Direct Observation of Nanometer-Sized Steps of Single Myosin VI Molecules in Living Cells.

Figures

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  1. Labeling HaloTag-fused myosin VI in living cells with CA-SiR-d12. (A) Chemical structure of deuterium (d12) congeners of tetramethyl­(silicon)­rhodamine (SiR-d12) with a chloroalkane (CA) linker handle on the 6-carboxy position (CA-SiR-d12) and its binding to HaloTag. CA-SiR-d12 binds to Asp106 in HaloTag through nucleophilic substitution, displacing chloride as the leaving group. HaloTag is shown in gray color based on the published X-ray crystal structure (pdb: 6u32) for TMR-bound HaloTag and modified in PyMOL. (B) Schematic domains of HaloTag-fused full-length myosin VI. Following the N-terminal motor domain, myosin VI contains a distinctive insert (Insert 2), an IQ motif, a three-helix bundle, and a stable single α helix (SAH) domain and the cargo-binding domain. The C-terminus of full-length myosin VI was engineered to incorporate a HaloTag for site-specific labeling with CA-SiR-d12. (C) Schematic of labeling HaloTag-fused myosin VI with CA-SiR-d12. Cells were transiently transfected with a plasmid encoding myosin VI containing HaloTag in the C-terminus. Following transfection, cells were incubated with CA-SiR-d12, which binds to HaloTag-fused myosin VI. Unbound CA-SiR-d12…

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  2. No nonspecific binding of CA-SiR-d12 in living cells. (A) Representative fluorescence images of cells transfected with Lifeact-GFP alone (top) and with Lifeact-GFP along with HaloTag-fused myosin VI (bottom) after incubation of CA-SiR-d12. Signals from Lifeact-GFP (green) and SiR-d12 (red) were acquired simultaneously. No SiR-d12 signal was detected without transfection of HaloTag-fused myosin VI, whereas numerous fluorescence puncta of SiR-d12 were observed in cells transfected with HaloTag-fused myosin VI. A scale bar represents 5 μm. (B) Percentage of cells exhibiting fluorescence signals of SiR-d12 without and with expressed HaloTag-fused myosin VI. Three independent experiments were performed; 93 ± 6.7 (SEM)% of cells transfected with HaloTag-fused myosin VI showed fluorescence signals of SiR-d12, whereas 0% of cells transfected with Lifeact-GFP alone showed fluorescence signals of SiR-d12, indicating that there was no nonspecific binding of CA-SiR-d12 in living cells.

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  3. Measurement of nanometer-sized steps of single wild-type myosin VI in living cells. (A) Representative fluorescence image of SiR-d12-labeled myosin VI in a living cell taken at 6.1 s after the initiation of imaging. An expanded field of view shows a SiR-d12-labeled myosin VI exhibiting unidirectional motion. A scale bar represents 5 μm. A color bar represents elapsed time. (B) Fluorescence intensity of SiR-d12-labeled myosin VI showed one-step photobleaching, indicating that this myosin VI contained one SiR-d12 molecule. (C) Representative trace of a SiR-d12-labeled myosin VI exhibiting stepwise motion. (D) Step size histogram of SiR-d12-labeled myosin VI in living cells. The histogram was fitted with a sum of three Gaussian functions, each centered at −40.0 ± 12.9 (±SD), 32.5 ± 7.1, and 61.2 ± 18.9 nm (n = 308 steps). (E) Dwell time histogram of SiR-d12-labeled myosin VI in living cells. The histogram was well fitted with a single exponential function (f(t) = A × k × e–kt ) with k = 0.82/s (n = 308). (F) Velocity histogram of SiR-d12-labeled myosin VI exhibiting stepwise motion (N = 75 myosin VI motors) in living cells. The average was 90.7 ± 56.8 nm/s. (G) Run length histogram…

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  4. Measurement of nanometer-sized steps of single myosin VI L310G mutant in living cells. (A) Schematic of HaloTag-fused myosin VI L310G mutant. This myosin VI mutant harbors a leucine-to-glycine substitution at position 310 within the motor domain of the wild-type full-length myosin VI. Following the N-terminal motor domain, myosin VI L310G mutant contains a distinctive insert (Insert 2), an IQ motif, a three-helix bundle, a stable single α helix (SAH) domain and the cargo-binding domain containing HaloTag to label mutant myosin VI with CA-SiR-d12. (B) Representative trace of single SiR-d12-labeled myosin VI L310G mutant exhibiting stepwise motion in living cells. (C) Step size histogram of single SiR-d12-labeled myosin VI L310G mutant in living cells. The histogram was fitted with a sum of three Gaussian functions, each centered at −37.7 ± 5.9 (±SD), 37.3 ± 10.1 and 72.4 ± 22.8 nm (n = 236 steps). (D) Dwell time histogram of single SiR-d12-labeled myosin VI L310G mutant. The histogram was well fitted with a single exponential distribution function (f(t) = A × k × e–kt ) with k = 0.53/s (n = 236). (E) Velocity histogram of myosin VI L310G mutant exhibiting stepwise motion in living…