Fig. 3: Single-molecule imaging of ATP-ATTO-488 bound to G-actin and actin filaments by total internal fluorescence microscopy (TIRFM).
From: A functional family of fluorescent nucleotide analogues to investigate actin dynamics and energetics
ATP adenosine triphosphate, FRAP fluorescence recovery after photobleaching. Scale bars: 10 µm. a Single-molecule imaging of individual ATP-ATTO-488 molecules (green) and Alexa-568-labeled G-actin (magenta). Prior to imaging, ATP-ATTO-488 (0.2 µM) and G-actin (2 µM; 7.5% labeled) were pre-incubated for exchange for 15 min in NFG + MEI buffer before a 105-fold dilution in imaging buffer 1 to isolate single molecules. White arrowheads highlight visible binding. This experiment was repeated independently three times. b Time-course of a simultaneous exchange of ATP-ATTO-488 (0.25 µM; green) and polymerization of actin (0.4 µM; 7.5% Alexa-568-labeled; magenta) in the presence of profilin (0.8 µM) in imaging buffer 2. Blue and orange arrowheads indicate the barbed and pointed ends, respectively, of an actin filament, which assembled exclusively from ATP-G-actin monomers at early timepoints, before integrating progressively increasing numbers of ATP-ATTO-488-bound actin subunits. This experiment was repeated independently three times. c Filament length vs. time kymograph of the filament indicated with blue and orange arrowheads in b. Dotted line shows that slope appears constant over time. d Elongation rates of actin filaments polymerized from actin monomers bound to 39% or 95% of ATP-ATTO-488, relative to their elongation rates when bound fully to ATP, indicating that actin subunits bound to ATP-ATTO-488 do not visibly impact actin polymerization. 0, 5 µM, or 125 µM of ATP-ATTO-488 were pre-incubated with 5 µM actin (10% Alexa-568-labeled) for 2 h at room temperature in NFG buffer to reach respectively 0%, 39%, or 95% occupancy. Polymerization was then induced between slides and coverslips by 5-fold dilution in imaging buffer 2 supplemented with 3 µM profilin. Red dots indicate average values. n = 20. Statistical significance is given with p-values from one-factor ANOVA tests. e Photobleaching of a segment of an actin filament labeled with ATP-ATTO-488 demonstrating absence of fluorescence recovery within 900 s, providing evidence that nucleotide exchange does not occur in filamentous form of actin. White arrowhead indicates the growing barbed end. This experiment was repeated independently three times. f Quantification of d. Source data are provided as a Source Data file.
