Dissection of molecular assembly dynamics by tracking orientation and position of single molecules in live 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.
Eugene Bell Center for Regenerative Biology and Tissue Engineering, Marine Biological Laboratory, Woods Hole, MA 02543.
Shalin B
0000-0002-2542-3582
SB
Mehta
Department of Biological Sciences, Dartmouth College, Hanover, NH 03755; Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599.
Molly
M
McQuilken
Department of Radiology, University of Chicago, Chicago, IL 60637.
Patrick J
PJ
La Riviere
Department of Biological Sciences, Dartmouth College, Hanover, NH 03755; Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599.
Patricia
P
Occhipinti
Eugene Bell Center for Regenerative Biology and Tissue Engineering, Marine Biological Laboratory, Woods Hole, MA 02543.
Amitabh
A
Verma
Eugene Bell Center for Regenerative Biology and Tissue Engineering, Marine Biological Laboratory, Woods Hole, MA 02543; Physics Department, Brown University, Providence, RI 02912.
Rudolf
R
Oldenbourg
Eugene Bell Center for Regenerative Biology and Tissue Engineering, Marine Biological Laboratory, Woods Hole, MA 02543; Department of Biological Sciences, Dartmouth College, Hanover, NH 03755; Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599.
Amy S
AS
Gladfelter
Eugene Bell Center for Regenerative Biology and Tissue Engineering, Marine Biological Laboratory, Woods Hole, MA 02543; ttani@mbl.edu.
Tomomi
T
Tani
D000199:Actins / Q000378:metabolism; D015415:Biomarkers; D003627:Data Interpretation, Statistical; D005454:Fluorescence Polarization; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008856:Microscopy, Fluorescence; D056004:Molecular Dynamics Simulation; D012680:Sensitivity and Specificity; D058112:Septins / Q000378:metabolism; D000072760:Single Molecule Imaging / Q000379:methods
D000199:Actins; D015415:Biomarkers; D049452:Green Fluorescent Proteins; D058112:Septins
actin; live cell imaging; polarized fluorescence; septin; single-molecule orientation
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