Figure 4. Single-particle motility assays with different SHE complexes visualized by TIRFM. (A) Single-particle motility of SHE complexes in the presence of ASH1-3′ RNA containing one E3 zip code element. 281 processive runs were analyzed. (B) Single-particle motility assay of the SHE complex in the absence of zip code RNA. 292 processive runs were analyzed. The presence of zip code RNA does not greatly change the motility properties of the SHE complex. (C) Single-particle motility assay of SHE complex with the RNA binding–deficient She2p (N36S, R63K) mutant confirms that the RNA cargo is dispensable for processive movement. 283 processive runs were analyzed. (D) Single-particle motility assays with RNA containing two zip code elements fail to show enhanced processivity. Mean run length and velocity of a tRNA fusion construct with two ASH1-E3 zip code elements are even slightly reduced compared with particles with only a single zip code RNA. (E) Box plots representing data distributions for run lengths and velocities of each experiment, except for ASH1-3′ and IST2 RNA together. Here, the number of particles is too low for valid statistical analyses. Outliers (open circles, for details see Materials and methods) were determined to confirm the homogeneity in the collected data. Only a small number (n < 28) represent outliers; thus, all the events herein analyzed were considered representative for the respective experimental condition. The bold black lines show the median, the boxed regions indicate the first and third quartiles, and the whiskers above and below show the limits for outliers. (F) Table summarizing results from the different single-particle motility assays. The mean values for run length (left) and velocity (right) are derived from single-exponential and Gaussian fits to the distributions, respectively. All data derive from TIRFM motility assays, in which the GFP from the GFP-She2p fusion protein was visualized (as indicated by the boxed molecule name in the complex descriptions), unless stated otherwise. Besides the motor and SHE proteins, particles were assembled in the presence of calmodulin, myosin light chain, and the myosin chaperone She4p.
Figure 4.

Single-particle motility assays with different SHE complexes visualized by TIRFM. (A) Single-particle motility of SHE complexes in the presence of ASH1-3′ RNA containing one E3 zip code element. 281 processive runs were analyzed. (B) Single-particle motility assay of the SHE complex in the absence of zip code RNA. 292 processive runs were analyzed. The presence of zip code RNA does not greatly change the motility properties of the SHE complex. (C) Single-particle motility assay of SHE complex with the RNA binding–deficient She2p (N36S, R63K) mutant confirms that the RNA cargo is dispensable for processive movement. 283 processive runs were analyzed. (D) Single-particle motility assays with RNA containing two zip code elements fail to show enhanced processivity. Mean run length and velocity of a tRNA fusion construct with two ASH1-E3 zip code elements are even slightly reduced compared with particles with only a single zip code RNA. (E) Box plots representing data distributions for run lengths and velocities of each experiment, except for ASH1-3′ and IST2 RNA together. Here, the number of particles is too low for valid statistical analyses. Outliers (open circles, for details see Materials and methods) were determined to confirm the homogeneity in the collected data. Only a small number (n < 28) represent outliers; thus, all the events herein analyzed were considered representative for the respective experimental condition. The bold black lines show the median, the boxed regions indicate the first and third quartiles, and the whiskers above and below show the limits for outliers. (F) Table summarizing results from the different single-particle motility assays. The mean values for run length (left) and velocity (right) are derived from single-exponential and Gaussian fits to the distributions, respectively. All data derive from TIRFM motility assays, in which the GFP from the GFP-She2p fusion protein was visualized (as indicated by the boxed molecule name in the complex descriptions), unless stated otherwise. Besides the motor and SHE proteins, particles were assembled in the presence of calmodulin, myosin light chain, and the myosin chaperone She4p.

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