(Georgia Institute of Technology, 2008-05-05)
Fay, Nicole C.
Transcytosis is the process by which macromolecules are transported across a polarized cell. It is one of the main methods of nutrient absorption from the blood stream as well as the only means past the blood brain barrier. Insight into the transcytic route has enormous medical potential where drug delivery methods stand to be significantly optimized. Despite the importance of this fundamental biological process, the mechanism of transcytosis is not well understood. Previously, fluorescence microscopy has allowed the tracking of fluorescently labeled low-density lipoprotein (LDL) intracellularly. However, this labeled LDL is not able to distinguish LDL particles undergoing transcytosis versus degradation. LDL is an ideal cargo for observing intracellular processes due to its vital role in transporting cholesterol for cell membrane fluidity. In order to differentiate transcytosis from degradation, I have produced two probes sensitive to degradation. The two probes are based on the principles of 1.) Förster resonance energy transfer (FRET) and 2.) fluorescence quenching. LDL degradation of the FRET-based or the fluorescence-quenching-based probe results in a significant increase in fluorescent activity. I have used several methods to assess the functionality of these probes including fluorescence measurements of detergent and enzyme degradations, SDS-PAGE analysis of degradations, and in vivo flow cytometry. Additionally, I have optimized the growth conditions for maintaining polarity in a cell line (MDCK) known to undergo transcytosis. The time and location of LDL degradation in a cell can be resolved through the use of these probes. Future work includes single-particle tracking of LDL as it is being degraded versus actively transcytosing across a cell layer. This process is critical to understanding transcytosis as well as LDL regulation in the human body.