The dynamics of calmodulin signaling revealed using optical methods

The Ca2+-binding protein calmodulin (CaM) is a key transducer of Ca2+ oscillations by virtue of its ability to bind Ca 2+ selectively and then interact specifically with a large number of downstream enzymes and proteins. It remains unclear whether Ca2+ -dependent signaling alone can activate the full range of Ca 2+/CaM regulated processes or whether other regulatory schemes in the cell exist that allow specific targeting of CaM to subsets of Ca 2+/CaM binding sites or regions of the cell. Here we investigate the possibility that alterations of the availability of CaM may serve as a potential cellular mechanism for regulating the activation of CaM-dependent targets. By utilizing sensitive optical techniques with high spatial and temporal resolution, we examine the intracellular dynamics of CaM signaling at a resolution previously unattainable. After optimizing and characterizing both the optical methods and fluorescently labeled probes for intracellular measurements, the diffusion of CaM in the cytoplasm of HEK293 cells was analyzed. It was discovered that the diffusion characteristics of CaM are similar to that of a comparably sized inert molecule. Independent manipulation of experimental parameters, including increases in total concentrations of CaM and intracellular Ca2+ levels, did not change the diffusion of CaM in the cytoplasm. However, changes in diffusion were seen when the concentration of Ca2+/CaM-binding targets was increased in conjunction with elevated Ca2+. This indicates that CaM is not normally limiting for the activation of Ca 2+/CaM-dependent enzymes in HEK293 cells but reveals that the ratio of CaM to CaM-dependent targets is a potential mechanism for changing CaM availability. Next we considered whether cellular compartmentalization may act to regulate concentrations of available Ca2+/CaM in hippocampal neurons. We discovered changes in diffusion parameters of CaM under elevated Ca2+ conditions in the soma, neurite and nucleus which suggest that either the composition of cytoplasm is different in these compartments and/or they are composed of unique families of CaM-binding proteins. Finally, we return to the HEK293 cell and for the first time directly show the intracellular binding of CaM and CaMKII, an important target for CaM critical for neuronal function and plasticity. Furthermore, we analyzed the complex binding stoichiometry of this molecular interaction in the basal, activated and autophosphorylated states of CaMKII and determined the impact of this binding on CaM availability in the cell. Overall these results demonstrate that regulation of CaM availability is a viable cellular mechanism for regulating the output of CaM-dependent processes and that this process is tuned to the specific functional needs of a particular cell type and subcellular compartment. ^

Evaluation of Polymer Gel Dosimeters for Measurements of Dose and LET in Proton Beams

This project assessed the effectiveness of polymer gel dosimeters as tools for measuring the dose deposited by and LET of a proton beam. A total of three BANG® dosimeter formulations were evaluated: BANG®-3-Pro-2 BANGkits™ for dose measurement and two BANG®-3 variants, the LET-Baseline and LET-Meter dosimeters, for LET measurement. All dosimeters were read out using an OCT scanner. The basic characteristics of the BANGkits™ were assessed in a series of photon and electron irradiations. The dose-response relationship was found to be sigmoidal with a threshold for response of approximately 15 cGy. The active region of the dosimeter, the volume in which dosimeter response is not inhibited by oxygen, was found to make up roughly one fourth of the total dosimeter volume. Delivering a dose across multiple fractions was found to yield a greater response than delivering the same dose in a single irradiation. The dosimeter was found to accurately measure a dose distribution produced by overlapping photon fields, yielding gamma pass rates of 95.4% and 93.1% from two planar gamma analyses. Proton irradiations were performed for measurements of proton dose and LET. Initial irradiations performed through the side of a dosimeter led to OCT artifacts. Gamma pass rates of 85.7% and 89.9% were observed in two planar gamma analyses. In irradiations performed through the base of a dosimeter, gel response was found to increase with height in the dosimeter, even in areas of constant dose. After a correction was applied, gamma pass rates of 94.6% and 99.3% were observed in two planar gamma analyses. Absolute dose measurements were substantially higher (33%-100%) than the delivered doses for proton irradiations. Issues encountered while calibrating the LET-Meter gel restricted analysis of the LET measurement data to the SOBP of a proton beam. LET-Meter overresponse was found to increase linearly with track-average LET across the LET range that could be investigated (1.5 keV/micron – 3.5 keV/micron).