Studies of molecular events inside of cells have provided novel and important insights into many questions in physiology and pathology: How do cellular motors work? How does a proton pump generate ATP? How is cargo secreted from cells? How do viruses enter a cell and, once in, how do they assemble. This proposal has three aims that will significantly advance ability to follow single molecular events in living cells. The first specific aim develops the technology for loading probes into cells. The technique is efficient and directly delivers probe into the cytosol rather than into the endocytic pathway. Of particular interest for us is the ability to deliver inteins. The second specific aim will generate inteins that will function inside of cells at physiological temperatures at faster rates and higher efficiencies. The third specific aim uses two biological questions to test the strengths and limitations of the results from the first three specific aims. The two questions relate to transport in and out of the nucleus: Can we follow the conformational changes of proteins in single nuclear pores and can we follow the transport of single molecules as they interact with the nuclear pore components and move through the pore. These questions are significant for understanding a fundamental process in cell biology. They will also provide insight as to further modifications that are needed in our technology for studying single molecular events in living cells.
Optical imaging has contributed very significant advances to our understanding of biology in the last few years. This is the consequence of a number of advantages of imaging: Imaging allows us to study biological systems that are still alive;Imaging allows us to study individual molecules, individual cells rather than the average behavior; Imaging allows us to study many different size scales from single molecules to whole individuals. Often a defect that occurs at the level of a single molecule represents itself as pathology at the level of the whole organism. Imaging allows us to follow the disease from the single molecule, to the molecular machine, to the whole cell, to the whole organ, to the individual patient. A major limitation of optical imaging has been the capability to get probes into cells to specifically and selectively label individual molecules. This proposal will contribute to our ability to study individual molecular events in the cell by giving us new methods of putting our probes into cells, without disturbing the cells and new methods of labeling cellular components with minimal, often no, effect on the cell.
|Atkinson, Claire E; Mattheyses, Alexa L; Kampmann, Martin et al. (2013) Conserved spatial organization of FG domains in the nuclear pore complex. Biophys J 104:37-50|
|Jaiswal, Jyoti K; Simon, Sanford M (2013) Belling the cat--tagging live cells with quantum dots. Clin Chem 59:995-6|
|Macro, Laura; Jaiswal, Jyoti K; Simon, Sanford M (2012) Dynamics of clathrin-mediated endocytosis and its requirement for organelle biogenesis in Dictyostelium. J Cell Sci 125:5721-32|
|Kampmann, Martin; Atkinson, Claire E; Mattheyses, Alexa L et al. (2011) Mapping the orientation of nuclear pore proteins in living cells with polarized fluorescence microscopy. Nat Struct Mol Biol 18:643-9|
|Mincer, Joshua S; Simon, Sanford M (2011) Simulations of nuclear pore transport yield mechanistic insights and quantitative predictions. Proc Natl Acad Sci U S A 108:E351-8|
|Mattheyses, Alexa L; Atkinson, Claire E; Simon, Sanford M (2011) Imaging single endocytic events reveals diversity in clathrin, dynamin and vesicle dynamics. Traffic 12:1394-406|
|Jouvenet, Nolwenn; Zhadina, Maria; Bieniasz, Paul D et al. (2011) Dynamics of ESCRT protein recruitment during retroviral assembly. Nat Cell Biol 13:394-401|
|Jouvenet, Nolwenn; Simon, Sanford M; Bieniasz, Paul D (2011) Visualizing HIV-1 assembly. J Mol Biol 410:501-11|
|Mattheyses, Alexa L; Simon, Sanford M; Rappoport, Joshua Z (2010) Imaging with total internal reflection fluorescence microscopy for the cell biologist. J Cell Sci 123:3621-8|
|Mattheyses, Alexa L; Kampmann, Martin; Atkinson, Claire E et al. (2010) Fluorescence anisotropy reveals order and disorder of protein domains in the nuclear pore complex. Biophys J 99:1706-17|
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