Dynamics Of Folate/Folate Receptor Complexes In KB Cells Observed Through Single Molecule Fluorescence Imaging

Abstract

Folate (vitamin B9) is essential in the synthesis of nucleotide bases and amino acids. Cellular uptake (endocytosis) of folate is mediated by the membrane bound folate receptor (FR), a glycosylphosphatidylinositol-anchored protein. FR has been found to be up-regulated and/or redistributed in the plasma membrane of specific types of carcinoma cells. Specifically, the over-expression in certain epithelial carcinoma cells can be as high as a thousand fold and thus FR provides an important possible target in efficient drug therapy design. In this study, we are probing the dynamics and interactions of folate/FR receptor complexes in the plasma membrane and through each part of its endocytotic cycle at the single molecule level. We are employing single molecule fluorescence microscopy to track individual fluorophore-labeled folate molecules bound to cell surface FR. We found an average diffusion constant of D = 2e-9 cm2/s on live human KB carcinoma cells when imaged at 30 fps. Trajectories of individual particles showed temporary stopping or confined motion. This anomalous diffusive behavior was quantified against Monte Carlo simulations of randomly diffusing particles. The frequency and duration of confinement was compared as the overall concentration of folate was incrementally increased from 1 fM to the physiological level of 1nM . The effect on the frequency and duration of single FR confinement due to cholesterol depletion, actin stabilization and actin depolymerization will be presented.