Fig. 3.

(A) Representative multiple tether force records from NGI3 cells transfected with EGFP (green) or EGFP-Myo1a-TH1 (red). Arrows indicate the beginning of the tethered phase in each case. Rapid drops in force during the tethered phase correspond to tether coalescence events. (B) Multiple representative examples of the tethered phases from records of NGI3 cells expressing EGFP (green) and EGFP-Myo1a-TH1 (red). Data were plotted with a fixed offset of 25 pN between all records. Visual inspection reveals that EGFP-Myo1a-TH1 records contain fewer observable coalescence events relative to EGFP control records of the same duration. (C) Box plots show the mean number of coalescence events or “steps” observed in EGFP or EGFP-Myo1a-TH1 records; cells expressing the TH1 dominant negative demonstrate significantly fewer coalescence events, relative to control cells (*P < 0.05). (D) Box plots of the force-time integrals calculated from the tethered phase of individual records; EGFP-Myo1a-TH1 expressing cells demonstrate significantly lower force-time integrals relative to EGFP-expressing controls (**P < 0.001). (E) Ensemble averages of tethered phase data from NGI3 cells transfected with EGFP (green) and EGFP-Myo1a-TH1 (red); open circles show average values at 0.3-s intervals; solid lines are single exponential fits to averaged data. Similar force decay kinetics were observed for EGFP (0.12 ± 0.01 s⁻¹, n = 28, R² = 0.99) and EGFP-Myo1a-TH1 (0.13 ± 0.01 s⁻¹, n = 30, R² = 0.98), respectively. (F) Model of the mechanism underlying the formation of multiple tethers in cells expressing EGFP (negative control) or EGFP-Myo1a-TH1 (dominant negative). These cartoons represent “snap shots” in the records shown in A, taken at the beginning of the tethered phases at the time point indicated by the black arrows.