The optimization of fluorescent proteins for subcellular environments
Costantini, Lindsey M.
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The use of fluorescent proteins (FP) has revolutionized fluorescence imaging and cellular biology. Qualitative and more importantly quantitative measurements can be made in live cells. The initial questions revealing when and where a protein of interest may be have evolved to real-time measurements of subcellular ion concentrations, localized protein activities, and macromolecule dynamics. However, in order to rely on FP technologies, FPs need to accurately report while not interfering with normal cellular functions or architecture. Therefore, both the intrinsic characteristics of FPs, including spectral properties, brightness, photostability, oligomeric state, and the impact of the local cellular environment need to be considered. For example, the endoplasmic reticulum (ER), unlike the cytoplasm, is an oxidizing environment and the site of two important post-translational modifications for secretory proteins: disulfide bond formation and N-glycosylation. We have found that when localized to the ER lumen, commonly utilized FPs are post-translationally modified. These modifications can grossly affect the sensitivity and reliability of these tools within the secretory pathway.;To optimize tools for studying events in this important cellular environment, we have modified the latest generation of FPs by mutating any cysteines and consensus N-glycosylation sites they may contain. In addition, current methods are insufficient at detecting the biologically relevant oligomeric state of FPs in a standardized manner. Several FPs are prone to form low affinity oligomers when expressed as integral membrane fusion proteins. Membrane and integral membrane FP fusions, by virtue of their constrained planar geometry, will achieve comparatively higher effective concentrations. We have determined that FPs, previously reported as monomeric, can robustly oligomerize when attached to ER membrane proteins and examined in live cells. Based on this observation, we have developed a set of practical assays to assess the tendencies of FPs to oligomerize in cells. Our research provides fundamental tools for the studies utilizing FPs in oxidizing environments, including the eukaryotic secretory pathway and the periplasm of gram-negative bacteria, and as membrane localized FP-fusions. Together our extensive studies and uses of FP have expanded the applications and further improved their utilities.
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