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Cell Division, Cell Morphogenesis, and Cell Fate Specification
Summary: Yixian Zheng's lab studies how eukaryotic cells divide and differentiate. 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, and mouse embryos.
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.
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 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 microtubule 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 microtubule nucleation from centrosomes. By characterizing one of these proteins, called Pontin, an AAA+ ATPase involved in diverse array of cellular functions, we have shown that Pontin interacts with γTuRC to promote microtubule assembly in mitosis.
Spindle assembly: Ran GTPase signaling and the membranous 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 uncover a Ran-signaling pathway that leads to activation of the 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 an 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 Ran GTPase and lamin B regulates spindle morphogenesis as one of the structural components of the membranous spindle matrix that tethers a number of SAFs. Our studies suggest that the GTP-bound Ran GTPase independently regulates the assembly of microtubules and lamin B, which reciprocally regulate each other by interacting with the SAFs, leading to spindle assembly.
Because membranes are part of the spindle matrix, proper organization of the membrane network may be important for spindle assembly. We have begun to explore this possibility by asking whether perturbing the ability of membranes to change their shapes would affect spindle morphology. Our studies showed that the endocytic accessory protein epsin, which can bend membranes, facilitates mitotic spindle organization in both tissue culture cells and Xenopus egg extracts. This revealed that proper membrane remodeling in mitosis affects spindle morphogenesis.
Spindle assembly: Ran GTPase 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 assembly 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, spindle disassembly, and cytokinesis. We found that two sets of membrane-associated enzymes, the ubiquitin-selective chaperone called the p97-Ufd1-Npl4 complex and the deubiquitination enzyme called 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 through a number of known spindle assembly factors. Mitsuhiro Yanagida and colleagues (Kyoto University) have shown that the p97 homolog in Schizosaccharomyces pombe is required to stabilize separase in anaphase. Since separase is required for chromosome separation and anaphase spindle morphogenesis, we asked whether separase could also regulate other aspects of anaphase functions. Using Caenorhabditis elegans, we demonstrated that separase regulates RAB-11-positive vesicles at the cleavage furrow and midbody and is required for successful cytokinesis in C. elegans early embryos.
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 microtubule 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 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.
We have explored this idea by performing 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 that could give rise to ESCs.
By analyzing cellular morphogenesis and gene expression profiles during TE differentiation from mouse ESCs, we have uncovered a new morphological regulator called binder of Rho GTPases 5 (Borg5). We found that differentiation of ESCs toward TE is accompanied by enhanced cell polarization and motility that requires up-regulation of Borg5. Using a cell-sorting assay, we showed that Borg5 is required for the sorting of differentiating TE cells from ESCs and for the TE cells to attract and enclose ESCs. Borg5 and Cdx2 are both up-regulated early, and they enhance each other's expression during TE differentiation. Borg5 interacts with the polarity regulator atypical protein kinase C (aPKC) and functions downstream of Cdc42 to enhance TE cell polarization and motility. Reduction of Borg5 in one-cell embryos disrupts aPKC localization, reduces Cdx2 expression, and inhibits TE development and blastocyst formation. We propose that the crosstalk between Borg5 and Cdx2 represents a general mechanism that couples gradual cell morphogenesis with progressive transcriptional changes in the building of tissues. Our study provides a starting point to define the coupling mechanism, which should help to decipher the steps of cell differentiation and tissue morphogenesis.
Last updated May 24, 2010
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