Progression through mitosis depends on perfectly choreographed alterations of the genetic and structural elements of the cell for it to divide and initiate a new cycle. It is during this critical time that a wide array of enzymatic activities function to ensure that this process is completed without fail - and it must do so repeatedly throughout the lifetime of an organism. Protein kinases, phosphatases, and other regulatory proteins are the major regulators of M phase. Mitotic onset and progression is governed primarily by cyclin- dependent kinase 1 (CDK1), and its activation requires the synthesis of M-phase cyclins as well as positive- feedback loops that arise through its stimulation of its activating phosphatase, Cdc25, and through its suppression of its inhibitory kinases, Wee1 and Myt1. In addition to CDK1, several other kinases play important roles in mitosis, including the regulation of centrosomes (Aurora A), the control of spindle assembly and the spindle checkpoint (Aurora B and MAPK), and the stimulation of enzymes that promote mitotic onset and exit (Polo kinase). There has been significant progress in realizing the separate roles that these proteins play in the cell, but there is still no comprehensive description of how their activities are coordinated as a system to drive the complex and dynamic process of mitotic progression. Mutation and/or overexpression of one or more of these regulators is often a hallmark of malignancies, so the pathological basis for many cancers requires understanding not only individual protein function, but also how subsets of proteins act together as a system to control cell division and cell cycle passage. It is the long-term goal of this project to elucidate the general organization of, and quantitate the dependencies between the signaling pathways that govern mitotic entrance, exit, and the cell cycle progression that follows.
The specific aims of this proposal are focused on learning how the networks of kinases and feedback loops that are responsible for M-phase progression coordinate their activities with CDK1, and how they operate as a unit to then modulate mitotic exit through the activity of the anaphase-promoting complex (APC). Dissecting the roles of individual M-phase kinases will be achieved by systematically studying their responses during an early embryonic oscillation of CDK1 activity, and identifying and testing the functional interactions between them using Xenopus laevis egg extracts. The role of positive feedback in CDK1 activation, as well as the effect that reducing this feedback has on M-phase kinase regulation and cell cycle progression will be investigated using single-cell methods in HeLa cells, such as flow cytometry and live-cell imaging. Through experimental and computational methods, we aim to attain a better understanding of how this network of proteins functions as a system to orchestrate precisely the timing and function of the machinery that controls mitotic entrance and exit in eukaryotic cells, from the earliest embryonic stages to adulthood.
The loss of proliferative controls in cells leads to malignancies and other deadly diseases that are caused by uncontrolled cell division. To understand this, it is important to explore how individual enzymes function together as a system to properly regulate the biochemical events that direct mitosis. This work will yield insights into how the actions of protein networks guide cells to make proper decisions as they proliferate, and may provide significant clues into how cells work to avoid making errors during this process.
