Extended Data Fig. 7: Tm1 does not affect dynein activity in osk RNPs.
From: Tropomyosin 1-I/C coordinates kinesin-1 and dynein motors during oskar mRNA transport
a, Kymographs showing motile behavior of DDBE (via tetramethylrhodamine (TMR)-dynein) and osk 3′ UTR 2 + 3 with or without unlabeled Tm1 FL. Co-translocation of Tm1 is observed when present in the assay. b, c, Velocity (b) and run length (c) of DDBE-osk 3′ UTR 2 + 3 complexes assembled with or without Tm1. In b, median ± interquartile range is shown and is derived from analysis of 211–1,295 individual complexes (N) from 4 imaging chambers and 2 independent experiments (n) per condition (see Supplementary Table 3). Median values for each independent experiment (large dots) are superimposed on a violin plot showing distribution of values from individual complexes. In c, 1-Cumulative frequency distributions of measured run lengths were fit as one-phase decays with the decay constant τ ± 95% confidence interval shown. Hollow triangles represent empirical bin values used for fitting derived from analysis of 1,277–1,295 individual complexes (N) from 2 independent experiments (n) per condition (see Supplementary Table 3). d-f, Fraction of dynein binding events that underwent processive motility (d) and frequencies of dynein binding to microtubules (e) and processive dynein movement (f) in the presence or absence of Tm1 FL. Mean ± s.d. is shown and is derived from 20 microtubules (N), representing analysis of 2,152–2,207 individual complexes from 3-4 imaging chambers and 2 independent experiments (n) per condition (see Supplementary Table 3). g, Kymographs showing reduced association of Tm1 with motile DDBE-osk 3′ UTR 2 + 3 RNPs after RNAse A treatment. h, Fraction of moving DDBE-osk 3′ UTR 2 + 3 RNPs that co-localized with Tm1 signal with or without RNase A. Mean ± s.d. is shown and is derived from 15 microtubules (N), representing analysis of 559–886 individual complexes from 2-3 imaging chambers and 2 independent experiments (n) per condition. Black or white circles indicate presence or absence of indicated components, respectively. In a and g, microtubule plus and minus ends are oriented toward the right and left of each kymograph, respectively. In panels b, d-f and h, mean values for each independent experiment (large dots) are superimposed on violin plots showing the distribution of values from individual complexes (b) or on values for individual microtubules (small dots; d-f, h); parallel experiments within each of these panels are shown by large dots of the same color. Statistical significance was determined by a nonparametric one-way Kruskal-Wallis ANOVA test with Dunn’s test for multiple comparisons (b), unpaired two-tailed Mann-Whitney test (c), unpaired two-tailed t-tests (d-f), or unpaired two-tailed t-test with Welch’s correction (h) using N values (total number individual complexes (b, c) or total number of microtubules (d-f, h)). ns: not significant (p > 0.05).
