Fig. 3: Directional movement of constitutively active MYO7A mutants. | Nature Communications

Fig. 3: Directional movement of constitutively active MYO7A mutants.

From: Single-molecule fluorescence microscopy reveals regulatory mechanisms of MYO7A-driven cargo transport in stereocilia of live inner ear hair cells

Fig. 3

a Structures of two constitutively active HaloTag-fused MYO7A mutants. HaloTag-MYO7A-RK/AA has two missense mutations, p.R2127A and p.K2130A, that we insert in the second MyTH4-FERM (M/F2) domain, referring to the study using human MYO7A70. HaloTag-MYO7A-ΔSH3-ΔM/F2 has a truncated tail. These mutations are introduced to remove the tail-mediated autoinhibition of the motor domain. b Representative confocal images of HaloTag-fused full-length MYO7A and HaloTag-MYO7A-RK/AA expressed in vestibular hair cells (P2–5). Full-length MYO7A diffusely distributes in stereocilia (arrowhead) while HaloTag-MYO7A-RK/AA accumulates at stereocilia tips (arrows) of some cells, indicating directional movement toward stereocilia tips. Bars, 5 µm. c Confocal images of MYO7A-ΔSH3-ΔM/F2 expressed in vestibular hair cells (P2–5). This mutant accumulates at stereocilia tips in a small number of cells (arrows) and localizes diffusely in stereocilia in other cells (arrowhead). Bars, 5 µm. d Single-molecule microscopy of HaloTag-MYO7A-RK/AA. Time-lapse images (lower panels) show a molecule directionally moving in stereocilia (magenta circles). The kymogram (upper right panel) illustrates the processive movement of this molecule (arrows) and another molecule (open arrows). JFX554, 0.3 nM. Single-plane time-lapse, every 1 s. Bars, 5 µm (time-lapse images); 2 µm and 20 s (kymogram). e Single-molecule microscopy of HaloTag-MYO7A-ΔSH3-ΔM/F2. Time-lapse images and the kymogram show the processive and directional movement of a molecule (magenta circles and arrows). A stationary molecule is indicated for comparison (cyan circles and open arrows). Imaging conditions and scale bars are the same as (d).

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