Discovery of ultrafast myosin, its amino acid sequence, and structural features

Abstract

Cytoplasmic streaming with extremely high velocity (∼70 μm s−1) occurs in cells of the characean algae (Chara). Because cytoplasmic streaming is caused by myosin XI, it has been suggested that a myosin XI with a velocity of 70 μm s−1, the fastest myosin measured so far, exists in Chara cells. However, the velocity of the previously cloned Chara corallina myosin XI (CcXI) was about 20 μm s−1, one-third of the cytoplasmic streaming velocity in Chara. Recently, the genome sequence of Chara braunii has been published, revealing that this alga has four myosin XI genes. We cloned these four myosin XI (CbXI-1, 2, 3, and 4) and measured their velocities. While the velocities of CbXI-3 and CbXI-4 motor domains (MDs) were similar to that of CcXI MD, the velocities of CbXI-1 and CbXI-2 MDs were 3.2 times and 2.8 times faster than that of CcXI MD, respectively. The velocity of chimeric CbXI-1, a functional, full-length CbXI-1 construct, was 60 μm s−1. These results suggest that CbXI-1 and CbXI-2 would be the main contributors to cytoplasmic streaming in Chara cells and show that these myosins are ultrafast myosins with a velocity 10 times faster than fast skeletal muscle myosins in animals. We also report an atomic structure (2.8-Å resolution) of myosin XI using X-ray crystallography. Based on this crystal structure and the recently published cryo-electron microscopy structure of acto-myosin XI at low resolution (4.3-Å), it appears that the actin-binding region contributes to the fast movement of Chara myosin XI. Mutation experiments of actin-binding surface loops support this hypothesis.

Genome sequence and cell biological toolbox of the highly regenerative, coenocytic green feather algaBryopsis

“…Cytoplasmic streaming in the giant cytoplasm of Acetabularia or in the internodal cell of Chara is inhibited by actin filament disassembly (Nagai and Fukui, 1981; Nagai and Kamiya, 1977). Consistent with this, multiple myosin-XIs, one of which is the fastest cytoskeletal motor (Haraguchi et al, 2022), are encoded by C. braunii (Fig. 4A, Table S4).…”

Motor crosslinking augments elasticity in active nematics

“…In the high-ATP limit the Chara myosin XI head has a low duty ratio, meaning it spends less than half of its catalytic cycle bound to an actin filament. [28][29][30] Because this duty ratio depends on [ATP], motor velocity and the distance a motor travels before dissociating (run length) on single filaments also depend strongly on [ATP]: at [ATP] = 10 mM, tetrameric clusters have single-filament velocities of 0.5 mm s À1 with run lengths of 4 mm, while at [ATP] = 500 mM, the velocity is 10 mm s À1 , and the run length is 0.5 mm (Fig. S2, ESI †).…”

Functional characterization of calmodulin-like proteins, CML13 and CML14, as novel light chains of Arabidopsis class VIII myosins

“…They were purified using nickel-affinity and FLAG-affinity resins as previously described (Haraguchi et al 2022).…”