RESUMEN
Kinesin-5 is an essential mitotic motor. However, how its spatial-temporal distribution is regulated in mitosis remains poorly understood. We expressed localization and affinity purification-tagged Eg5 from a mouse bacterial artificial chromosome (this construct was called mEg5) and found its distribution to be tightly regulated throughout mitosis. Fluorescence recovery after photobleaching analysis showed rapid Eg5 turnover throughout mitosis, which cannot be accounted for by microtubule turnover. Total internal reflection fluorescence microscopy and high-resolution, single-particle tracking revealed that mEg5 punctae on both astral and midzone microtubules rapidly bind and unbind. mEg5 punctae on midzone microtubules moved transiently both toward and away from spindle poles. In contrast, mEg5 punctae on astral microtubules moved transiently toward microtubule minus ends during early mitosis but switched to plus end-directed motion during anaphase. These observations explain the poleward accumulation of Eg5 in early mitosis and its redistribution in anaphase. Inhibition of dynein blocked mEg5 movement on astral microtubules, whereas depletion of the Eg5-binding protein TPX2 resulted in plus end-directed mEg5 movement. However, motion of Eg5 on midzone microtubules was not altered. Our results reveal differential and precise spatial and temporal regulation of Eg5 in the spindle mediated by dynein and TPX2.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Dineínas/metabolismo , Cinesinas/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Mitosis/fisiología , Proteínas Nucleares/metabolismo , Huso Acromático/metabolismo , Animales , Secuencia de Bases , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/genética , Recuperación de Fluorescencia tras Fotoblanqueo , Cinesinas/genética , Células LLC-PK1 , Ratones , Microscopía Fluorescente , Proteínas Asociadas a Microtúbulos/antagonistas & inhibidores , Proteínas Asociadas a Microtúbulos/genética , Microtúbulos/metabolismo , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/genética , ARN Interferente Pequeño/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , PorcinosRESUMEN
Mitotic spindle assembly requires the regulated activity of numerous spindle-associated proteins. In mammalian cells, the Kinesin-5 motor Eg5 interacts with the spindle assembly factor TPX2, but how this interaction contributes to spindle formation and function is not established. Using bacterial artificial chromosome technology, we generated cells expressing TPX2 lacking the Eg5 interaction domain. Spindles in these cells were highly disorganized with multiple spindle poles. The TPX2-Eg5 interaction was required for kinetochore fiber formation and contributed to Eg5 localization to spindle microtubules but not spindle poles. Microinjection of the Eg5-binding domain of TPX2 resulted in spindle elongation, indicating that the interaction of Eg5 with TPX2 reduces motor activity. Consistent with this possibility, we found that TPX2 reduced the velocity of Eg5-dependent microtubule gliding, inhibited microtubule sliding, and resulted in the accumulation of motor on microtubules. These results establish a novel function of TPX2 in regulating the location and activity of the mitotic motor Eg5.
Asunto(s)
Cromosomas de los Mamíferos/metabolismo , Cinesinas/metabolismo , Cinetocoros/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Mitosis/fisiología , Huso Acromático/metabolismo , Animales , Línea Celular , Cromosomas de los Mamíferos/genética , Cinesinas/genética , Proteínas Asociadas a Microtúbulos/genética , Microtúbulos/genética , Microtúbulos/metabolismo , Estructura Terciaria de Proteína , Huso Acromático/genética , PorcinosRESUMEN
In all eukaryotic cells, molecular motor proteins play essential roles in spindle assembly and function. The homotetrameric kinesin-5 motors in particular generate outward forces that establish and maintain spindle bipolarity and contribute to microtubule flux. Cell-cycle dependent phosphorylation of kinesin-5 motors regulates their localization to the mitotic spindle. Analysis of live cells further shows that kinesin-5 motors are highly dynamic in the spindle. Understanding the interactions of kinesin-5 motors with microtubules and other spindle proteins is likely to broaden the documented roles of kinesin-5 motors during cell division.
