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. 2014 Feb 3;204(3):343–357. doi: 10.1083/jcb.201307164

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© 2014 Higuchi et al.

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Figure 8. — Off-loading of polysomes from moving early endosomes. (A) Mean run-length of EEs (Rab5a), the large ribosomal subunit (Rpl25), and the RNA-binding protein Rrm4. Bars are given as mean ± SEM (error bars); sample size n is 68, 84, and 81, respectively, from 2–4 experiments. ***, statistical significance at P ≤ 0.0004 (Mann-Whitney test). Run length of Rrm4 and Rpl25 was not different; P = 0.90991 (Mann-Whitney test). (B) Image series showing off-loading of ribosomes (arrowheads), labeled with Rpl25-GFP (green), from moving EEs, labeled with mCherry-Rab5a (red). The photobleached area is indicated by “Bleach” and the red arrows. See also Video 8. Brightness, contrast, and gamma settings were adjusted. Time is indicated in milliseconds. (C) Off-loading of Rrm4-GFP (arrowheads; green in Merge) from moving EEs (Rab5a; red in Merge). Brightness, contrast, and gamma settings were adjusted. The photobleached area is indicated by “Bleach” and the red arrows. Bars, 2 s and 1 µm. (D) Off-loading of Rrm4-GFP (Rrm4) and Rps3-mCherry₃ (Rps3). Both markers are deposited together (arrowheads), which indicates that entire polysomes are released from the EEs. Images are contrast inverted, and brightness, contrast, and gamma settings were adjusted. The photobleached area is indicated by “Bleach” and the red arrows. (E) Anterograde motility of Rpl25-GFP subunits in a photobleached region (red arrows). Ribosome subunits are off-loaded before they reach the hyphal tip (yellow arrowheads). Images are contrast inverted, and brightness, contrast, and gamma settings were adjusted. The photobleached area is indicated by “Bleach” and the red arrows. Bars, 3 s and 2 µm. (F) “Off- and re-loading” of Rpl25-GFP onto EEs. The ribosome is indicated by a yellow dotted line (Rpl25); the two EEs involved in transport are indicated by a red (1, Rab5a) and green dotted line (2, Rab5a). The photobleached area is indicated by “Bleach” and the red arrows. Bars, 2 s and 1 µm.