Regulation of order, such as orientation and conformation, drives the function of most molecular assemblies in living cells but remains difficult to measure accurately through space and time. We built an instantaneous fluorescence polarization microscope, which simultaneously images position and orientation of fluorophores in living cells with single-molecule sensitivity and a time resolution of 100 ms. We developed image acquisition and analysis methods to track single particles that interact with higher-order assemblies of molecules. We tracked the fluctuations in position and orientation of molecules from the level of an ensemble of fluorophores down to single fluorophores. We tested our system in vitro using fluorescently labeled DNA and F-actin, in which the ensemble orientation of polarized fluorescence is known. We then tracked the orientation of sparsely labeled F-actin network at the leading edge of migrating human keratinocytes, revealing the anisotropic distribution of actin filaments relative to the local retrograde flow of the F-actin network. Additionally, we analyzed the position and orientation of septin-GFP molecules incorporated in septin bundles in growing hyphae of a filamentous fungus. Our data indicate that septin-GFP molecules undergo positional fluctuations within ∼350 nm of the binding site and angular fluctuations within ∼30°of the central orientation of the bundle. By reporting position and orientation of molecules while they form dynamic higher-order structures, our approach can provide insights into how micrometer-scale ordered assemblies emerge from nanoscale molecules in living cells.single-molecule orientation | live cell imaging | polarized fluorescence | actin | septin T he generation and dissolution of order within populations of biological molecules are central to almost all cellular processes. The emergence of ordered arrays of biological molecules is manifested in lipid membranes, DNA, the cytoskeleton, and many other molecular assemblies in the cytoplasm of the living cell. Polarization-resolved fluorescence imaging of densely labeled assemblies has successfully probed their net order and architectural dynamics (1-5). In addition, tracking of sparsely labeled cytoskeletal networks (6-9) has illuminated how turnover of individual molecules enables transitions in the spatial organization of cytoskeletal networks. We have combined tracking of both orientation and position of single fluorophores associated with cystoskeletal proteins in live cells to analyze the molecular order within cytoskeletal assemblies.Fluorophores, including the GFP, emit fluorescence by radiating light as dipoles. The light emitted from a single dipole is fully polarized, with most of the energy polarized along the dipole axis. For example, the absorption and emission dipole axes of GFP chromophores are fixed within the GFP molecule as was found in GFP crystals (10, 11). Therefore, fluorescent labels can report the orientation of biomolecules as long as the labels are rigidly bound to the biomolecules, ...
We have developed an imaging system for 3D time-lapse polarization microscopy of living biological samples. Polarization imaging reveals the position, alignment and orientation of submicroscopic features in label-free as well as fluorescently labeled specimens. Optical anisotropies are calculated from a series of images where the sample is illuminated by light of different polarization states. Due to the number of images necessary to collect both multiple polarization states and multiple focal planes, 3D polarization imaging is most often prohibitively slow. Our MF-PolScope system employs multifocus optics to form an instantaneous 3D image of up to 25 simultaneous focal-planes. We describe this optical system and show examples of 3D multi-focus polarization imaging of biological samples, including a protein assembly study in budding yeast cells.
Regulation of order, such as orientation and conformation, drives the function of most molecular assemblies in living cells but remains difficult to measure accurately through space and time. We built an instantaneous fluorescence polarization microscope, which simultaneously images position and orientation of fluorophores in living cells with single-molecule sensitivity and a time resolution of 100 ms. We developed image acquisition and analysis methods to track single particles that interact with higher-order assemblies of molecules. We tracked the fluctuations in position and orientation of molecules from the level of an ensemble of fluorophores down to single fluorophores. We tested our system in vitro using fluorescently labeled DNA and F-actin, in which the ensemble orientation of polarized fluorescence is known. We then tracked the orientation of sparsely labeled F-actin network at the leading edge of migrating human keratinocytes, revealing the anisotropic distribution of actin filaments relative to the local retrograde flow of the F-actin network. Additionally, we analyzed the position and orientation of septin-GFP molecules incorporated in septin bundles in growing hyphae of a filamentous fungus. Our data indicate that septin-GFP molecules undergo positional fluctuations within ∼350 nm of the binding site and angular fluctuations within ∼30°of the central orientation of the bundle. By reporting position and orientation of molecules while they form dynamic higher-order structures, our approach can provide insights into how micrometer-scale ordered assemblies emerge from nanoscale molecules in living cells.single-molecule orientation | live cell imaging | polarized fluorescence | actin | septin T he generation and dissolution of order within populations of biological molecules are central to almost all cellular processes. The emergence of ordered arrays of biological molecules is manifested in lipid membranes, DNA, the cytoskeleton, and many other molecular assemblies in the cytoplasm of the living cell. Polarization-resolved fluorescence imaging of densely labeled assemblies has successfully probed their net order and architectural dynamics (1-5). In addition, tracking of sparsely labeled cytoskeletal networks (6-9) has illuminated how turnover of individual molecules enables transitions in the spatial organization of cytoskeletal networks. We have combined tracking of both orientation and position of single fluorophores associated with cystoskeletal proteins in live cells to analyze the molecular order within cytoskeletal assemblies.Fluorophores, including the GFP, emit fluorescence by radiating light as dipoles. The light emitted from a single dipole is fully polarized, with most of the energy polarized along the dipole axis. For example, the absorption and emission dipole axes of GFP chromophores are fixed within the GFP molecule as was found in GFP crystals (10, 11). Therefore, fluorescent labels can report the orientation of biomolecules as long as the labels are rigidly bound to the biomolecules, ...
Septins are conserved filament-forming proteins that act in diverse cellular processes. They closely associate with membranes and, in some systems, components of the cytoskeleton. It is not well understood how filaments assemble into higher-order structures in vivo or how they are remodeled throughout the cell cycle. In the budding yeast S. cerevisiae, septins are found through most of the cell cycle in an hourglass organization at the mother-bud neck until cytokinesis when the collar splits into two rings that disassemble prior to the next cell cycle. Experiments using polarized fluorescence microscopy have suggested that septins are arranged in ordered, paired filaments in the hourglass and undergo a coordinated 90° reorientation during splitting at cytokinesis. This apparent reorganization could be due to two orthogonal populations of filaments disassembling and reassembling or being preferentially retained at cytokinesis. In support of this idea, we report a decrease in septin concentration at the mother-bud neck during cytokinesis consistent with other reports and the timing of the decrease depends on known septin regulators including the Gin4 kinase. We took a candidate-based approach to examine what factors control reorientation during splitting and used polarized fluorescence microscopy to screen mutant yeast strains deficient in septin interacting proteins. Using this method, we have linked known septin regulators to different aspects of the assembly, stability, and reorganization of septin assemblies. The data support that ring splitting requires Gin4 activity and an anillin-like protein Bud4, and normal accumulation of septins at the ring requires phosphorylation of Shs1. We found distinct regulatory requirements for septin organization in the hourglass compared to split rings. We propose that septin subpopulations can vary in their localization and assembly/disassembly behavior in a cell-cycle dependent manner at cytokinesis.
Polarized growth is critical for the development and maintenance of diverse organisms and tissues, but particularly central to fungi where nutrient uptake, communication, and reproduction all rely on cell asymmetries. To achieve polarized growth, fungi spatially organize both their cytosol and cortical membranes. The septins, a family of GTP-binding proteins, have emerged as key regulators of spatial compartmentalization in fungi and other eukaryotes. By forming higher-order structures on fungal plasma membranes, septins are thought to contribute to the generation of cell asymmetries by acting as molecular scaffolds and forming diffusional barriers. Here we discuss the links between septins and polarized growth and consider molecular models for how septins contribute to cellular asymmetry in fungi.
The measurement of not only the location but also the organization of molecules in live cells is crucial to understanding diverse biological processes. Polarized light microscopy provides a nondestructive means to evaluate order within subcellular domains. When combined with fluorescence microscopy and GFP‐tagged proteins, the approach can reveal organization within specific populations of molecules. This unit describes a protocol for measuring the architectural dynamics of cytoskeletal components using polarized fluorescence microscopy and OpenPolScope open‐access software (http://www.openpolscope.org). The protocol describes installation of linear polarizers or a liquid crystal (LC) universal compensator, calibration of the system, polarized fluorescence imaging, and analysis. The use of OpenPolScope software and hardware allows for reliable, user‐friendly image acquisition to measure and analyze polarized fluorescence. © 2015 by John Wiley & Sons, Inc.
This is an Accepted Manuscript for the Microscopy and Microanalysis 2020 Proceedings. This version may be subject to change during the production process.
PIFE, an acronym that stands for ''Protein Induced Fluorescence Enhancement'', is a term that has been coined to describe the enhancement of fluorescence intensity that the dye Cy3 experiences in the proximity of a protein. The approach has been used to study dynamic aspects of a large number of DNAand RNA-protein interactions at the single molecule level. We and others have hypothesized that the phenomenon results from a restriction in the photoisomerization deactivation pathway of the dye. Here, we present the results of a detailed spectroscopic study that aims to characterize PIFE at the molecular level. We used time-resolved fluorescence, fluorescence anisotropy and transient spectroscopy to fully characterize the deactivation pathways of the dye on DNA when next to a protein. Our results allowed us to confirm our hypothesis that the enhancement in fluorescence correlates with a decreased yield of photoisomer.
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