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. 2021 May 31;118(23):e2103383118. doi: 10.1073/pnas.2103383118

Fig. 2.

Fig. 2.

The dynein–dynactin interaction is required to initiate, but not to sustain motion. (A) Probability of motion (PMOTION expressed as %) is scored as the fraction of LPs that exhibit at least one excursion within 20 s when held above a MT using an optical trap. LPs were treated with 0.25 µM DIC-WT or 0.25 µM DIC-Mut. DIC-WT reduces PMOTION significantly. Experiments were done at 1 mM ATP in presence of the ATP regenerating system. Each experiment was repeated N times using different phagosome preparations, total of n LPs were used (both mentioned in the figure). Error bars, SEM. P values were calculated using Student’s t test. (B) Percentage of LPs that escape from the trap within 20 s when held above a MT. Error bars, SEM. P values were calculated using Student’s t test. (C) Representative stall force records for dynein on LPs. Experiments were done at 1 mM ATP in presence of the ATP regenerating system. LPs treated with 0.25 µM DIC-Mut displayed stalls of high force (Hi >9 pN; see next panel) and low force (Lo <9 pN). DIC-WT-treated LPs displayed only low-force stalls (<9 pN). (D) Stall forces (FSTALL) for untreated, DIC-Mut- and DIC-WT-treated LPs. DIC-Mut-treated LPs showed stalls from ∼2 pN to ∼14 pN (DICMut-ALL). This force range is divided into DIC-Mut-high (FSTALL >9 pN) and DICMut-low (FSTALL <9 pN) groups. DIC-WT-treated LPs always showed FSTALL <9 pN. FSTALL for DIC-Mut (low) and DIC-WT population is statistically the same. Mean, thick black line. The 95% confidence interval of means (thin black lines) was calculated using a bootstrap algorithm. Comparison of DIC-Mut (low) against DIC-WT: bootstrap P = 0.25; Kolmogorov–Smirnov (KS) test P = 0.14, D = 0.24; Mann–Whitney U test = 1,095, probability > U = 0.21. Phagosomes used: untreated 73; DIC-ALL 55; DIC-Mut high 20; DIC-Mut low 35; DIC-WT 54. (E) The time spent by an LP above half-maximal force (TSTALL) for the corresponding categories in D. TSTALL for DIC-Mut (low) and DIC-WT population is statistically the same. Mean, thick black line. The 95% confidence interval of means (thin black lines) was calculated using a bootstrap algorithm. Comparison of DIC-Mut (low) against DIC-WT: bootstrap P = 0.76; KS test P = 0.95, D = 0.10; Mann–Whitney U test = 982, probability > U = 0.76.