NuMA Recruits Dynein Activity to Microtubule Minus-Ends at Mitosis

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 g-TuRC, CAMSAP1, and KANSL1/3. Both NuMA's minus-endbinding 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.

Nu MA assemblies organize microtubule asters to establish spindle bipolarity in acentrosomal human cells

“…In addition, we confirmed that, in the absence of NuMA, p150 Glued was strongly displaced from the acentrosomal spindle poles (Appendix Fig S7H). Thus, it is likely that NuMA is essential for the recruitment of p150 Glued at the acentrosomal spindle pole, as was observed at centrosomal pole (Hueschen et al, 2017; Appendix Fig S7G). Collectively, these results suggest that NuMA is important for placing the monopolar spindle in the center of acentrosomal cells, in prior to formation of bipolar spindles.…”

“…We next addressed the mechanisms underlying NuMA assembly for acentrosomal spindle pole formation. It has been established that NuMA is transported toward the spindle poles in a microtubule‐based motor dynein‐dependent manner (Merdes et al , , ; Hueschen et al , ). Therefore, we validated the requirement of microtubules and dynein for NuMA assembly at acentrosomal spindle poles.…”

“…The functions of these proteins for spindle pole organization and spindle positioning have been well studied in meiotic and mitotic spindles. It has been shown that NuMA is recruited to the minus-ends of microtubules dependent on (Merdes et al, 2000) or independently of dynein (Hueschen et al, 2017). During prophase, NuMA begins to form aggregates in the nucleus (Kisurina-Evgenieva et al, 2004).…”

“…Specific inhibition of NuMA or dynein disrupts spindle pole formation (Merdes et al, 1996(Merdes et al, , 2000. Thus, the spindle pole is organized by NuMA and the dynein-dynactin complex (Merdes et al, 1996(Merdes et al, , 2000Hueschen et al, 2017). In addition to their functions in spindle pole organization, NuMA and the dynein-dynactin complex cooperate to regulate spindle positioning by forming dynein-dynactin-NuMA clusters at the mitotic cell cortex through the clustering activity of NuMA (Okumura et al, 2018).…”

“…The microtubule organization activity of NuMA has been studied in detail (Nachury et al, 2001;Wiese et al, 2001). It has been shown that NuMA possesses clustering properties through its C-terminus (Harborth et al, 1999) and forms microtubule-independent insoluble complexes that are resistant to nocodazole treatment (Dionne et al, 1999;Merdes et al, 2000;Kisurina-Evgenieva et al, 2004;Hueschen et al, 2017). It has also been reported that the C-terminus of NuMA interacts with microtubules (Du et al, 2002;Haren & Merdes, 2002), presumably mediating centrosome-independent aster formation (Gaglio et al, 1995;Du et al, 2002;Haren & Merdes, 2002).…”