Rapid and Quantitative Imaging of Excitation Polarized Fluorescence Reveals Ordered Septin Dynamics in Live Yeast

Abstract

We report an imaging method for fast, sensitive analysis of the orientation of fluorescent molecules by employing a liquid-crystal based universal polarizer in the optical path of a wide-field light microscope. We developed specific acquisition and processing algorithms for measuring the anisotropy and for correcting artifacts caused by fluorescence bleaching, background light, and differential transmission of optical components. We call this approach the Fluorescence LC-PolScope and we used it to analyze the architectural dynamics of septin-green fluorescent protein (septin-GFP) constructs in the neck region of budding yeast. We describe three different states of highly anisotropic septin arrays in which the prevailing orientation of GFP dipoles was either parallel or perpendicular to the mother-bud axis. The transitions between these ordered states were characterized by transient isotropic states. To analyze the patterns of polarized fluorescence, we modeled the alignment of septin-GFP constructs in different stages of septin ring formation. Based on our model, our experimental data are consistent with the formation of paired rather than single filaments and the axis of the α-helical septin terminus linked to a GFP molecule is likely oriented normal to the cell surface. The Fluorescence LC-PolScope combines the molecular specificity of fluorescence tagging with the structural specificity of polarized light analysis.

Septin assemblies form by diffusion-driven annealing on membranes

“…We speculate that flexibility of filaments and their capacity to anneal may accelerate assembly, rearrangement, and disassembly of higher-order structures. Previous work using polarization microscopy has demonstrated that the septin hour-glass structure in yeast is anisotropic, suggesting it is highly ordered both before and after cytokinesis (13,36,37). We imagine that the order emerges from a septin binding or bundling partner that constrains and contains the flexible filaments.…”

“…If present, these lateral associations either form in a concentration-dependent manner or require a cytosolic factor because we did not obtain any evidence for lateral associations on membranes in vitro. Despite appearing highly anisotropic through most of the cell cycle, the septin hourglass displays a notably more "fluid" or isotropic transition at cytokinesis (1,13,36,37). The flexibility, annealing, and fragmentation properties are likely important for transit through the isotropic phase.…”

Mitotic Exit and Separation of Mother and Daughter Cells

“…As diagrammed in Figure 1, this septin filament system eventually forms a focused band that extends around the bud neck, close to the plasma membrane (Longtine and Bi 2003;Kinoshita 2006;Oh and Bi 2011). The septin lattice is initially highly dynamic, but reorganizes into a more stable structure as bud growth proceeds (Dobbelaere et al 2003;Dobbelaere and Barral 2004;Vrabioiu and Mitchison 2006;Demay et al 2011). The septin ring at the bud neck functions as a barrier that prevents diffusion of membrane proteins and other cell cortex material (Barral et al 2000;Dobbelaere and Barral 2004;Vrabioiu and Mitchison 2006;Caudron and Barral 2009).…”

Thioflavine-T and Congo Red Reveal the Polymorphism of Insulin Amyloid Fibrils When Probed by Polarization-Resolved Fluorescence Microscopy

“…However, orientational information was only provided in sample areas where the average orientation could be a priori known unambiguously [22][23][24][25] . A more complete and flexible measurement scheme has been proposed recently, which circumvents these limitations.…”