RESUMO
The microtubule motor protein kinesin-5 (Eg5) provides an outward force on centrosomes, which drives bipolar spindle assembly. Acute inhibition of Eg5 blocks centrosome separation and causes mitotic arrest in human cells, making Eg5 an attractive target for anti-cancer therapy. Using in vitro directed evolution, we show that human cells treated with Eg5 inhibitors can rapidly acquire the ability to divide in the complete absence of Eg5 activity. We have used these Eg5-independent cells to study alternative mechanisms of centrosome separation. We uncovered a pathway involving nuclear envelope (NE)-associated dynein that drives centrosome separation in prophase. This NE-dynein pathway is essential for bipolar spindle assembly in the absence of Eg5, but also functions in the presence of full Eg5 activity, where it pulls individual centrosomes along the NE and acts in concert with Eg5-dependent outward pushing forces to coordinate prophase centrosome separation. Together, these results reveal how the forces are produced to drive prophase centrosome separation and identify a novel mechanism of resistance to kinesin-5 inhibitors.
Assuntos
Centrossomo/fisiologia , Dineínas/metabolismo , Cinesinas/metabolismo , Mitose/fisiologia , Membrana Nuclear/fisiologia , Prófase/fisiologia , Fuso Acromático/fisiologia , Western Blotting , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Ensaio de Unidades Formadoras de Colônias , Dineínas/genética , Citometria de Fluxo , Células HeLa , Humanos , Cinesinas/antagonistas & inibidores , Cinesinas/genética , Osteossarcoma/metabolismo , Osteossarcoma/patologia , RNA Interferente Pequeno/genéticaRESUMO
During mitosis, all chromosomes must attach to microtubules of the mitotic spindle to ensure correct chromosome segregation. Microtubule attachment occurs at specialized structures at the centromeric region of chromosomes, called kinetochores. These kinetochores can generate microtubule attachments through capture of centrosome-derived microtubules, but in addition, they can generate microtubules themselves, which are subsequently integrated with centrosome-derived microtubules to form the mitotic spindle. Here, we have performed a large scale RNAi screen and identify cyclin G-associated kinase (GAK) as a novel regulator of microtubule generation at kinetochores/chromatin. This function of GAK requires its C-terminal J-domain, which is essential for clathrin recycling from endocytic vesicles. Consistently, cells lacking GAK show strongly reduced levels of clathrin on the mitotic spindle, and reduction of clathrin levels also inhibits microtubule generation at kinetochores/chromosomes. Finally, we present evidence that association of clathrin with the spindle is promoted by a signal coming from the chromosomes. These results identify a role for GAK and clathrin in microtubule outgrowth from kinetochores/chromosomes and suggest that GAK acts through clathrin to control microtubule outgrowth around chromosomes.
Assuntos
Clatrina/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Microtúbulos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fuso Acromático/metabolismo , Linhagem Celular Tumoral , Centrômero/metabolismo , Segregação de Cromossomos/fisiologia , Cromossomos Humanos/genética , Cromossomos Humanos/fisiologia , Cromossomos Humanos/ultraestrutura , Clatrina/genética , Clatrina/fisiologia , Células HeLa , Humanos , Cinetocoros/fisiologia , Microtúbulos/ultraestrutura , Mitose , RNA Interferente Pequeno , Fuso Acromático/genética , Tubulina (Proteína)/metabolismoRESUMO
BACKGROUND: The formation of a bipolar spindle is an essential step during cell division. Bipolar spindle assembly is driven by the highly conserved microtubule motor Eg5 (kinesin-5), which can slide antiparallel microtubules apart to drive centrosome separation. However, it is currently unclear whether and how additional motors can contribute to centrosome separation and bipolar spindle formation. RESULTS: We have developed a novel assay to identify motors involved in spindle bipolarity; via this assay, we identify Kif15/Hklp2 (kinesin-12, hereafter referred to as Kif15). Kif15 is not required for spindle bipolarity in cells with full Eg5 activity but becomes essential when Eg5 is partially inhibited. We show that the primary function of Kif15 is to promote spindle elongation and to ensure maintenance of spindle bipolarity. Nonetheless, ectopic expression of Kif15 can fully reconstitute bipolar spindle assembly in the absence of Eg5 activity, demonstrating that Kif15 can replace all essential functions of Eg5 in bipolar spindle assembly. Importantly, this activity of Kif15 depends on its interaction with the microtubule-associated protein TPX2, indicating that a Kif15-TPX2 complex promotes centrosome separation. CONCLUSIONS: These findings show that, similar to Eg5, Kif15 can drive centrosome separation during bipolar spindle assembly. For this activity, Kif15 requires both its motor domain and its interaction with TPX2. Based on these data, we propose that a complex of Kif15 and TPX2 can crosslink and slide two antiparallel microtubules apart, thereby driving centrosome separation.
