NuMA Interacts With And Targets Astrin At The Spindle During Cell Division

Mitosis is the essential process through which chromosomes are accurately segregated into two daughter cells. Chromosome segregation is mediated by a complex called mitotic spindle that pulls the sister chromatids apart and moves a complete set of chromosomes to each pole of the cell. The mitotic spindle is based on a bipolar array of microtubules, each of which is a polarized protein polymer. The microtubules of the spindle are organized with their less dynamic minus ends tightly focused into two spindle poles. Each chromatid carries a kinetochore, a multi-protein complex that attaches the chromatid to microtubules connected to a spindle pole. Kinetochores are responsible for maintaining attachment to growing and shrinking microtubules resulting in chromosome movements that lead to alignment during metaphase and segregation during anaphase, A proper kinetochore-microtubule association is one in which each sister kinetochore is stably associated with a microtubule bundle associated with an opposite spindle pole.In all eukaryotes construction of a bipolar spindle depends in large part on the ability of the spindle components to self-organize. This behavior is regulated by many different proteins that bind to the sides or ends of microtubules. These include the motor proteins and non-motor microtubule-regulatory proteins. These proteins show diverse localization on mitotic spindles including centrosomes, spindle poles, spindle body, central spindle, and kinetochores where they participate in both microtubule organization and nucleation. Complicating the understanding of these functions, many of these proteins cooperate or interact with each other in some activities.Loss of function studies combined with in vitro biochemical experiments demonstrate that many non-motor spindle proteins mechanically cross link and stabilize microtubules to provide structural support to the mitotic spindle. NuMA, a220kDa protein with a large coiled-coil domain that is capable of extensive oligomerization, binds microtubules directly, and localizes to microtubule minus ends at spindle poles. Perturbation of NuMA function leads to splaying of microtubule minus ends at spindle poles indicating that NuMA combines self association and microtubule binding activities to mechanically cross link microtubules at spindle poles. These roles of NuMA in spindle formation are both structural and mechanical, as NuMA acts as a necessary motor accessory. NuMA associates with the minus-end directed microtubule motor cytoplasmic dynein and by simultaneously binding microtubules, allows the translation of dynein motor activity into microtubule minus end focusing activity. In addition, the combination of microtubule binding and oligomerization of NuMA reinforces and helps to maintain spindle pole focusing.To better understand the role of NuMA in the regulation of spindle assembly and mitotic progression in human cells, we used Yeast two-hybrid system to search for proteins that specifically interact with NuMA. We report here the identification of the human spindle-associated protein Astrin as a novel interactor and substrate of NuMA. We show that the C-terminal of NuMA directly binds to the C terminus of Astrin and can recruit Astrin to microtubules. In particular, we show that the associate of Astrin with spindle pole requires NuMA. Knock-down of NuMA resulted in significantly reduced association of Astrin with spindle poles and spindle microtubules. Blocking the microtubule binding of NuMA by overexpression of LGN or blocking the transport of NuMA by delete of Dynein also led to similar results. Ectopic expression of NuMA C terminus, the binding fragment of Astrin, led to accumulation of Astrin at spindle pole in metaphase cells. Moreover, If Astrin levels are reduced, NuMA does not concentrate at the spindle pole efficiently and the spindle microtubules appear to be destabilized. These findings indicate that the NuMA-Astrin interaction contributes to the mitosis-specific recruitment of Astrin to spindle poles, and NuMA may balance the localization of Astrin between spindle poles and kinetochores. Our data suggest NuMA-Astrin complex may cooperate with each other contribute to the assembly and stabilization of K-fibers and alignment of chromosome during mitosis.