STED nanoscopy combined with optical tweezers reveals protein dynamics on densely covered DNA.
Dense coverage of DNA by proteins is a ubiquitous feature of cellular processes such as DNA organization, replication and repair. We present a single-molecule approach capable of visualizing individual DNA-binding proteins on densely covered DNA and in the presence of high protein concentrations. Our approach combines optical tweezers with multicolor confocal and stimulated emission depletion (STED) fluorescence microscopy. Proteins on DNA are visualized at a resolution of 50 nm, a sixfold resolution improvement over that of confocal microscopy. High temporal resolution (<50 ms) is ensured by fast one-dimensional beam scanning. Individual trajectories of proteins translocating on DNA can thus be distinguished and tracked with high precision. We demonstrate our multimodal approach by visualizing the assembly of dense nucleoprotein filaments with unprecedented spatial resolution in real time. Experimental access to the force-dependent kinetics and motility of DNA-associating proteins at biologically relevant protein densities is essential for linking idealized in vitro experiments with the in vivo situation.
Department of Physics and Astronomy, Vrije Universiteit (VU University) Amsterdam, Amsterdam, The Netherlands.
Iddo
I
Heller
Gerrit
G
Sitters
Onno D
OD
Broekmans
Géraldine
G
Farge
Carolin
C
Menges
Wolfgang
W
Wende
Stefan W
SW
Hell
Erwin J G
EJ
Peterman
Gijs J L
GJ
Wuite
D004247:DNA / Q000032:analysis / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000032:analysis* / Q000378:metabolism; D004867:Equipment Design; D007091:Image Processing, Computer-Assisted; D018613:Microscopy, Confocal / Q000379:methods; D008856:Microscopy, Fluorescence / Q000295:instrumentation* / Q000379:methods*; D057054:Molecular Imaging / Q000379:methods*; D036103:Nanotechnology / Q000379:methods; D009698:Nucleoproteins / Q000032:analysis / Q000378:metabolism; D052898:Optical Tweezers
D004268:DNA-Binding Proteins; D009698:Nucleoproteins; D004247:DNA