INTRINSIC FLAVIN AS A BIOMARKER FOR MONITORING PATHOLOGICALLY-INDUCED CHANGES IN CELLULAR ENERGY METABOLISM
Open Access
- Author:
- Lutes, Andrew Tyler
- Graduate Program:
- Bioengineering
- Degree:
- Master of Science
- Document Type:
- Master Thesis
- Date of Defense:
- None
- Committee Members:
- Ahmed A Heikal, Thesis Advisor/Co-Advisor
- Keywords:
- FLIM
two-photon
FAD
flavin adenine dinucleotide
flavin
anisotropy
breast cancer - Abstract:
- Flavin adenine dinucleotide (FAD) exists as a metabolic cofactor for numerous metabolic pathways in living cells. Conventional biochemical techniques on this and other cofactors require the destruction of cells and therefore the morphological context is lost. Here, we used integrated, non-invasive microscopy and spectroscopy techniques to quantify the concentration and molecular conformation (i.e., free versus enzyme-bound) of natural flavins as a function of cell pathology. In these studies, we used metastatic breast cancer (Hs578T) and normal (Hs578Bst) cells as a model system. These cells exhibited distinct morphological differences, as revealed by confocal microscopy, such as perinuclear crowding of mitochondria in transformed cells due to variation in calcium signaling and apoptotic pathways. Prior to converting intensity images of natural flavins, it was essential to quantify the variation of their fluorescence quantum yield in cellular environment using two-photon fluorescence lifetime imaging microscopy (FLIM) in a calibrated microscope. These measurements, and simple image processing, indicated that the natural flavin concentrations in the normal breast cells (144 ± 38 μM) were significantly larger than in their transformed counterpart (21 ± 10 μM). In addition, steady-state and time-resolved fluorescence polarization anisotropy measurements, the first to our knowledge at this spectral window, indicated a restrictive environment and mostly protein-bound flavins. As a control, these cellular flavin autofluorescence were compared with free FAD in solution along with a representative flavoprotein (namely, LipDH) that exists in abundance in cells. To examine the sensitivity of our experimental approaches and universality of our findings, preliminary studies were carried out on the non-metastatic breast cancer cells (MCF7) and normal counterpart (MCF10A) as complementary models. These preliminary studies will open the door for using molecular dynamics imaging of natural cofactors as biomarkers for energy metabolism and health problems that are related to mitochondrial anomalies such as Alzheimer’s disease, cancer, diabetes, and other neurodegenerative diseases. The experimental and analytical approaches developed here provide an alternative to biochemical techniques on cell lysates and will be applicable to a wide range of molecular and cellular biology studies.