Life is dynamic, local and directional. For example, neuronal morphogenesis requires precise spatial and temporal control of sub-processes such as growth cone guidance, branching and synapse formation and consolidation to build a functional nervous system and maintain complex cognitive functions. Such complex, sequential biological processes require precise control of protein activities in time and space. Yet, current genetic approaches are inadequate to dissect protein functions with high spatial and temporal accuracy, which fundamentally limits our understanding of complex biology. At the apex of the genomic era nearly the entire proteome has been catalogued and high resolution structures and reliable predictions are available for almost half. However, for a large percentage of proteins, we only have a vague or no idea of what they are doing in order to build and maintain functional, living, dynamic cells and organisms. This widening gap between the ever-growing wealth of genomic data and our inadequate functional understanding of the proteome represents a central challenge of the post-genomic era. In order to deeply and mechanistically probe how specific protein activities enable complex biology in the various tissue types and developmental stages of an organism, we must have technologies to acutely, locally, and reversibly modulate protein activities in cells and organisms, we must be able to watch biological responses in real time, and we must be able to do so repeatedly to generate statistically robust data and insights into underlying dynamic molecular mechanisms. Such a technology would bridge the gap between our vast genomic datasets and our need for a thorough functional understanding of the proteome, and has the potential to spark a revolution in how cellular processes are studied, and ultimately how diseases are treated. The central goal of this exploratory project is to develop and demonstrate acute and reversible photo-inactivation of endogenous proteins under endogenous transcriptional control as such innovative and transformative technology to probe or screen protein activities within cells or whole organisms with unsurpassed spatial and temporal accuracy. Our team is currently at the cusp of developing a compact and simple protein photo-inactivation module encoded in a single genetic element. We refer to this as a ?-element, and we will test the central idea that this module can be employed to replace endogenous proteins with light-controlled variants by: Developing technology for inserting light-sensitive ?-elements into endogenous genes in mammalian tissue culture cells; and defining molecular rules for reversibly disrupting protein activities by ?-element-induced photo-dissociation. As a proof of concept, we further plan to test this approach by screening ?-variants of exemplary cytoskeleton proteins with pleiotropic functions in an in vitro neuronal morphogenesis model system.

Public Health Relevance

A principle of biology is that although genes are the basis of inheritance, it is the activities of the encoded proteins that fulfil spatially and temporally controlled functions to dynamically build and maintain complex cells and organisms. To enable the required spatial and temporal granularity in the analysis of protein functions in real time in living cells or organisms, we propose to develop acute and reversible photo-inactivation of endogenous proteins under endogenous transcriptional control as an entirely new approach to probe or screen in vivo protein activities. This is not currently possible with even the most sophisticated genetic tools that are essentially static compared to the time scale of most cellular and organismal processes. Our long-term vision is that this idea will transform discovery of protein activities in development, neurobiology and other complex, sequential biological processes, and ultimately how we understand and treat disease.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Exploratory/Developmental Grants (R21)
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Special Emphasis Panel (ZRG1-IMST-D (02)M)
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Knowlton, John R
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University of California San Francisco
Anatomy/Cell Biology
Schools of Dentistry
San Francisco
United States
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