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

Abstract

High RanGTP around chromatin is important for governing spindle assembly during meiosis and mitosis by releasing the inhibitory effects of importin α/β. Here we examine how the Ran gradient regulates Kinesin-14 function to control spindle organization. We show that Xenopus Kinesin-14, XCTK2, and importin α/β form an effector gradient that is highest at the poles and diminishes toward the chromatin, which is opposite the RanGTP gradient. Importin α and β preferentially inhibit XCTK2 antiparallel microtubule cross-linking and sliding by decreasing the microtubule affinity of the XCTK2 tail domain. This change in microtubule affinity enables RanGTP to target endogenous XCTK2 to the spindle. We propose that these combined actions of the Ran pathway are critical to promote Kinesin-14 parallel microtubule cross-linking to help focus spindle poles for efficient bipolar spindle assembly. Furthermore, our work illustrates that RanGTP regulation in the spindle is not simply a switch, but rather generates effector gradients where importins α and β gradually tune the activities of spindle assembly factors.

Aneuploidy in human eggs: contributions of the meiotic spindle

“…Importantly, the Ran-GTP pathway not only promotes microtubule assembly, but also regulates the activity of several motor proteins. For instance, the Ran-GTP gradient directly controls the activity of HSET, a motor protein important for spindle pole focusing, and Kid, another motor protein necessary for chromosome alignment on the metaphase plate [27,28]. Interestingly, microtubules are not the only structural filaments in the oocyte spindle.…”

Importin α/β Promote Kif18B Microtubule Association to Spatially Control Microtubule Destabilization

“…However, the NLS1/2 mutations caused ∼50% reduction in maximal MT binding and ∼4-fold increase in the K d for MT binding relative to YPet- Kif18B(605-828) (Figures, 1G and 2D), suggesting a moderate effect on MT affinity. Interestingly, these results are similar to our previous work with the Kinesin-14 XCTK2, wherein importin α /β regulate the interaction of the XCTK2 tail with MTs to control its ability to cross-link MTs (Ems-McClung et al ., 2004; Ems-McClung et al ., 2020), suggesting a common regulatory mechanism for importin-regulated MT binding of kinesin tails.…”

“…Another emerging theme from our studies is that importin α/β may be a major mechanism to control the affinity of MT binding. Spindle assembly factors regulated by Ran include the molecular motors XCTK2 (Ems- McClung et al ., 2004; Ems-McClung et al ., 2020) and Kif18B (this study) as well as microtubule binding proteins NuMA (Wiese et al ., 2001) and TPX2 (Gruss et al ., 2001; Schatz et al ., 2003a; Schatz et al ., 2003b). For the molecular motor proteins, each of these motors has a second microtubule binding site in the tail that is used for either cross-linking or for motor processivity.…”

“…To express His6-EB1-CyPet, full length human EB1 was amplified by PCR from pET30a-EB1 ((Stout et al ., 2011), a gift from Jennifer Tirnauer) using the primers in Table S1. The resulting PCR product was cloned into the Xba I /Xho I sites of pRSETB-CyPet (Ems-McClung et al ., 2020) to create pRSETB-EB1-CyPet. For expression and purification of the C-terminal tail proteins of human Kif18B (Kif18B) fused to YPet, we generated plasmids with an N-terminal YPet tag and a C-terminal His6 tag in two steps.…”

“…For the expression of His6-EB1-CyPet, His6-EB1 (Stout et al ., 2011), His6-importin α-CyPet (Ems-McClung et al ., 2020), His6-importin β, and importin α-His6 (Ems-McClung et al ., 2004) recombinant proteins, plasmids were transformed into BL21(DE3)pLysS bacteria and induced with 0.1 mM isopropyl β-D-1- thiogalactopyranoside (IPTG) at 16°C for 24 h. For the expression of YPet-Kif18B-His6 recombinant proteins, plasmids were transformed into SHuffle T7 Express LysY bacteria (NEB Cat# C3030J) and induced with 0.4 mM of IPTG at 16°C for 24h. Cells were pelleted, frozen in liquid nitrogen, and stored at -80°C.…”