Mobility of prostate-specific membrane antigen (PSMA) on LNCaP cells
Abstract
Proteins have been observed to diffuse with different modes of motion on cell membrane. Four modes of motion seen at 30 frames per second (fps) of camera speed were reported: stationary, simple Brownian, directed, and confined. PSMA is a type-II integral glycoprotein on LNCaP cells with an N-terminus in extracellular region, a C-terminus in cytoplasmic region, and a transmembrane domain. Prostate-Specific Membrane Antigen (PSMA) has been revealed to constitutively internalize from cell surface via clathrin mediated endocytosis (CME). In this study, we observed mobility of PSMA, in vivo, under varying concentrations of its ligand—Dupa. In the first part of this study, we apply single molecule Tracking (SMT) and Total Internal Reflection Fluorescent Microscopy (TIRMF) to track movement of single PSMA molecule. We report that the diffusion coefficient is sensitive to the concentration of Dupa, suggesting a cooperative mechanism among bound PSMA on cell membrane. Significantly, we find that two motion modes exist--random motion (Brownian motion) and "tunnel-like" motion. We single out "tunnel-like" motion by using K-means clustering. We find that in the 50nM concentration group, near one third of the trajectories present "tunnel-like" motion pattern. We then show that "tunnel-like" may be involved in the process of endocytosis. Results from clathrin siRNA experiment and intracellular potassium depletion experiment suggest two possible mechanisms: 1) "tunnel-like" motion could be involvedin the assembly of clathrin before CME; 2) "tunnel-like" motion may be motion of vesicle inside LNCaP cell after PSMA has been internalized.
Degree
Ph.D.
Advisors
Ritchie, Purdue University.
Subject Area
Biochemistry|Biomechanics
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