Characterization of Nuclear Envelope Proteins by Fluorescence Fluctuation Spectroscopy

University of Minnesota Ph.D. dissertation. November 2019. Major: Physics. Advisor: Joachim Mueller. 1 computer file (PDF); xiv 199 pages.The nuclear evelope (NE) consists of two concentric membranes surrounding the nucleus of eukaryotic cells. The two nuclear membranes are separated by a ~40 nm wide fluid layer, known as the lumen. While the NE is well known as a physical barrier separating the genetic material-containing nucleus from the cytoplasm, the NE has also been recognized as a central hub for cellular signaling. Proteins found in the lumen or at the nuclear membranes are critical to the NE’s role in cellular functions, with mutations in these proteins associated with human diseases such as muscular dystrophy and cancer. Despite their significance to human health, the interactions of NE proteins have been limited to in vitro studies. This thesis lays the foundation for investigating the interactions and assembly of NE proteins in their native environment by bringing fluorescence fluctuation spectroscopy to the NE of the living cell. After adapting the fluorescence fluctuation technique to the challenging environment of the NE, we succeeded in quantifying the self-oligomerization of proteins within the NE as well as determining their mobility. Our application focuses mainly on constituent proteins of the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex, which are important in nucleocytoplasmic force transduction, with the goal to determine their assembly states spectroscopically. The results of these studies demonstrate the power of the fluorescence fluctuation methods described in this thesis and provide a starting point for extending this work to other NE proteins with the goal to characterize their assembly within their native cellular environment