The N-terminal region of Hok1 mediates dynein binding to EEs. (A) GFP₃-labeled dynein heavy chain (dynein) and mCherry-Rab5a–labeled endosomes (EEs) in Δhok1 mutants. Dynein (asterisk) accumulates at the cell tip (tip), from where it leaves without EEs. Cells were photobleached (bleach) to reduce signal interferences, which did not affect EE or motor motility (Schuster et al., 2011c). See also Video 4. (B) Estimated dynein numbers in moving GFP₃-Dyn2 signals in control and Δhok1 cells. Estimation used an internal calibration standard, assuming that the GFP₃-labeled dynein heavy chain forms dimers (Schuster et al., 2011c). Data represent two experiments. The red line shows a normal distribution curve. (C) Retrograde dynein flux in control and Δhok1 cells at ∼10 µm behind the tip. Bars represent data from two experiments and are means ± SE; sample size is indicated. No significant difference was found, P = 0.09. (D) Images and linescan plot of dynein and EE colocalization in Δhok1 hyphal tips after disruption of the MTs (+benomyl). The dotted line in merged image indicates the region of intensity scan. a.u., arbitrary unit. (E) Motility of Hok1^1–624 and mCherry-Rab5a–labeled EEs in Δhok1. Hok1^1–624 moves in retrograde direction, whereas EEs remain stationary. See also Video 5. (F) Retrograde flux of Hok1 and Hok1^1–624 at ∼10 µm behind the cell tip. Bars represent data from two experiments and are means ± SE; sample size is indicated. Significant difference is indicated: ***, P < 0.0001. (G) Co-migration of Hok1^1–624 and mCherry₃-labeled dynein heavy chain. Kymographs were slightly misaligned to better show colocalization. See also Video 6. (H) Domain organization of proteins used in immunoprecipitation and mass spectrometry experiments. Images in A, D, E, and G were adjusted in brightness, contrast, and γ settings. Horizontal bars are in micrometers, and vertical bars are in seconds.