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Cell Division, Cell Morphogenesis, and Cell Fate Specification
Summary: Yixian Zheng's lab studies how eukaryotic cells divide, differentiate, and evolve. Specifically, the lab studies how eukaryotic cells orchestrate their division and differentiation through the morphogenesis of the mitotic spindle in a number of systems, including embryonic stem cells. The lab also uses hybrid yeasts from the Saccharomyces sensu stricto group to study eukaryotic genome evolution.
My lab is interested in understanding how the cytoskeleton, the nuclear lamina, and the membrane network coordinate with one another to regulate cell division and differentiate. We employ a variety of model systems to study the mechanism of cell division. Using embryonic stem cells (ESCs) and mouse embryos, we also study how cell migration, cellular morphogenesis, and cell division are coupled to cell fate specifications during development. I am also carrying out a collaborative project with Douglas Koshland (HHMI, Carnegie Institution of Washington) and Maitreya Dunham (University of Washington) to use hybrid yeasts to study genome evolution.
Cell Division: Mitotic Spindle Assembly and Chromosome Segregation
Spindle morphogenesis ensures equal segregation of mitotic chromosomes and proper partitioning of other cellular components important for the survival, proliferation, and differentiation of daughter cells. Spindle assembly and organization are orchestrated by a large array of structural and regulatory proteins.
Centrosome and microtubule nucleation. Centrosomes play important roles in mitotic spindle assembly by providing the major microtubule (MT) nucleation and organization sites. Through the purification and study of the ~36S γ-tubulin ring complex (γTuRC; Figure 1), we showed that this complex is essential not only for MT nucleation at the centrosome but also for mitotic spindle assembly.
Using a centrosome-complementation assay, biochemical fractionations, and mass spectrometry (in collaboration with John Yates [Scripps Research Institute], we have identified additional candidate factors that regulate MT nucleation from centrosomes. We have shown that one of these proteins, called Pontin, an AAA+ ATPase involved in a diverse array of cellular functions, interacts with γTuRC to promote MT assembly in mitosis.
Spindle assembly: RanGTPase signaling and the mitotic spindle matrix. We have shown that the nuclear, small GTPase Ran regulates multiple aspects of spindle assembly in mitosis by modulating the interaction between spindle assembly factors (SAFs) containing nuclear localization signals (NLSs) with nuclear transport receptors importin-α and -β. Additional analyses have led us to an important Ran signaling pathway that leads to activation of the important mitotic kinase Aurora A (AurA; Figure 2). By coupling AurA to magnetic beads (AurA beads), we developed an efficient spindle assembly assay, which has offered us the opportunity to study not only AurA kinase but also spindle assembly (Figure 3).
Aided by this assay, we uncovered a mitosis-specific function for lamin B, a type V intermediate filament protein with a well-established role in nuclear organization and gene regulation in interphase. We have shown that lamin B is a downstream target of RanGTPase and that it regulates spindle morphogenesis as one of the structural components of the membranous spindle matrix that tethers a number of SAFs. Our studies suggest that RanGTP independently regulates the assembly of MTs and lamin B, which reciprocally regulate each other by interacting with the SAFs, leading to spindle assembly.
Spindle assembly: RanGTPase signaling and the cell cycle. Our studies have shown that the cell cycle machinery directly regulates the Ran-signaling pathway by phosphorylating the NLS of RCC1, the nucleotide exchange factor for Ran. Phosphorylated RCC1 does not bind to importin-α and -β; consequently, it is able to produce a high concentration of RanGTP on chromosomes to guide spindle assembly toward the chromosomes. By manipulating a Ran-binding protein called RanBP1 in combination with computational simulation (collaboration with Pablo Iglesias [Johns Hopkins University]), we showed that the highest RanGTP concentration gradient could be achieved when all mitotic chromosomes have aligned to the metaphase plate. This elevated RanGTP level facilitates metaphase-to-anaphase transition by aiding the inactivation of the spindle checkpoint. Our studies have demonstrated that the Ran-signaling pathway and the cell cycle machinery reciprocally regulate each other to control both spindle assembly and mitotic progression.
Chromosome segregation and spindle disassembly. We found that two sets of membrane-associated enzymes, the ubiquitin-selective chaperone p97-Ufd1-Npl4 complex and the deubiquitination enzyme FAM, regulate mitosis. The p97-Ufd1-Npl4 complex and FAM interact with and regulate Survivin, a subunit of the chromosome passenger complex required for chromosome segregation and cytokinesis. Whereas FAM removes the K63-ubiquitin linkage on Survivin in mitosis, p97-Ufd1-Npl4 is required for Survivin to acquire such linkages. A balanced K63 ubiquitination level on Survivin in turn regulates the dynamic binding of Survivin to centromeres, which is important for chromosome alignment and segregation. Our further study of p97-Ufd1-Npl4 has demonstrated that this chaperone also regulates spindle disassembly at the end of mitosis.
Lineage Specification During Development and Stem Cell Differentiation
Our studies of mitosis have shown that a component of the nuclear lamina (lamin B) functions with the MT cytoskeleton to help chromosome organization and movement on the mitotic spindle. By analogy, the interphase nuclear lamina might also function with cytoskeletons to organize chromatin and to affect gene expression. Indeed, the interphase nuclear envelope and lamina make extensive contacts with chromatin and the cytoskeleton in the nucleus and cytoplasm, respectively. Therefore, cytoskeleton reorganization occurring during cell migration and cell-shape change could influence chromatin organization through the nuclear lamina, which in turn could affect gene regulation. The changes in transcription may also affect cytoskeleton assembly dynamics. This kind of reciprocal regulation could play an important role in development, because specification of cell lineages relies not only on transcriptional regulation but also on cell division and gradual cellular morphological changes.
To explore this idea, we have performed live imaging of the behavior of pluripotent ESCs as they differentiate into different cell lineages. We found that distinct cell behavior accompanied different differentiation pathways. We have focused our study on the first lineage specification in preimplantation mammals, which is known to involve cell sorting and transcriptional changes. Successful specification of the first lineage results in the formation of a blastocyst containing the outer trophectoderm (TE) cells expressing the transcription factor Cdx2 and the inner cell mass (ICM) that could give rise to ESCs.
By analyzing cellular morphogenesis and gene expression profiles during TE differentiation from mouse ESCs, we have uncovered a role for a protein, which we named Recem (regulator of cell morphogenesis), in first-lineage specification. We show that differentiation of ESCs toward TE is accompanied by an enhanced cell motility that requires up-regulation of Recem. We have developed a cell-sorting assay to show that Recem is required for sorting together the differentiating TE cells from ESCs and for the TE cells to attract and enclose ESCs. The Recem protein localizes to the nuclei of precompaction embryos, becomes enriched in the cytoplasm and at cell-cell contacts in morula, and exhibits stronger expression in TE than in ICM in blastocysts. Since reduction of Recem blocks blastocyst formation and since Cdx2 and Recem facilitate each other's expression, we propose that the two proteins function in coupling cell sorting with transcriptional changes to regulate first-lineage specification.
Genome Evolution of the Hybrid Yeast
Genome duplication, involving individual genes, individual chromosomes, or a whole genome, is thought to be advantageous for evolutionary innovations. In plants, whole-genome duplication (WGD)—either from a diploid genome or following interspecies hybridization—has long been recognized as a common phenomenon that drives the evolution of new species. Although genome duplication is a widespread evolutionary phenomenon, little is known about the forces that drive the duplication or the molecular mechanism that underlies the genome reorganization/gene expression changes following duplication. Studies of gene paralogs in S. cerevisiae suggest that WGD also occurred in yeasts. In collaboration with Koshland and Dunham, I have used the fully sequenced yeast sensu stricto species (S. cerevisiae and S. bayanus), which cross-hybridize naturally but produce mostly unviable gametes, to study genome evolution. By studying the telomere behavior in the hybrids, I found evidence that the telomere systems of S. cerevisiae and S. bayanus have evolved apart significantly to become partially incompatible. This incompatibility could be one of the driving forces for chromosome rearrangements, gene deletion, or duplication in hybrid yeasts. The hybrid yeast may thus offer an opportunity to study genome evolution as a result of interspecific hybridization.
Last updated December 04, 2008
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