Research Interests in the Sible Lab
The early cell cycle of Xenopus laevis
Our laboratory investigates the regulation of the eukaryotic cell cycle during the early development of the South African clawed frog Xenopus laevis. In the Xenopus embryo, the first twelve mitotic divisions after fertilization are rapid, synchronous, and are characterized by alternating phases of DNA synthesis (S phase) and mitosis (M phase) with no intervening gap phases (G phases). There is no transcription during these cycles, and thus, the cell cycle is regulated by maternally supplied products. After the twelfth cleavage, the embryos begin the midblastula blastula transition (MBT) during which transcription is initiated and cells become motile, events required for gastrulation. The simple, embryonic cell cycles gain complexity after the MBT as cell divisions become asynchronous and slower, and G phases are acquired.
This period of cell cycle remodeling during early development of Xenopus is the focus of research in our laboratory. We and others have come to appreciate that cell cycle regulators play unique roles at this critical stage of early development. By discovering how the cell cycle functions in this dynamic context, we hope to learn more about how the cell cycle is misregulated in disease states, such as cancer, and how it can be redirected through novel therapeutic interventions.
We are also interested in identifying fundamental properties of the cell cycle at a systems-level. Using cell-free egg extracts from Xenopus, as well as embryos, we collaborate with the Tyson Group to combine computational and experimental cell biology to develop mathematical models that accurately describe and predict cell cycle behaviors.
Cell cycle checkpoints in the early embryo
Checkpoints in the eukaryotic cell cycle maintain the integrity of the genome by arresting cell cycle progression when DNA is damaged or incompletely replicated (Novak et al. 2002). Checkpoint pathways converge upon the activities of cyclin-dependent kinases (Cdks), the enzymes that drive cell cycle.
The Xenopus embryo does not engage cell cycle checkpoints prior to the MBT. Instead, the embryo continues rapid cleavage divisions until the early gastrula stage when cells with damage die by a maternally regulated program of apoptosis (Sible et al. 1997). However, we have demonstrated that both checkpoint kinases Chk1 and Chk2 are expressed and that signaling pathways downstream of these kinases can be activated even prior to the MBT (Kappas et al., 2000).
Even though cell cycle checkpoints are not activated before the MBT, Chk1 is transiently activated at the MBT, even in the absence of damaged or unreplicated DNA (Sagata lab). We have shown that overexpresssion of Chk1 interferes with cell cycle remodeling (Petrus et al. 2004), and inhibition of either Chk1 or Chk2 kinases in the early embryo leads to activation of the maternal program of apoptosis (Carter and Sible, 2003; Wroble and Sible, 2005). Therefore, checkpoint kinase pathways, and Chk1 in particular, are essential players in the transition from rapid cleavage divisions, to slower cell cycles with checkpoints in the early embryo. In these studies, we also discovered that modest overexpression of Chk2 in Xenopus protected embryos from radiation-induced apoptosis. This discovery may have implications for radiation therapy and minimizing its side effects.
Recently, we have made some interesting discoveries as we have probed further into the regulatory mechanism that triggers cell cycle remodeling in the early embryo. One compelling hypothesis is that a threshold nucleocytoplasmic (N/C) ratio, achieved at the MBT, is the trigger for cell cycle remodeling. However, we have discovered that the transient activation of Chk1 at the MBT occurs independent of the N/C ratio (Adjerid et al. 2008). We have also discovered a link between the cyclin E/Cdk2 timer and the maternal program of apoptosis that functions at the MBT (Wroble et al. 2007). In ongoing studies, we are exploring the developmental regulation of Claspin, a regulator of Chk1 kinase. Not surprisingly, we have identified distinct regulatory features for Claspin in the dynamic context of the early Xenopus cell cycles. Stay tuned for more information!
Toward a systems-level view of the eukaryotic cell cycle
Our laboratory is also collaborating with the Tyson Research Group, fellow members of the Department of Biological Sciences at Virginia Tech. Tyson and colleagues have developed mathematical equations based on classic biochemical kinetics and modern dynamic systems theory to model the eukaryotic cell cycle. These equations make specific predictions about the behavior cycling cell-free extracts derived from Xenopus eggs.
Our laboratory tested the most fundamental predictions of the Novak-Tyson mathematical model (Sha et al., 2003). Cells progressing through the cell cycle must commit irreversibly to mitosis without slipping back to interphase before properly segregating their chromosomes. The Novak-Tyson model predicts that irreversible transitions into and out of mitosis are driven by hysteresis in the molecular control system. Hysteresis refers to toggle-like switching behavior in a dynamical system. In the mathematical model, the toggle switch is created by positive feedback in the phosphorylation reactions controlling the activity of Cdc2, a protein kinase bound to its regulatory subunit, cyclin B. To determine whether hysteresis underlies entry into and exit from mitosis in cell-free egg extracts, we tested three predictions of the Novak-Tyson model. (1) The minimal concentration of cyclin B necessary to drive an interphase extract into mitosis is distinctly higher than the minimal concentration necessary to hold a mitotic extract in mitosis, evidence for hysteresis. (2) Unreplicated DNA elevates the cyclin threshold for Cdc2 activation, indication that checkpoints operate by enlarging the hysteresis loop. (3) A dramatic "slowing-down" in the rate of Cdc2 activation is detected at concentrations of cyclin B marginally above the activation threshold. All three predictions were validated. These observations confirm hysteresis as the driving force for cell cycle transitions into and out of mitosis.
We are expanding our studies to provide a more quantitative analysis of DNA replication and DNA damage checkpoints. Our model of the DNA replication checkpoint in frog egg extracts fits a large body of experimental data and verifies distinct roles for the cell cycle inhibitory kinases Wee1 and Myt1. This project was also an opportunity to test newly developed Parameter Optimization Tools (PET) from the Tyson group and to develop a protocol for using PET for medium-sized data sets. The manuscript describing our model was just accepted in Journal of Theoretical Biology and will be available here soon.
We have also paired modeling and experimentation to study the less well characterized oscillations of the cyclin E/Cdk2 developmental timer in Xenopus embryos (Ciliberto et al. 2003). The collaboration between molecular cell biologists and kinetic theorists was a novel approach when we began 10 years ago, but we are now proud to be part of a rapidly growing group of colleagues in computational cell biology.
Research in STEM Education
In addition to our cell biology program, we also engage in research in science education, including investigation of pedagogies to better engage women and minorities in the practice of science (Sible et al. 2006). Current projects include an NSF-funded program to training economically challenged students for careers in biotechnology, an NIH-funded post-baccalaureate program for underrepresented minorities, and implementation of Scientific Teaching methods in a large freshman biology class.