Figure 1. A novel in vitro assay to study mRNA motility.
(A) Schematic of assay; see text for details. SA, streptavidin; Aptamer, streptavidin-binding RNA aptamer. (B and C) Stills generated from time-lapse series of motile unidirectional (B) and bidirectional (C) Cy3-h wild-type (hWT) RNPs (magenta). Double-headed arrow indicates orientation of microtubule (− and +, minus and plus end); plus end (black arrowhead above top still) is marked by greater incorporation of HiLyte 647-tubulin (green). (D and E) Kymographs (time-distance plots) of examples of unidirectional (D) and bidirectional (E) RNPs; t, time.
Figure 1—figure supplement 1. Assessing RNA copy number in hWT RNPs and tracking accuracy in the RAT-TRAP assay.
(A and B) Motile RNPs assembled in the presence of equimolar Alexa488 (A488)-labelled h and Cy3-labelled h contained only A488 or Cy3 dyes, but never both. Thus, single fluorescent h molecules were present in each RNP. Kymographs of two examples of RNPs (A) and overall quantification from the experimental series (B) are shown. d, distance; −, minus end; +, plus end; N, number of motile RNPs analysed. For the experiments in A and B the Alexa488-labelled RNAs and Cy3-labelled RNAs were mixed and incubated with the streptavidin bead matrix before the addition of extract. (C) Representative kymograph of h RNPs in the presence of 20 U·ml−1 apyrase (which is used to deplete ATP and ADP from the chamber [Higuchi et al., 1997; Ma and Taylor, 1997]). Note that RNPs appear static. (D) Distribution of instantaneous frame-to-frame displacements generated from automatic tracking of h RNPs on microtubules in the presence of 20 U·ml−1 apyrase. In accordance with previous studies (Hendricks et al., 2010), our tracking accuracy was defined as one standard deviation of these displacements. Dashed line shows Gaussian fit from which standard deviation was calculated. R2, goodness of fit; N, total number of individual displacements analysed (from 15 RNPs).