Sequence and functional analyses of HookA. (A) A sequence alignment of the N-terminal putative microtubule-binding domain of HookA (A. nidulans), Hook (Drosophila), and Hook3 (human_HK3). The alignment was performed using CLUSTALW (Pôle BioInformatique Lyonnais Network Protein Sequence Analysis). Residues that are identical (asterisks), strongly similar (double dots), or weakly similar (single dots) are shown as red, green, and blue characters, respectively. Also see Fig. S1. (B) Colony phenotype of the ΔhookA mutant. (C) Distributions of mCherry-RabA–labeled early endosomes in the ΔhookA mutant (also see Videos 1 and 2) and the ΔclipA mutant. An obvious accumulation of mCherry-RabA signals is found at ∼80% of the hyphal tips in the ΔhookA mutant (n = 326), whereas none of the hyphal tips in wild type (n = 240) or the ΔclipA mutant (n = 73) show the same accumulation. (D, top) GFP–dynein HC (GFP-HC) signals in wild type and the ΔhookA mutant. Maximal signal intensities (arbitrary units) of the plus-end GFP-HC comets in wild-type cells and in ΔhookA cells are 276 ± 262 (n = 25) and 268 ± 225 (n = 30), respectively, and there is no statistical difference between the values at P = 0.05. (Bottom) Images of nuclei stained by DAPI in wild type and the ΔhookA mutant. The pattern of nuclear distribution in the ΔhookA mutant is normal, as none of the mutant cells shows any cluster of four or more nuclei when grown under the same conditions that allow us to see the hyphal tip mCherry-RabA accumulation (n > 100 for wild type, and n > 100 for the mutant). Bars, 5 µm.