Ran is a small GTPase required for nucleocytoplasmic trafficking, spindle assembly, nuclear assembly and cell cycle control. The nucleotide exchange factor for Ran, RCC1, is a chromatin-associated protein. The GTPase activating protein for Ran, RanGAP1, is cytoplasmic during interphase. During mitosis, the bulk of RanGAP1 is broadly distributed, although a significant fraction of RanGAP1 becomes associated with kinetochores (see Z01 HD001902-09). Ran-GTP nucleotide hydrolysis also requires a family of accessory proteins. The best-characterized member of this family is mammalian RanBP1, which is distributed to the cytosol during interphase. RanBP1 accelerates the rate of RanGAP1-mediated Ran-GTP hydrolysis by about an order of magnitude in vitro. RanBP1 also promotes dissociation Ran-GTP from transport receptors, whose binding would otherwise block RanGAP-mediated GTP hydrolysis. The distribution of Ran's regulators has been widely hypothesized to modulate local concentrations of Ran-GTP within the cell, spatially directing the many processes in which Ran has been implicated. Ran's primary known effectors are a set of Ran-GTP binding proteins that were originally described as nuclear transport receptors. Ran-GTP binding regulates association between these proteins and their transport cargoes. Defects in the Ran pathway disrupt both the onset and the completion of mitosis, although Ran's function in cell cycle progression had not been clearly distinguished from its roles in nuclear transport and spindle assembly. We were therefore interested in examining Ran's role in mitotic regulation more closely. Mitosis is tightly controlled in eukaryotes by the activity of Cyclin B and Securin. Both Cyclin B and Securin are ubiquitinated at the metaphase-anaphase transition by an E3 ligase called the anaphase-promoting complex/cyclosome (APC/C), working in association with its activators Cdc20/FZY and Cdh1/FZR. In the presence of misassembled spindles, with kinetochores that are unattached or that lack tension from spindle microtubules, the onset of anaphase is delayed through activation of a spindle assembly checkpoint. This checkpoint pathway prevents APC/CFZY activation and thereby stabilizes APC/CFZY substrates. After all of the chromosomes have become attached and aligned within the mitotic spindle, the checkpoint is turned off, APC/CFZY becomes active and anaphase commences. Components of the spindle assembly checkpoint include: Mad1, Mad2, Mps1, Bub1, Bub3, BubR1 and CENP-E. We have examined the role of Ran in regulating mitotic checkpoints using Xenopus egg extracts, a well-established model system for checkpoint control. During normal cell cycles in cycling egg extracts, we find that the amount of chromatin-associated RCC1 increases dramatically at the onset of Cyclin B destruction. Moreover, moderate levels of exogenous RCC1 protein abrogate mitotic spindle checkpoint arrest and allow Cyclin B destruction in extracts containing nuclei plus the nocodazole, a microtubule depolymerizing agent. We find that the spindle assembly checkpoint in Xenopus is characterized by decreased APCFZY activity and addition of RCC1 to extracts with the activated checkpoint restores APCFZY activity to control levels. In order to determine the precise mechanism through which RCC1 abrogates checkpoint arrest, we examined the localization of mitotic regulators, including Mad2, CENP-E, Bub1 and Bub3. We find that these proteins are mislocalized away from kinetochores in nocodazole-treated extracts after the addition of high levels of RCC1 protein. Interestingly, displacement of Bub1 and Bub3 from kinetochores could be reversed by the addition of recombinant RanGAP1 protein, suggesting that their behavior responds directly by Ran-GTP levels. Taken together, our results indicate that the Ran pathway is normally regulated in a highly dynamic manner during mitosis. The transitions of the Ran pathway can be mimicked by addition of exogenous RCC1 protein, triggering the metaphase-anaphase transition prematurely in the presence of unattached kinetochores. Our observations suggest that changes in RCC1's chromosomal dynamics may be a key link in the chain of events between completion of metaphase spindle assembly and mitotic exit.
| Dasso, Mary; Fontoura, Beatriz M A (2018) Editorial overview: The cell nucleus: Dynamic interplay of shape and function. Curr Opin Cell Biol 52:iv-vi |
| Dasso, Mary; Fontoura, Beatriz M A (2016) Gating Immunity and Death at the Nuclear Pore Complex. Cell 166:1364-1366 |
| Dasso, Mary (2016) Kar9 Controls the Cytoplasm by Visiting the Nucleus. Dev Cell 36:360-1 |
| Zhang, Michael Shaofei; Arnaoutov, Alexei; Dasso, Mary (2014) RanBP1 governs spindle assembly by defining mitotic Ran-GTP production. Dev Cell 31:393-404 |
| Mukhopadhyay, Debaditya; Arnaoutov, Alexei; Dasso, Mary (2010) The SUMO protease SENP6 is essential for inner kinetochore assembly. J Cell Biol 188:681-92 |
| Mishra, Ram Kumar; Chakraborty, Papia; Arnaoutov, Alexei et al. (2010) The Nup107-160 complex and gamma-TuRC regulate microtubule polymerization at kinetochores. Nat Cell Biol 12:164-9 |
| Boyarchuk, Yekaterina; Salic, Adrian; Dasso, Mary et al. (2007) Bub1 is essential for assembly of the functional inner centromere. J Cell Biol 176:919-28 |
| Orjalo, Arturo V; Arnaoutov, Alexei; Shen, Zhouxin et al. (2006) The Nup107-160 nucleoporin complex is required for correct bipolar spindle assembly. Mol Biol Cell 17:3806-18 |
| Dasso, M (2006) Ran at kinetochores. Biochem Soc Trans 34:711-5 |
| Rundle, Natalie T; Nelson, Jim; Flory, Mark R et al. (2006) An ent-kaurene that inhibits mitotic chromosome movement and binds the kinetochore protein ran-binding protein 2. ACS Chem Biol 1:443-50 |
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