Calcium ions regulate a number of processes essential for the growth and development of an individual. Cell division, cellular architecture, and the early stages in development are all regulated by increases in the intracellular concentration of calcium signalled by extracellular messengers. Calmodulin is a ubiquitous calcium-binding protein believed to mediate cellular responses to calcium fluxes. Upon binding Ca2+, calmodulin undergoes conformational transitions that trigger recognition and regulation of target proteins. Potential targets include enzymes of cyclic nucleotide metabolism, the Ca2+- pumping ATPase, cytoskeletal proteins, protein kinases and a protein phosphatase. All of these proteins are regulated by calmodulin in a calcium-dependent manner in vitro, but it has been difficult to study the regulation in vivo. Furthermore, the interactions between calmodulin and its targets are not fully understood at the molecular level. A powerful approach for characterizing calmodulin and its interactions with cellular targets is a combined genetic and biochemical analysis. For this purpose, the yeast Saccharomyces cerevisiae offers considerable experimental advantage because it is amenable to genetic manipulations. Furthermore, calmodulin is essential for the growth of yeast cells. Vertebrate calmodulin can substitute for yeast calmodulin in vivo, therefore an analysis of yeast calmodulin will provide information about vertebrate calmodulin. First, yeast mutants that carry conditionally lethal defects in calmodulin will be isolated. Then, mutations in the genes encoding the target proteins will be isolated by their ability to suppress the conditionally lethal phenotype of the calmodulin mutants or because they confer a dependence on overproduction of calmodulin. (Yeast cells that overproduce calmodulin 80-fold are viable.) Substantial information about the function of calmodulin will be obtained by characterizing the mutants physiologically. The essential structural determinants of calmodulin will be identified by correlating the sequence changes in the mutant proteins with their altered biochemical properties. Special emphasis will be placed on a study of the interactions between mutant calmodulins and mutant and wild type target proteins. In this way, the details of the interactions between calmodulin and target proteins will be elucidated. In the last two years of the proposal, the target proteins will be more thoroughly characterized and a mutational analysis of their function begun.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
Project #
Application #
Study Section
Molecular Cytology Study Section (CTY)
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
University of Washington
Schools of Medicine
United States
Zip Code
Kim, Jae Ook; Zelter, Alex; Umbreit, Neil T et al. (2017) The Ndc80 complex bridges two Dam1 complex rings. Elife 6:
Kudalkar, Emily M; Deng, Yi; Davis, Trisha N et al. (2016) Coverslip Cleaning and Functionalization for Total Internal Reflection Fluorescence Microscopy. Cold Spring Harb Protoc 2016:pdb.prot085548
Kudalkar, Emily M; Davis, Trisha N; Asbury, Charles L (2016) Single-Molecule Total Internal Reflection Fluorescence Microscopy. Cold Spring Harb Protoc 2016:pdb.top077800
Hsia, Yang; Bale, Jacob B; Gonen, Shane et al. (2016) Design of a hyperstable 60-subunit protein dodecahedron. [corrected]. Nature 535:136-9
Kudalkar, Emily M; Davis, Trisha N; Asbury, Charles L (2016) Preparation of Reactions for Imaging with Total Internal Reflection Fluorescence Microscopy. Cold Spring Harb Protoc 2016:pdb.prot085563
Kollman, Justin M; Greenberg, Charles H; Li, Sam et al. (2015) Ring closure activates yeast ?TuRC for species-specific microtubule nucleation. Nat Struct Mol Biol 22:132-7
Kudalkar, Emily M; Scarborough, Emily A; Umbreit, Neil T et al. (2015) Regulation of outer kinetochore Ndc80 complex-based microtubule attachments by the central kinetochore Mis12/MIND complex. Proc Natl Acad Sci U S A 112:E5583-9
Zelter, Alex; Bonomi, Massimiliano; Kim, Jae ook et al. (2015) The molecular architecture of the Dam1 kinetochore complex is defined by cross-linking based structural modelling. Nat Commun 6:8673
Tien, Jerry F; Umbreit, Neil T; Zelter, Alex et al. (2014) Kinetochore biorientation in Saccharomyces cerevisiae requires a tightly folded conformation of the Ndc80 complex. Genetics 198:1483-93
Umbreit, Neil T; Miller, Matthew P; Tien, Jerry F et al. (2014) Kinetochores require oligomerization of Dam1 complex to maintain microtubule attachments against tension and promote biorientation. Nat Commun 5:4951

Showing the most recent 10 out of 57 publications