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Chromosome Segregation in Higher Eukaryotes
- Mary Dasso, PhD, Head, Section on Cell Cycle Regulation
- Maia Ouspenskaia, DVM, Biologist
- Alexei Arnaoutov, PhD, Visiting Fellow
- Ming-Ta Lee, PhD, Visiting Fellow
- Maria Lianguzova, PhD, Visiting Fellow
- Sarine Markossian, PhD, Visiting Fellow
- Min Mo, PhD, Visiting Fellow
- Hyunju Ryu, PhD, Visiting Fellow
- Shaofei Zhang, BA, Graduate Student
We are interested in mechanisms of chromosome segregation. Defects in chromosome segregation lead to aneuploidy, the condition of having an abnormal number of chromosomes. Several common birth defects, such as Down's syndrome, result from aneuploidy arising during meiotic cell divisions. Moreover, aneuploidy arising from mitotic divisions is a hallmark of many types of solid tumors. During interphase, chromosomes are enclosed within nuclei, and exchange of all molecules between this compartment and the rest of the cell occurs through nuclear pore complexes (NPCs). Surprisingly, NPC proteins and proteins involved in trafficking of molecules into and out of the nucleus have important roles in chromosome segregation; we are investigating these roles at a molecular level. Our studies concentrate on a GTPase called Ran and on a family of small ubiquitin-like modifiers (SUMOs), which are indispensable for mitotic chromosome segregation. The ultimate goals of our studies are to understand how these proteins enable accurate chromosome segregation and to discover how they are coordinated with each other and with other aspects of cell physiology.
Mitotic roles of nuclear pore complex proteins
Trafficking between the nucleus and cytoplasm occurs through nuclear pore complexes (NPCs), which consist of ca. thirty distinct proteins called nucleoporins. Kinetochores are proteinaceous structures that assemble at the centromere of each sister chromatid during mitosis and serve as sites of spindle microtubule attachment. The relationship between NPCs and mitotic kinetochores is both surprisingly intimate and poorly understood. During interphase, several kinetochore proteins stably bind to NPCs (e.g., Mad1, Mad2, Mps1). During mitosis, metazoan NPCs disassemble, and at least a third of nucleoporins associate with kinetochores, including the RanBP2 complex and the Nup107-160 complex. We showed that these complexes play important roles in kinetochore function. Several other nucleoporins that do not associate with kinetochores have also been shown to have important mitotic roles, including Nup214, Nup98, and TPR. Much of our current work concerns the RanBP2 complex, which consists of RanBP2 (a large nucleoporin that is also known as Nup358), SUMO-1–conjugated RanGAP1 (the activating protein for the Ran GTPase), and Ubc9 (the conjugating enzyme for the SUMO family of ubiquitin-like modifiers).
The RanBP2 complex associates with kinetochores in a microtubule-dependent manner that also requires Crm1, a Ran-dependent nuclear export receptor. Disruption of RanBP2 association with kinetochores causes defective mitotic spindle assembly. Additional observations suggest an in vivo role of RanBP2 in interphase microtubule organization. Our current studies on this complex focus on functional dissection of the multiple domains within this large protein and on interacting proteins that may be essential for the function of the RanBP2 complex. We developed cell lines in which RanBP2 levels can be controlled through the induction of short interfering RNAs (siRNAs). We are currently assessing cellular phenotypes that result from the loss of RanBP2 and will further determine whether physiological defects can be reversed through expression of RanBP2 mutants lacking one or more functional domains. As a comparative system, we are analyzing the association of RanBP2 and RanGAP1 in invertebrate species, particularly the fly Drosophila melanogaster. Flies mediate the association of RanBP2 and RanGAP1 through a biochemically distinct set of interactions that do not require SUMOylation or Ubc9. Elucidation of the mechanism of this association will not only allow us to test the importance of RanBP2 complex formation in a non-vertebrate system, but also provide a convenient alternative mechanism for formation of this complex that can be used to understand its importance in vertebrate cells.
Mitotic regulation of the Ran GTPase
Ran is a Ras-family GTPase that plays critical roles in multiple cellular processes including nucleo-cytoplasmic transport, nuclear envelope assembly and mitotic spindle assembly. Ran alternates between GDP– and GTP–bound forms. In interphase cells, GTP–bound Ran (Ran-GTP) is the major form in nucleus while GDP–bound Ran (Ran-GDP) is the predominant form in cytoplasm. The asymmetrical distribution of Ran-GTP and Ran-GDP drives cargo transport between the nucleus and cytoplasm through karyopherins, a family of nuclear transport carrier proteins that bind to Ran-GTP. In mitosis, after nuclear envelope breakdown, Ran-GTP is concentrated in the region close to mitotic chromatin, while Ran-GDP is the major form distal to chromatin. The Ran-GTP gradient guides mitotic spindle assembly by releasing spindle assembly factors (SAFs) from karyopherins based on local Ran-GTP concentrations. The conversion of Ran-GDP to Ran-GTP in cells is catalyzed by a Ran-specific guanine exchange factor (RanGEF), called RCC1 (Regulator of chromosome condensation 1) in vertebrates. The capacity of RCC1 to bind to chromatin establishes the asymmetrical distribution of Ran-GTP in interphase as well as the chromatin-centered Ran-GTP gradient in mitosis. Interestingly, RCC1’s association to chromatin is not static during the cell cycle and is regulated in a particularly dramatic fashion during anaphase in vertebrate systems. The regulation has not been correlated with posttranslational modifications of RCC1, and its underlying molecular mechanism has not been reported.
RanBP1 is a highly conserved Ran-GTP–binding protein that acts as co-activator of RanGAP1 and can form a heterotrimeric complex with Ran and RCC1 in vitro. We have found that RCC1 not associated with chromosomes during mitosis is sequestered and inhibited in RCC1/Ran/RanBP1 heterotrimeric complexes and that the sequestration is crucial for normal mitotic spindle assembly. In addition, RanBP1 complex formation competes with chromatin binding to regulate the distribution of RCC1 between the chromatin-associated and soluble fractions. Moreover, we identified a cell cycle–dependent phosphorylation on RanBP1 that modulates RCC1/Ran/RanBP1 heterotrimeric complex assembly and releases RCC1 to bind to chromatin; the phosporylation is directly responsible for controlling RCC1 dynamics during anaphase. Together, our findings demonstrate novel roles of RanBP1 in spindle assembly and RCC1 regulation in mitosis. We are currently examining the details of this mechanism, how it facilitates chromosome segregation and mitotic exit, and how it may modulate changes in the Ran pathway as cells re-establish interphase nuclear structures.
SUMO–family small ubiquitin-like modifiers in higher eukaryotes
SUMOs are ubiquitin-like proteins (Ubls) that become conjugated to substrates through a pathway that is biochemically similar to ubiquitination. SUMOylation is involved in many cellular processes, including DNA metabolism, gene expression, and cell cycle progression. Vertebrate cells express three major SUMO paralogs (SUMO-1–3): mature SUMO-2 and SUMO-3 are 95% identical to each other, while SUMO-1 is 45% identical to SUMO-2 or SUMO-3. (Where they are functionally indistinguishable, we collectively call SUMO-2 and SUMO-3 SUMO-2/3.) Like ubiquitin, SUMO-2/3 can be assembled into polymeric chains through the sequential conjugation of SUMOs to each other. Many SUMOylation substrates have been identified. SUMOylation promotes a variety of fates for individual targets, dependent upon protein itself, the conjugated paralog, and whether the conjugated species contains a single SUMO or SUMO chains.
SUMOylation is dynamic owing to rapid turnover of conjugated species by SUMO proteases. Both post-translational processing of SUMO polypeptides and deSUMOylation are mediated by the same family of proteases, which play a pivotal role in determining the spectrum of SUMOylated species. This group of proteases is called Ubl–specific proteases (Ulp) in yeast and Sentrin-specific proteases (SENP) in vertebrates. There are two yeast Ulps (Ulp1p and Ulp2p/Smt4p), and six mammalian SENPs (SENP1, SENP2, SENP3, SENP5, SENP6, and SENP7). SENP1, SENP2, SENP3 and SENP5 form a Ulp1p–related sub-family, while SENP6 and SENP7 are more closely related to Ulp2p. Yeast Ulps have important roles in mitotic progression and chromosome segregation. We defined the enzymatic specificity of the vertebrate SENP proteins and analyzed their key biological roles.
Ulp1p localizes to NPCs and is encoded by an essential gene; it is important for SUMO processing, nucleocytoplasmic trafficking, and late steps in the ribosome biogenesis pathway. Humans possess two NPC–associated SENPs: SENP1 and SENP2. While SENP2 is dispensable for cell division, mammalian SENP1 was recently shown to play an essential role in mitotic progression. Notably, we found that frogs possess a single NPC–bound SUMO protease, xSENP1, and we are currently exploiting this fact to analyze the function of SUMO proteases at the NPC.
We determined the interaction partners of this enzyme throughout the cell cycle using Xenopus egg extracts (XEEs). We found that xSENP1 associates strongly with Psmd1, the largest subunit of the proteasome 19S regulatory particle (19S-RP). Proteasomes are complex ATP–dependent proteases that mediate the degradation of many cellular proteins typically targeted for destruction by ubiquitination. Ubiquitinated degradation substrates are fed into the proteasome’s catalytic 20S core particle (20S-CP) through the 19S-RP. Psmd1 plays a key structural role in the 19S-RP, and acts as a binding site for the recruitment of other proteasome subunits, including Adrm1. Adrm1 is one of two subunits that directly recruit ubiquitinated substrates to the proteasome. While many proteasomal subunits have been found in proteomic screens for SUMOylation substrates, no role of these modifications has been reported. We mapped SUMOylation sites within Psmd1 and found that modification of a critical lysine adjacent to the Adrm1–binding domain regulates Adrm1 association with Psmd1. Our findings suggest that Psmd1 SUMOylation controls proteasome composition and function, providing a new mechanism for the regulation of ubiquitin-mediated protein degradation through the SUMO pathway.
Yeast Ulp2p is nucleoplasmic and not essential for vegetative growth but important for chromosome segregation. Ulp2p acts particularly in disassembly of poly-SUMO chains. We demonstrated that human SENP6 is a vertebrate Ulp2p–related enzyme that similarly prefers substrates containing multiple SUMO-2/3 moieties. We analyzed the mitotic role of SENP6 and found that it is essential for accurate chromosome segregation. Mitotic defects observed in the absence of SENP6 reflected the loss of inner kinetochore proteins, including components of the CENP-H/I/K and CENP-O complexes. The findings demonstrate a novel function of the SUMO pathway in inner kinetochore assembly, which finely balances the incorporation and degradation of components of the inner plate. We are currently analyzing other aspects of SENP6 mitotic function, including its role in chromosome morphology.
- Chow K-H, Elgort S, Dasso M, Powers M, Ullman K. The SUMO proteases, SENP1 and SENP2, play a critical role in nucleoporin homeostasis and nuclear pore complex function. Mol Biol Cell 2013;in press.
- Neyret-Kahn H, Benhamed M, Ye T, Le Gras S, Cossec JC, Lapaquette P, Bischof O, Ouspenskaia M, Dasso M, Seeler J, Davidson I, Dejean A. Sumoylation at chromatin governs coordinated repression of a transcriptional program essential for cell growth and proliferation. Genome Res 2013;23:1563-1579.
- O'Rourke JG, Gareau JH, Ochaba J, Song W, Raskó T, Reverter D, Lee J, Monteys AM, Pallos J, Mee L, Vashishtha M, Apostol BL, Nicholson TP, Illes K, Zhu YZ, Dasso M, Bates GP, Difiglia M, Davidson B, Wanker EE, Marsh J, Lima CD, Steffan JS, Thompson LM. SUMO-2 and PIAS1 modulate insoluble mutant huntingtin protein accumulation. Cell Rep 2013;4:362-375.
- Sharma P, Yamada S, Lualdi M, Dasso M, Kuehn MR. Senp1 is essential for desumoylating sumo1-modified proteins but dispensable for SUMO2 and SUMO3 deconjugation in the mouse embryo. Cell Rep 2013;3:1640-1650.
- Dasso M. A Mad that wears two hats: Mad1's control of nuclear trafficking. Dev Cell 2013;24:121-122.
- Anne Dejean, PhD, Institut Pasteur, Paris, France
- Michael R. Kuehn, PhD, Laboratory of Protein Dynamics and Signaling, Center for Cancer Research, NCI, Frederick, MD
- Leslie M. Thompson, PhD, University of California, Irvine, Irvine, CA
- Katharine S. Ullman, PhD, Huntsman Cancer Institute, University of Utah, Salt Lake City, UT