Control of E2F transcription factor activity, regulated by the action of the retinoblastoma tumor suppressor, is critical for determining cell cycle entry and cell proliferation. However, an understanding of the precise determinants of this control, including the role of other cell cycle regulatory activities, has not been clearly defined.

Recognizing that the contributions of individual regulatory components could be masked by heterogeneity in populations of cells, we made use of an integrated system to follow E2F transcriptional dynamics at the single cell level and in real time. We measured and characterized E2F temporal dynamics in the first cell cycle where cells enter the cell cycle after a period of quiescence. Quantitative analyses revealed that crossing a threshold of amplitude of E2F transcriptional activity serves as the critical determinant of cell-cycle commitment and division.

By using a developed ordinary differential equation model for Rb/E2F network, we performed simulations and predicted that Myc and cyclin D/E activities have distinct roles in modulating E2F transcriptional dynamics. Myc is critical in modulating the amplitude whereas cyclin D/E activities have little effect on the amplitude but do contribute to the modulation of duration of E2F transcriptional activation. These predictions were validated through the analysis of E2F dynamics in single cells under the conditions that cyclin D/E or Myc activities are perturbed by small molecule inhibitors or RNA interference.

In an ongoing study...

Cell division is a biological process fundamental to all life. One aspect of the process that is still under investigation is whether or not cells in a lineage are correlated in their cell-cycle progression. Data on cell-cycle progression is typically acquired either in lineages of single cells or in synchronized cell populations, and each source of data offers complementary information on cell division. To formally assess dependence in cell-cycle progression, I develop a hierarchical statistical model of single-cell measurements and extend a previously proposed model of population cell division in the budding yeast, Saccharomyces cerevisiae. Both models capture correlation and cell-to-cell heterogeneity in cell-cycle progression, and parameter inference is carried out in a fully Bayesian manner. The single-cell model is fit to three published time-lapse microscopy datasets and the population-based model is fit to simulated data for which the true model is known. Based on posterior inferences and formal model comparisons, the single-cell analysis demonstrates that budding yeast mother and daughter cells do not appear to correlate in their cell-cycle progression in two of the three experimental settings. In contrast...

The cell cycle is a series of ordered events that culminates in a single cell dividing into two daughter cells. These events must be properly coordinated to ensure the faithful passage of genetic material. How cell cycle events are carried out accurately remains a fundamental question in cell biology. In this dissertation, I investigate mechanisms orchestrating cell-cycle events in the yeast, Saccharomyces cerevisiae.

Cyclin dependent kinase (CDK) activity is thought to both form the fundamental cell-cycle oscillator and act as an effector of that oscillator, regulating cell-cycle events. By measuring transcript dynamics over time in cells lacking all CDK activity, I show that transcriptional oscillations are not dependent on CDK activity. This data indicates that CDKs do not form the underlying cell-cycle oscillator. I propose a model in which a transcription factor network rather than CDK activity forms the cell-cycle oscillator. In this model, CDKs are activated by the periodic transcription of cyclin genes and feedback on the network increasing the robustness of network oscillations in addition to regulating cell-cycle events.

I also investigate CDK-dependent and -independent mechanism regulating the duplication of the yeast centrosome...

The cell cycle is one of the fundamental processes in all living organisms, and all cells arise from the division of existing cells. To better understand the regulation of the cell cycle, synchrony experiments are widely used to monitor cellular dynamics during this process. In such experiments, a large population of cells is generally arrested or selected at one stage of the cycle, and then released to progress through subsequent division stages. Measurements are then taken in this population at a variety of time points after release to provide insight into the dynamics of the cell cycle. However, due to cell-to-cell variability and asymmetric cell division, cells in a synchronized population lose synchrony over time. As a result, the time-series measurements from the synchronized cell populations do not accurately reflect the underlying dynamics of cell-cycle processes.

In this thesis, we introduce a deconvolution algorithm that learns a more accurate view of cell-cycle dynamics, free from the convolution effects associated with imperfect cell synchronization. Through wavelet-basis regularization, our method sharpens signal without sharpening noise, and can remarkably increase both the dynamic range and the temporal resolution of time-series data. Though it can be applied to any such data...

The cell division cycle is the process in which the entirety of a cell's contents is duplicated completely and then equally segregated into two identical daughter cells. The order of the steps in the cell cycle must be followed with fidelity to guarantee two viable cells. Understanding the regulatory mechanisms that control cell-cycle events remains to be a fundamental question in cell biology. In this dissertation, I explore the mechanisms that coordinate and regulate cell-cycle progression in the budding yeast, Saccharomyces cerevisiae.

Cell-cycle events have been shown to be triggered by oscillations in the activity of cyclin dependent kinases (CDKs) when bound to cyclins. However, several studies have shown that some cell-cycle events, such as periodic transcription, can continue in the absence of CDK activity. How are periodic transcription and other cell-cycle events coupled to each other during a wild-type cell cycle? Currently, two models of cell-cycle regulation have been proposed. One model hypothesizes that oscillations in CDK activity controls the timing of cell-cycle events, including periodic transcription. The second model proposes that a transcription factor (TF) network oscillator controls the timing of cell-cycle events...

Within individual cells, the opposing processes of proliferation and apoptosis are precisely regulated. When this regulatory balance is interrupted, cells may become abnormal or even transformed. Understanding how to reverse or avoid these detrimental transformative processes begins with an intimate knowledge of the processes governing the cell cycle and apoptosis. Cell proliferation is governed by the cell cycle machinery. The cell cycle is driven by Cyclin-dependent kinase (Cdk) activity, which is dependent on the availability of specific Cyclin binding partners. The amount of available Cyclin is tightly controlled by a ubiquitin ligase protein complex called the anaphase promoting complex/cyclosome (APC/C.) This complex mediates the timely ubiquitylation and degradation of cell cycle regulators in order to control mitotic exit, the G1/S transition and to respond to signals emanating from spindle assembly checkpoint.

Given the importance of the APC/C, cells develop many ways to regulate APC/C activity. Post-translational modifications of the APC/C have been shown to alter its functionality, and many pseudosubstrate-based inhibitors have been discovered. Moreover, inhibitors such as Emi1 and Emi2, have been showed to inhibit the APC/C through their own intrinsic ubiquitin E3 ligase activities. Utilizing the Xenopus egg extract system...