Dynamics of Polymeric Protein Assemblies in Live Cells Revealed by Fluorescence Polarization Imaging of Single Molecules

Abstract

Fluorescence single molecule imaging is a powerful means of investigating the distribution and dynamics of individual biological molecules and their assemblies. Molecular orientation underpins the biophysical properties of the biopolymers (e.g., DNA, cytoskeleton, extracellular matrix and misfolded protein oligomers). Therefore, methods that reveal the molecular orientation have yielded new insights in the structure and function of biomolecular assemblies. However, current methods are limited to measurement of orientation of single fluorophores and have not permitted robust imaging of orientation of many single molecules in parallel. We have developed a new fluorescence polarization microscope that instantaneously and efficiently sorts the emitted fluorescence along four polarization orientations (separated by 45 degrees) to provide instantaneous imaging of position and orientation of single fluorescent molecules and their assemblies. With aid of computational algorithms, the microscope provides diffraction limited spatial resolution, 10fps temporal resolution, and 10 degree of angular resolution in living cells.Our technique has enabled analysis of molecular position and orientation in vitro and in living cells. We found that phalloidin-Alexa Fluor 488 reports local orientation of sparsely labeled actin filaments. Taking advantage of this signal, we have measured changes in the orientation of local actin filaments as they undergo retrograde flow at the leading edge of migrating human keratinocytes. We also used our system to study organization of septins, a highly conserved cytoskeleton critical for cytokinesis and intracellular compartmentalization. We found that individual septin molecules labeled with constrained GFP attach to the cell cortex with consistent orientation. The consistent alignment of single septin-GFP suggested the presence of highly ordered scaffold.Our single molecule fluorescence orientation imaging technique is also promising to explore conformational changes of single molecules or mechanisms of protein assembly in living cells.