Ultrastructural Studies by Correlative Stochastic Optical Reconstruction Microscopy and Electron Microscopy

Fluorescence light microscopy (LM) and electron microscopy (EM) are two of the most widely used imaging modalities for probing cellular structures. In this dissertation I present our works in both developing methods of several correlative super-resolution fluorescence light microscopy (LM) and electron microscopy (EM) assays by combining stochastic optical reconstruction microscopy (STORM), a super-resolution imaging technique with several different EM imaging modalities and applying super-resolution microscopy to investigate the distributions and interactions of purine biosynthetic enzymes organization complex called purinosomes within the cell. The first work contained in this dissertation is to develop Correlative fluorescence light microscopy and electron microscopy allows the imaging of spatial distributions of specific biomolecules in the context of cellular ultrastructure. Recent development of super-resolution fluorescence microscopy allows the location of molecules to be determined with nanometer-scale spatial resolution. However, correlative super-resolution fluorescence microscopy and electron microscopy (EM) still remains challenging because the optimal specimen preparation and imaging conditions for super-resolution fluorescence microscopy and EM are often not compatible. Here, we have developed several experiment protocols for correlative stochastic optical reconstruction microscopy (STORM) and EM methods, both for un-embedded samples by applying EM-specific sample preparations after STORM imaging and for embedded and sectioned samples by optimizing the fluorescence under EM fixation, staining and embedding conditions. We demonstrated these methods using a variety of cellular targets. In the second part of this dissertation, I focus on the study of dynamic purine biosynthetic enzymes organization complex called purinosomes. Purine biosynthetic enzymes are assembled into dynamic multi-enzyme complex called purinosomes. However, spatial or temporal control of these structures remains unknown. Here, we explored the endogenous purinosomes in medically important HGPRT-deficient LND fibroblasts in order to understand the de novo purine biosynthesis. Using super-resolution microscopy we investigated the interaction of purinosomes and mitochondria or microtubules using photoactivatable fluorescent protein, mMaple3 and LND fibroblast as an ideal model system for the endogenous purinosomes formation in order to avoid possible protein aggregation problems. The STORM images with this ideal model system revealed a highly correlated spatial distribution of endogenous purinosomes with mitochondria or microtubules, suggesting direct physical associations between two structures. In addition to identifying endogenous purinosome association with other cellular components, we also demonstrated that mTOR directly influenced the purinosome association with mitochondria. Inhibition of mTOR decouples spatial correlation of purinosomes with mitochondria. These data provide strong evidences for physical and functional association of endogenous purinosomes with mitochondria and microtubules.Chemistry and Chemical Biolog