(A) Examples of traces of GFP fluorescent signals (after background subtraction) from individual hWT RNPs assembled in the presence of tagged Dlic (i, ii) or Dmn (iii, iv). Traces show stepwise decreases in fluorescence, indicative of photobleaching of single GFP molecules. (i) and (iii) are the raw traces, with (ii) and (iv) showing fitting with the Stepfinder algorithm (Kerssemakers et al., 2006). (B) Distributions and means (± SEM) of number of GFP decay steps for GFP::Dlic (top) and GFP::Dmn (bottom) for RNPs associated with hΔLE, hWT, or hSL1x3 RNAs. (C) Distributions and means (± SEM) of total GFP fluorescence at the beginning of imaging (after background subtraction) for GFP::Dlic (top) and GFP::Dmn (bottom) for RNPs associated with hΔLE, hWT or hSL1x3 RNAs. In B and C, means ± SEM were determined from raw data and N represents the number of RNPs analysed. ***p<0.001, compared to hWT values (Mann–Whitney non-parametric t test). Due to differences in fluorescence illumination in the TIR field in x, y, and z (Axelrod, 1989; Stout and Axelrod, 1989), measurements of total GFP fluorescence are likely to be less accurate than quantification of the number of stepwise decay events. However, the total GFP signal measurements provide further evidence for alterations in Dlic and Dmn copy number following manipulation of the number of SL1 elements. Note that the maximum number of dynein motors recruited by one localisation element and the proportion of recruited motors that are actively engaged during RNP transport is unknown. (D) Fluorescent Western Blot with α-Dlic antibodies revealing the levels of GFP-labelled Dlic compared to unlabelled, endogenous Dlic in the GFP::Dlic extracts used for photobleaching analysis. Predicted molecular weights of Dlic and GFP::Dlic are 54.5 kDa and 81.4 kDa, respectively. The mean percentage of total Dlic that was GFP-labelled was 0.49 ± 0.01 (mean ± SEM). Note that we previously showed that GFP::Dlic is incorporated in microtubule-associated motor complexes in accordance with its abundance in extracts relative to unlabelled, endogenously expressed Dlic (Amrute-Nayak and Bullock, 2012). Estimation of the ratio of GFP::Dmn to endogenous Dmn could not be made due to the unavailability of previously published α-Drosophila Dmn antibodies. (E) Table illustrating the calculations used to estimate dynein copy number per RNP based on stepwise photobleaching.