Mitosis is one of the most fascinating processes in biology, and is often described as a well-choreographed dance. During mitosis, sister chromatids attach to dynamic microtubules in the mitotic spindle, align and oscillate at the metaphase plate, separate synchronously at the metaphase–anaphase transition, and are partitioned equally into the two daughter cells. Chromosome missegregation leads to aneuploidy and genomic instability, which can drive tumorigenesis under certain contexts. Combining structural biology, biochemistry, and cell biology methods, Bao et al. (2017) show that the acetylation of the small GTPase Ran switches its preference for binding partners, and acts as an important molecular switch for proper chromosome alignment and mitotic progression in human cells (Figure 1).

Ran acetylation switches its binding from Mog1 to RCC1. In mitosis, Cdk1 phosphorylates and activates the acetyltransferase TIP60, which acetylates Ran on K134. This acetylation event disrupts Mog1 binding to Ran and promotes RCC1 binding. This partner switch promotes Ran-GTP generation and spindle assembly. The acetylation process is presumably dynamic. Perturbing the dynamics of Ran acetylation alters Ran-GTP levels and causes spindle and mitotic defects.
View largeDownload slide
Ran acetylation switches its binding from Mog1 to RCC1. In mitosis, Cdk1 phosphorylates and activates the acetyltransferase TIP60, which acetylates Ran on K134. This acetylation event disrupts Mog1 binding to Ran and promotes RCC1 binding. This partner switch promotes Ran-GTP generation and spindle assembly. The acetylation process is presumably dynamic. Perturbing the dynamics of Ran acetylation alters Ran-GTP levels and causes spindle and mitotic defects.
The canonical function of Ran is to regulate nuclear transport of proteins (Cautain et al., 2015). For example, importins bind to cargoes through their nuclear localization signals (NLS) and transport them through the nuclear pore. Because RCC1, the guanine nucleotide-exchange factor for Ran, is bound to chromatin, there is a higher concentration of Ran-GTP in the nucleus. Binding of Ran-GTP to importins triggers the release of cargoes, thus driving cargo import into the nucleus. Pioneering work by Nachury et al. (2001) and Wiese et al. (2001) initially identified the Ran–importin system as a major regulator of spindle assembly during mitosis. They showed that RCC1 remains bound to mitotic chromosomes and generates a Ran-GTP gradient in the vicinity of chromosomes. The local release of importin cargoes, many of which are spindle-assembly factors, promotes microtubule formation around mitotic chromosomes. This process is critical for proper spindle assembly. The spatiotemporal regulation of the Ran-GTP gradient in mitosis is not completely understood (Clarke and Zhang, 2008). The study of Bao et al. (2017) significantly advances our understanding of how Ran-GTP gradient is fine-tuned during mitosis, and how different post-translational modifications cooperate to ensure faithful cell division.

Bao et al. (2017) began their study by charactering the molecular interactions between Ran and its binding partner Mog1 with nuclear magnetic resonance (NMR) spectroscopy and site-directed mutagenesis (Baker et al., 2001). Their structural model of the Ran–Mog1 complex explains the preference of Mog1 for nucleotide-free Ran. They also found that the Mog1-binding surface of Ran partially overlapped with its RCC1-binding surface. Indeed, Mog1 and RCC1 competed for binding to Ran, and Mog1 binding to nucleotide-free Ran prevented RCC1 from binding. Altering the levels of Mog1 in human cells by RNA interference (RNAi) or overexpression delayed the onset of anaphase and caused a multitude of mitotic defects, including abnormal spindle length, multipolar spindles, and chromosome misalignment. These results suggest that the balanced Mog1–RCC1 antagonism might be critical for mitotic progression (Figure 1).

Consistent with a recent study implicating the acetyltransferase TIP60 in acetylating Ran (de Boor et al., 2015), Bao et al. (2017) went on to show that K134 of Ran is a bona fide acetylation site of TIP60 by mass spectrometry and immunoblotting with antibodies specific for acetylated Ran K134. The acetyltransferase activity of TIP60 is enhanced by Cdk1-dependent phosphorylation (Lemercier et al., 2003). Consistent with the mitotic activation of TIP60, the acetylation of Ran on K134 is greatly elevated in mitosis. Moreover, addition of NU9056, a chemical inhibitor of TIP60, rescued the lagging chromosome phenotype in Mog1 RNAi cells. GST pull-down assays further demonstrated that Ran K134 acetylation prevented Mog1 from binding to Ran (Bao et al., 2017). Consistent with an increase of Ran acetylation in mitosis, binding of Mog1 to Ran is suppressed during mitosis whereas binding of Ran to RCC1 is enhanced. Finally, expression of either the acetylation-deficient Ran K134R mutant or the acetylation-mimicking Ran K134Q mutant failed to rescue the lagging chromosome phenotype in human cells depleted of Ran by RNAi, suggesting that the dynamic acetylation–deacetylation cycle on K134 is functionally important for proper chromosome segregation. Taken together, these findings have established another layer of Ran regulation by acetylation. Mitosis-specific Ran acetylation by TIP60 enables Ran to switch binding partners from Mog1 to RCC1. This molecular switch in turn promotes the generation of the Ran-GTP gradient in the vicinity of chromosomes.

The exciting findings of Bao et al. (2017) raise many interesting questions and suggest several avenues of future investigation. For example, does Ran acetylation regulate its canonical functions in nucleocytoplasmic transport or its other proposed functions? Because the acetylation-mimicking mutant of Ran fails to rescue the mitotic defects caused by Ran depletion, timely deacetylation of Ran is likely critical for maintaining the proper levels of Ran-GTP. What is the enzyme responsible for Ran deacetylation? Is the deacetylation of Ran regulated and coordinated with cell cycle progression?

The mitotic spindle is a highly dynamic structure. Its assembly and maintenance require a complex regulatory network that consists of many components. Reversible post-translational modifications of these components can modify their functions at a relatively fast timescale and are ideally suited for fine-tuning spindle and chromosome dynamics. This elegant study by Bao et al. (2017) adds to the repertoire of spindle regulatory mechanisms, and establishes Ran acetylation as an important molecular signal that orchestrates the intricate dance moves of spindle components and chromosomes during mitosis.