Endosomes, peroxisomes, and nuclei are cargos of dynein and kinesin-3. (A) Bar graphs showing the mean velocities of anterograde- and retrograde-moving endosomes and peroxisomes in wild-type hyphae. Endosomes moved with mean anterograde and retrograde velocities of 2.62 ± 0.83 µm/s (SD) and 2.66 ± 0.77 µm/s, respectively (n = 250). Peroxisomes moved with mean anterograde and retrograde velocities of 1.66 ± 0.77 µm/s (n = 50) and 1.63 ± 0.87 µm/s (n = 48), respectively. (B) Bar graph showing the mean sizes of endosomes and peroxisomes in wild-type hyphae. Organelles were measured along their longest axes. Typical endosomes were round, with a mean diameter of 0.45 ± 0.07 µm (SD), whereas peroxisomes were more elongated, with a mean length of 1.03 ± 0.43 µm (n = 200 for both endosomes and peroxisomes). Endosomes were statistically smaller than peroxisomes (unpaired t test, P < 0.0001). (C and D) Localization of GFP-Rab5/RabA–labeled endosomes, Pex11/PexK-GFP–labeled peroxisomes, and histone H1–mCherry-labeled nuclei after targeted deletion of cargo-transporting motors. Loss of the dynein heavy chain gene resulted in hyphal tip accumulation of both endosomes and peroxisomes and failure of nuclei to distribute properly. In kinesin-1Δ strains, both endosomes and peroxisomes accumulated at the hyphal tip; a slight nuclear misdistribution phenotype was also observed. Deletion of kinesin-3 did not affect nuclei distribution but caused accumulation of endosomes near nuclei and accumulation of peroxisomes in the growing hyphal tip and subapical region. Dashed lines indicate the outline of the hyphae. Bars, 5 µm.