Dissection of molecular assembly dynamics by tracking orientation and position of single molecules in live cells

Abstract

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, ...

Actin retrograde flow actively aligns and orients ligand-engaged integrins in focal adhesions

“…S1 B and C). We validated results obtained by EA-TIRFM using Instantaneous FluoPolScope where the sample is illuminated with isotropic polarization and anisotropy is determined using detectors sensitive to four different emission polarization orientations (14) (Fig. 1B, Bottom).…”

“…1E and Table S1). The orientation of the GFP dipole with respect to the FA long axis of αV-GFP-constrained derived from EA-TIRFM was verified using Instantaneous FluoPolScope (14), which indicated a dipole orientation of −19.5°± 3.85°relative to the FA long axis (Fig. 1F).…”

Super-resolution fluorescence polarization microscopy

“…Four detection channels could enable 3D orientation detection of°uorescent dipoles instead of only in-plane measurement, 41 or could provide an unbiased measurement of dipole orientations. 16 Based on emitted°uorescence polarization of radiating dipoles, the°uorescent intensity distribution outside the objective image space could also provide orientational information. 42,43 When the dipole lies at an orientation unparalleled to the optical axis, the polarized emission would lead to rotationally asymmetric distribution in the annular image.…”

“…Some researchers hold the view that four polarizations are enough for sampling the sinusoidal curve and for an unbiased measurement of the in-plane dipole orientation. 16,28 Eight combinations of polarization could allow unambiguous 3D measurement of dipole orientations. 47 Most FPM could only measure in-plane dipole orientations while some could access out-of-plane tilting angle of dipoles as well.…”

“…Actin was¯rstly to be found to have an organized distribution of probed dipole orientations 13 and was imaged by various FPM techniques. [14][15][16][17][18] Myosin is an important motor protein moving along actin. It exhibits interesting walking pattern with steps and rotation and is an extensively studied system.…”