on the collective activities of motor proteins. The minus-end-directed dynein motor complex generates spindle inward forces and plays a major role in spindle pole focusing. The dynactin complex regulates many dynein functions, increasing its processivity and force production. Here, we show that DnaJB6 is a novel RanGTP-regulated protein. It interacts with the dynactin subunit p150Glued (also known as DCTN1) in a RanGTP-dependent manner specifically in M-phase, and promotes spindle pole focusing and dynein force generation. Our data suggest a novel mechanism by which RanGTP regulates dynein activity during M-phase.
Assuntos
Complexo Dinactina/metabolismo , Proteínas de Choque Térmico HSP40/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Fuso Acromático/metabolismo , Proteínas de Xenopus/metabolismo , Proteína ran de Ligação ao GTP/metabolismo , Animais , Linhagem Celular Tumoral , Proteínas de Choque Térmico HSP40/genética , Células HeLa , Humanos , Proteínas Associadas aos Microtúbulos/genética , Mitose/fisiologia , Chaperonas Moleculares/genética , Proteínas do Tecido Nervoso/genética , Interferência de RNA , RNA Interferente Pequeno/genética , Proteínas de Xenopus/genética , Xenopus laevisRESUMO
The Aurora-A kinase has well-established roles in spindle assembly and function and is frequently overexpressed in tumours. Its abundance is cell cycle regulated, with a peak in G2 and M phases, followed by regulated proteolysis at the end of mitosis. The microtubule-binding protein TPX2 plays a major role in regulating the activity and localisation of Aurora-A in mitotic cells. Here, we report a novel regulatory role of TPX2 and show that it protects Aurora-A from degradation both in interphase and in mitosis in human cells. Specifically, Aurora-A levels decrease in G2 and prometaphase cells silenced for TPX2, whereas degradation of Aurora-A is impaired in telophase cells overexpressing the Aurora-A-binding region of TPX2. The decrease in Aurora-A in TPX2-silenced prometaphases requires proteasome activity and the Cdh1 activator of the APC/C ubiquitin ligase. Reintroducing either full-length TPX2, or the Aurora-A-binding region of TPX2, but not a truncated TPX2 mutant lacking the Aurora-A-interaction domain, restores Aurora-A levels in TPX2-silenced prometaphases. The control by TPX2 of Aurora-A stability is independent of its ability to activate Aurora-A and to localise it to the spindle. These results highlight a novel regulatory level impinging on Aurora-A and provide further evidence for the central role of TPX2 in regulation of Aurora-A.