Fluorescent Single Molecule Orientation Imaging for Exploring 3D Architectural Dynamics of Cytoskeletal Protein Assembly in Living Cells

Abstract

Fluorescent single molecule imaging is a powerful tool to investigate the distribution and dynamics of individual biological molecules with nanometer precision and millisecond timing in living cells. However, it is difficult to detect small conformational changes within individual proteins or slight angular rearrangements of functional proteins inside living cells. We are proposing a light microscopy to detect changes in intra-molecular structure or inter-molecular organization based on orientation imaging of fluorescent single molecules. We have developed robust instrumentation for polarized fluorescence imaging exhibiting the speed and sensitivity required to monitor 3D angular changes of individual fluorophores that are rigidly connected to proteins of interest.While developing the optical arrangement and required acquisition and processing algorithms, we use the system to monitor the organization of septin molecules in a filamentous fungus, Ashbya gossypii, and in budding yeast. The septins are a highly conserved component of the cytoskeleton that are critical for cytokinesis and intracellular compartmentalization. Important insights have been gained about the steady state organization of septins using polarized fluorescence imaging approaches but never at the single molecule level.In this presentation we describe our single-molecule approaches of polarized fluorescence imaging in vivo which include instrumentation, image acquisition and processing algorithms, and methods for rigidly linking fluorescent markers to protein molecules. We then propose applications of these methods to answer biological questions pertaining to the mechanisms of spatially organized protein assembly in living cells using septins as model systems.