Abstract
To build the spindle at mitosis, motors exert spatially regulated forces on microtubules. We know that dynein pulls on mammalian spindle microtubule minus-ends, and this localized activity at ends is predicted to allow dynein to cluster microtubules into poles. How dynein becomes enriched at minus-ends is not known. Here, we use quantitative imaging and laser ablation to show that NuMA targets dynactin to minus-ends, localizing dynein activity there. NuMA is recruited to new minus-ends independently of dynein and more quickly than dynactin; both NuMA and dynactin display specific, steady-state binding at minus-ends. NuMA localization to minus-ends involves a C-terminal region outside NuMA's canonical microtubule-binding domain and is independent of minus-end binders γ-TuRC, CAMSAP1, and KANSL1/3. Both NuMA's minus-end-binding and dynein-dynactin-binding modules are required to rescue focused, bipolar spindle organization. Thus, NuMA may serve as a mitosis-specific minus-end cargo adaptor, targeting dynein activity to minus-ends to cluster spindle microtubules into poles.
Highlights
Each time a cell divides, molecular motors help remodel the microtubule cytoskeleton into a bipolar assembly of microtubules called the spindle
Dynactin and NuMA display mitosis-specific, steady-state binding at microtubule minus-ends
To determine whether dynein-dynactin and NuMA localize to individual microtubule minus-ends, we treated mammalian PtK2 and RPE1 cells with the microtubule-depolymerizing drug nocodazole and fixed cells 6–8 min after drug washout to capture acentrosomal microtubules with clearly visible plus- and minus-ends (Figure 1A). p150Glued (p150, a dynactin subunit) and NuMA strongly co-localized at one end of these individual microtubules (Figure 1A), with a clear binding preference for minus-ends over the microtubule lattice or the plus-end when polarity was marked by EB1 (Figure 1B)
Summary
Each time a cell divides, molecular motors help remodel the microtubule cytoskeleton into a bipolar assembly of microtubules called the spindle. Dynein is a minus-end-directed motor which slides parallel spindle microtubules to focus their minus-ends into spindle poles (Heald et al, 1996; Verde et al, 1991), working in complex with its adaptor dynactin and the microtubule-binding protein NuMA (Gaglio et al, 1996; Merdes et al, 1996). NuMA is thought to require dynein activity to carry it to minus-ends and spindle poles (Merdes et al, 2000), where it anchors spindle microtubules (Dionne et al, 1999; Gaglio et al, 1995; Heald et al, 1997; Silk et al, 2009) It remains unclear whether dynein-dynactin and NuMA have specific binding
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