Cell Division, Cell Morphogenesis, and Cell Fate Specification

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 (ESC) and mouse embryos, we also study how cell migration, cellular morphogenesis, and cell division are coupled to development and tissue homeostasis.

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 is 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), 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 Dr. John Yates), we have identified additional candidate factors that regulate MT nucleation from centrosomes. By further 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 MT assembly in mitosis.

Spindle assembly—the 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 (NLS) with nuclear transport receptors importin α and β. Additional analyses have led us to uncover an important Ran signaling pathway that leads to activation of the important mitotic kinase Aurora A (AurA). By coupling AurA to magnetic beads (AurA-beads), we developed an efficient spindle assembly assay, which has offered us with a great opportunity to study not only AurA kinase but also spindle assembly.

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 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 spindle matrix, which reciprocally regulate each other through interacting with the SAFs, leading to spindle assembly.

Spindle Assembly—the 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 importins α and β, consequently 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 Dr. Pablo Iglesias), we showed that the highest RanGTP concentration gradient could be achieved when all mitotic chromosomes have congressed to the metaphase plate.  This elevated RanGTP level facilitates metaphase to anaphase transition by aiding the inactivation of the spindle checkpoint.  Together, 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 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 K-63 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.

The coupling of cell morphogenesis and cell division with development and tissue homeostasis

Our studies of mitosis have shown that the 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 organization 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 re-organization 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 pre-implantation 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 during TE differentiation from mouse embryonic stem cells (ESC), we have uncovered a role for the protein, binder of RhoGTPases 5 (Borg5), in blastocyst formation.  We find that differentiation of ESCs toward TE is accompanied by enhanced cell polarization and motility that requires up-regulation of Borg5.  Borg5 functions down-stream of Cdc42 and it interacts with the atypical protein kinase C (aPKC) to regulate the sorting of differentiating TE cells from ESCs and for the TE cells to attract and enclose ESCs.  In developing embryos, Borg5 protein localizes to cell-cell contacts and the cytoplasm after compaction.  It exhibits higher levels of expression in outer cells than in inner cells in morula and blastocysts.  Reduction of Borg5 disrupts aPKC localization and inhibits blastocyst formation.  Since Borg5 and Cdx2, a transcription factor essential for TE development, facilitate each other’s expression, we propose that the two proteins regulate first lineage differentiation and blastocyst formation by coupling gradual cell morphogenesis with progressive transcriptional changes.

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