The formation of organisms from a fertilized egg depends on the coordinated expression of genes, whose protein products interact with and influence each other. One of these genes encodes a regulatory protein known as CaM kinase II. This protein is 94% identical between humans and simple vertebrate organisms, such as the zebrafish. In fact, defects in the development of the heart caused by mutations in the zebrafish CaM kinase II gene, can be reversed by introducing the human CaM kinase II protein. This protein normally influences proper heart development by binding to and/or modifying other proteins. Previous studies from this laboratory have used a biochemical technique (peptide mass spectrometry) to identify such protein partners in cells in culture. Proteomic studies like these are ultimately required to define downstream targets and thus functions of regulatory proteins like CaM kinase II. This interdisciplinary project will link molecular tools with proteomics in a whole animal model to identify downstream proteins in specific tissues at any time during development. In particular, such proteins will be identified at specific developmental stages in the embryonic heart. It is anticipated that this project will shed light on the unsolved puzzle of cardiac chamber formation and looping and will begin to identify new binding partners and substrates of the CaM kinase II family. This project will influence our understanding of the development of the cardiovascular system and will develop a template approach for other investigators interested in evaluating tissue-specific changes in the nature of any protein complex during development. Graduate and undergraduate students will be trained.
Robert M. Tombes (IOS-0817658) "Tissue-specific Identification of CaM Kinase II Binding Partners and Substrate" The formation of a vertebrate animal from a fertilized egg involves changes in cells and tissues in three-dimensional space. This is referred to as morphogenesis. Morphogenesis depends on the coordinated expression of genes, whose protein products interact with and influence each other. One of these proteins regulates cell behaviors in response to calcium (Ca2+) signals. This protein, known as CaM kinase II, is 94% identical between humans and simple vertebrate organisms, such as the zebrafish. CaMK-II influences the morphogenesis of specific tissues by acting on separate proteins in each tissue. This study defined the role of CaMK-II in the development of a specific tissue, the heart, and characterized some of the CaMK-II target proteins. We used a new biochemical technique (peptide mass spectrometry) to identify the protein partners of CaMK-II in zebrafish embryos. Together with more traditional approaches, we made a large number of other exciting discoveries about the importance of Ca2+ signaling in early development. Most of these discoveries were completely unexpected. 1. CaMK-II influences cardiac development and function. Our studies revealed that CaMK-II is activated by evolutionarily conserved genes that influence heart development. In the absence of CaMK-II, heart chambers do not form, but the heart remains in an earlier developmental stage where it appears as a contractile tube. The heart rate also slows, demonstrating the continued requirement for this enzyme even after the heart has formed. 2. CaMK-II is activated in many ciliated tissues in the developing embryo (See Figure below). Cilia are cellular protrusions, which beat to cause fluid flow but also function as antennae, enabling communication between cells. Some of these communication chains depend on small changes in the concentration of calcium. 3. CaMK-II is Necessary for Left-Right Asymmetry. One of the locations where CaMK-II is Expressed (See Figure Below) is in the "KV." The KV is a ciliated organ which ensures that the heart is placed on the left side of the body and the liver on the right. Without CaMK-II, organs are placed randomly. 4. CaMK-II is Necessary for Kidney Development. Another location where CaMK-II appears (See Figure Below) is in the Kidney. In the absence of CaMK-II, kidney cysts form. This discovery was heralded by other scientists as filling in a "missing link" in this field of study. 5. CaMK-II is Required for Ear Development. CaMK-II is intensely activated at the base of sensory cilia in the ear (See Figure Below). We have discovered that this activation is necessary for proper ear development and cilia function. 6. CaMK-II protein targets included p53 and HDAC. These proteins have already been linked to calcium signals and to development, but our results suggested that CaMK-II may be their link. Future research will test the role of these and other proteins in zebrafish embryo development. Such studies can only be conducted in model organisms like the zebrafish and will impact our understanding of how all vertebrate animals develop normally.
