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Browsing Theses and Dissertations by Subject "actin cytoskeleton"

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    Super-resolution imaging and modeling of murine sperm during capacitation process
    (Colorado State University. Libraries, 2019) Xu, Xinran, author; Krapf, Diego, advisor; Munsky, Brian, committee member; Pezeshki, Ali, committee member; Tamkun, Michael, committee member
    The effort to achieve better spatial resolution beyond the diffraction limit has been dedicated for many years. In the past decade, super-resolution microscopy methods have successfully advanced into extremely powerful tools to reveal hidden three-dimensional structures and properties in various biological complex systems. Here we use single-molecule localization based three-dimensional super-resolution microscopy to study the mouse sperm capacitation process, a critical step in gaining the fertilization ability. On top of that, we construct a stochastic model to represent this signaling pathway in order to be able to predict the cellular event within the capacitation. The major subjects we are interested in can be categorized into two parts: actin-based cytoskeleton and capacitation-associated signaling proteins. In the midpiece, we discovered that F-actin forms a highly specialized double helical structure, which has been the very first observation among species and has disappeared in the principal piece. Similarly, the distinctive compartments regarding actin-binding proteins have also been visualized in the mouse sperm tail. Additionally, the structure as well as localization of capacitation central mediator, protein kinase A have been investigated to address the significance of spatial positioning during the capacitation event. As the capacitation end point reporter, tyrosine phosphorylation localization has been studied to help identify its real upstream kinase among other candidates. As in many regulating processes, second messenger Ca2+ plays a vital role in the capacitation process, which needs to be conveyed by the sperm specific calcium channel CatSper. We show the structural relation of a small GTPase Cdc42 to CatSper, implying its key role in transporting Ca2+. Considering that major critical signaling molecules are well characterized in the complex capacitation network, we choose a different method–stochastic modeling, other than experimental studies, surpassing the need for probing the behavior of a large number of individual cells over time, to describe capacitation process and furthermore to predict the behavior of sperm. With the known pathways of those signaling molecules in hand, we are able to build a stochastic model by utilizing chemical master equations. A couple sets of experimental measurements are used to assist in quantifying the model.