We are interested in the systems required for maintenance of mitotic arrest. In order to complete division and exit mitosis, cells must degrade the cyclin B protein and thereby inactivate the cdc2 kinase. Cyclin B is targeted for degradation by the anaphase-promoting complex (APC/C) with the help of FZY, an adapter protein whose binding to APC/C is necessary for cyclin ubiquitination. After ubiquitination, Cyclin B is rapidly degraded by proteasomes. Cells can be arrested in M phase in response to developmental cues (i.e., Xenopus laevis frog eggs remain arrested in second meiotic metaphaseuntil fertilization, called CSF arrest) or in response to checkpoint signals (i.e., most vertebrate cells will arrest in M phase if one or more unattached kinetochores are present, called checkpoint arrest). Both of these types of arrest can be mimicked in extracts prepared from Xenopus eggs. Addition of calcium eliminates CSF arrest in egg extracts. The mechanism of this release is very similar to the calcium wave triggered by fertilization that promotes destruction of cyclin B and CSF activities and releases the eggs into first interphase. A microtubule-dependent checkpoint can be generated in egg extracts by addition of the microtubule destabilizing agent nocodazole and chromosomes. This microtubule-dependent checkpoint is not sensitive to calcium. To understand molecular mechanisms that are responsible for generation of these two types of arrest, we followed the fate of an I-125 labeled fragment of cyclin B1 in Xenopus egg extract, combined with in vitro reactions with purified components reconstructing ubiquitination by APC/C and FZY, deubiquitination by isopeptidases and degradation by proteasomes. We found that exit from mitosis and therefore degradation of Cyclin B was always accompanied by activation of ubiquitination through APC/C while isopeptidase and proteasomal activities were not changed. After checkpoint activation, APC/C activity was blocked by sequestration of FZY into an inactive complex with checkpoint proteins, thus preventing association of FZY with APC/C. Cyclin B degradation could be restored in checkpoint-arrested extracts with physiological levels of baculovirus-produced FZY. While CSF arrest is also mediated by inhibition of APC/C, we found that it cannot be restored by addition of FZY. To the contrary, our data currently suggest that CSF arrest results from direct inhibition of APC/C by phosphorylation. Taken together, our results indicate that different types of mitotic arrest utilize different approaches to stabilize Cyclin B. We are also interested in examining how Ran interacts with these mitotic regulatory pathways. Ran is a GTPase that is required for nuclear transport, cell cycle control, mitotic spindle formation and post-mitotic nuclear assembly. Ran is regulated by a cytosolic GTPase activating protein, RanGAP1, and by a chromatin-bound nucleotide exchange factor, RCC1. The distribution of Ran-GTP provides important spatial information that directs cellular activities throughout the cell cycle. During interphase, the localization of RCC1 and RanGAP1 predicts that nuclear Ran is GTP-bound and cytosolic Ran is GDP-bound. This compartmentalization determines the direction of nuclear transport by promoting the loading and unloading of transport receptors in a manner that is appropriate to the nucleus or cytosol. In mitosis, chromatin-bound RCC1 protein generates a high concentration of Ran-GTP in the vicinity of the chromosomes. Ran-GTP acts to stabilize microtubules (MTs) near the chromosomes, in a manner that is essential for the formation of a correct bipolar spindle. We have observed that addition of bacterially expressed RCC1 but not an inactive RCC1 mutant override the spindle-assembly checkpoint in a dose-dependent manner. We have further documented that exogenous RCC1 changes the behavior of checkpoint pathway components. Notably, increased concentrations of RCC1 do not release extracts from CSF-mediated arrest. Our results suggest that the mitotic function of Ran GTPase lies not only in formation of the microtubule spindle but also in regulating the checkpoint pathway.
| 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 |
| 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 |
| Prunuske, Amy J; Liu, Jin; Elgort, Suzanne et al. (2006) Nuclear envelope breakdown is coordinated by both Nup358/RanBP2 and Nup153, two nucleoporins with zinc finger modules. Mol Biol Cell 17:760-9 |
Showing the most recent 10 out of 23 publications
