A major challenge in biology today is to integrate our knowledge of biomolecules to explain complex cell behavior. This challenge is part of the broader challenge in science and engineering of integrating from the nanoscale to the microscale. A prime example of such integration is the microtubule, whose nanometer-sized components, heterodimers of alpha and beta tubulin monomer proteins, self-assemble to form tubes that are 25 nm in diameter and several micrometers long. Once formed, microtubules mediate the transport of vital subcellular cargoes, including mitochondria, membrane-bound secretory and other vesicles, and chromosomes. Understanding the fundamental mechanisms of microtubule assembly and disassembly is therefore crucial to understanding how cells reorganize their cytoplasm during cellular growth, mitosis, and responses to external signals. Microtubules exhibit a highly unusual and complex self-assembly behavior known as "dynamic instability," where individual microtubules switch stochastically between alternate phases of growth and shortening. Various theories have been offered to explain the molecular origin of dynamic instability, but they all fail to explain the mechanics and structure of the microtubule tip. What is required is a computational framework that integrates chemical kinetics and thermodynamics (associated with tubulin addition or loss and GTP hydrolysis) with mechanics (associated with conformational changes in tubulin). Given the importance of mechanical force in microtubule dynamics, it then becomes essential to identify the origin and magnitude of external forces exerted on, and by, microtubules in the cytoplasm. Microtubules are often highly curved in the cytoplasm, and it has been shown that actomyosin contractility can cause microtubule bending. However, it is not clear whether this is the dominant mechanism, or whether other mechanisms might also contribute significantly to bending. Preliminary studies suggest that microtubule polymerization causes bending, even when the plus end is not in contact with the leading edge of the cell. These studies also suggest that unbending, which has not been considered before in the literature, may result from depolymerization and from actomyosin contractility. Mechanical regulation of microtubule dynamics could be a major means of regulating microtubule access to the cortical or peripheral regions of the cell's cytoplasm, which in turn can be a determinant of cell shape and polarity during directed movement or polarized (vectorially directed) cell growth. This project has three specific aims: 1, to understand the mechanochemical basis of microtubule dynamic instability; 2, to understand the mechanisms of microtubule bending and unbending in living cells; and 3, to develop educational and outreach programs in cellular systems biology. These questions will be addressed using an integrated theoretical, computational and experimental approach. The mechanochemical computational modeling will provide specific, quantitative predictions that will be tested directly against experimental results, including GFP fluorescence imaging of single microtubules in living cells and application of controlled forces on the nanonewton scale (using a high precision magnetic bead force application system). This integrated systems approach is necessary in order to deal with the inherent complexity of microtubule dynamics, including both its chemical and mechanical components.
Intellectual merit of the proposed activity: this project will advance our understanding of how chemical kinetics, thermodynamics and mechanics interact to mediate microtubule assembly and disassembly, leading to a theoretically based computational framework that can be used to understand how microtubule-associated proteins control microtubule behavior. It will also advance our understanding of how mechanical forces are imposed on and self-generated by microtubules in living cells.
Broader impacts resulting from the proposed activity: as part of this project, Dr. Odde will develop a "Collaborative Modeling in Cell Biology Initiative" through which cell biologists will collaborate with with teams of engineering seniors and graduate students in the University of Minnesota BMEn 5351 "Cell Engineering" course who will develop computer simulations of cellular processes. In addition, Dr. Odde will continue to run his successful "Future Faculty Program" for biomedical engineering students from underrepresented groups.
