The Ran-GTP Gradient Spatially Regulates XCTK2 in the Spindle

Abstract

Summary Ran is a small GTP binding protein that was originally identified as a regulator of nucleocytoplasmic transport [1], and subsequently found to be important for spindle formation [2-5]. In mitosis, a gradient of Ran-GTP emanates from chromatin and diminishes toward spindle poles [6]. Ran-GTP promotes spindle self-organization through the release of importin-bound spindle assembly factors (SAFs), which stimulate microtubule (MT) nucleation, organization and regulate MT dynamics [7-9]. Although many SAFs are non-motile MT-associated proteins, such as NuMA, TPX2, and HURP [7, 10-12], Ran also controls motor proteins, including Kid and HSET/XCTK2 [13, 14]. The Kinesin-14, XCKT2, is important for spindle assembly and pole organization [15-20], and Ran-GTP is proposed to promote XCKT2 MT crosslinking activity by releasing importin α/β from a bipartite nuclear localization signal (NLS) located in the tail domain [14]. Here we show that the Ran-GTP gradient spatially regulates XCTK2 within the spindle. A flattened Ran-GTP gradient blocked the ability of excess XCTK2 to stimulate bipolar spindle assembly and resulted in XCTK2-mediated bundling of free MTs. These effects required the XCTK2 tail, which promoted the motility of XCTK2 within the spindle independent of the Ran-GTP. In addition, the turnover kinetics of XCTK2 were spatially controlled: they were faster near the poles relative to the chromatin, but not with a mutant XCTK2 that cannot bind to importin α/β. Our results support a model in which the Ran-GTP gradient spatially coordinates motor localization with motility to ensure efficient spindle formation.

Molecular mechanisms of kinesin-14 motors in spindle assembly and chromosome segregation

“…When the physical Ran-GTP gradient gradually diminishes from the chromosomes to the spindle pole (Kalab et al, 2002), importin α/β competitively binds to XCTK2, reducing its spindle anchoring and increasing its turnover kinetics during spindle formation (Weaver et al, 2015).…”

KIFC1: a promising chemotherapy target for cancer treatment?

“…KIFC1 has already been transported into the nucleus. B. Ran-GTPase probably will spatially regulate the binding of KIFC1 with microtubules by changing its gradient [61]. From the dark color to the light color in this figure, the Ran-GTPase gradient is gradually reduced.…”

“…In addition, KIFC1 [59] and XCTK2 [60] is known for being a vital target of Ran. More specifically, the entrance of one of the homologs of KIFC1, XCTK2, into the nucleus has been verified to be controlled by the Ran gradient in Xenopus egg extracts [61]. HsKIFC1 and XCTK2 were both observed to bind to alpha/beta importin [18] as well (Figure 3).…”

“…HsKIFC1 is also observed to function as a replacement of XCTK2 when added into Xenopus egg extracts [52]. The Ran-GTP gradient is testified to be significant when XCTK2 stimulates spindle assembly by mediating the XCTK2 microtubule turnover after the release of importin from the tail of XCTK2 [61]. The understanding of the transportation regulation process relating to XCTK2 has been widely studied while that relating to HsKIFC1 still remains unclear.…”

RanGTP induces an effector gradient of XCTK2 and importin α/β for spindle microtubule cross-linking

“…These SAFs include a number of MT associated proteins, such as TPX2 (Gruss et al, 2001), NuMA (Nachury et al, 2001;Wiese et al, 2001), HURP (Koffa et al, 2006), and NuSAP (Ribbeck et al, 2006), as well as molecular motor proteins, Kid (Tahara et al, 2008;Trieselmann et al, 2003) and XCTK2 (Ems-McClung et al, 2004) that contain binding sites for either importin α and/or importin β. XCTK2 is a minus-end directed Kinesin-14 motor that cross-links and slides both parallel and anti-parallel MTs (Hentrich and Surrey, 2010) and contributes to proper spindle assembly, spindle length, and spindle pole formation (Cai et al, 2009;Walczak et al, 1997;Walczak et al, 1998). XCTK2 association within the spindle is spatially controlled by the RanGTP gradient through the tail domain (Hallen et al, 2008;Weaver et al, 2015), suggesting that the RanGTP gradient may specifically regulate the MT binding properties of XCTK2 to the spindle. How the RanGTP gradient regulates the localization of SAFs to the spindle is unknown.…”