Kinetic adaptation of human Myo19 for active mitochondrial transport to growing filopodia tips

Figures

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  Figure 1. Myo19-3IQ expression, purification and its steady state ATPase activity. (A) The actomyosin ATPase cycle. The minimal reaction mechanism for the myosin’s conserved chemo mechanical energy transduction pathway, in boxes is the conserved predominate pathway, A-actin, M- Myosin, K′/k′ and K/k are the equilibrium/rate constants for the attached state (bound to actin) and detached states (unbound to actin), respectively. (B) Schematic representation of the expressed construct. (C) Coomassie Blue stained SDS-polyacrylamide gel of purified Myo19-3IQ heavy chain (97.3 kDa) (upper bands) and the associated calmodulin light chain (16.3 kDa) (lower bands). (D) The active-head titration measured by fluorescence increase of mT binding to myosin under single turnover conditions. The solid line through the data points is the best fit to Eq. 7 yielding active site concentration of 95 ± 6 nM. Error bars are within the symbols. Inset, time courses of mT binding by mixing constant amount of Myo19-3IQ and 15.63 (a) 31.25 (b), 46.88 (c), 62.5 (d), 93.75 (e), 125 (f) 187.5 (g), 250 (h) and 500 (i) nM mT. The solid lines to initial fluorescence increases are best fit to single exponential functions.…

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  Figure 2. ATP binding to Acto·Myo19-3IQ. (A) Time course of light scattering decrease after mixing 0.25 µM Acto·Myo19-3IQ with increasing ATP concentration from 0 (upper trace) to 2, 4, 8, 16, 31, 63, 150 and 500 µM (bottom trace). Data are averaged transients (n = 3–5). The smooth lines through the data represent best fit to double exponential function. (B) [ATP]-dependence of the fast-observed rate constant of ATP binding to Acto·Myo19-3IQ as measured by light scattering. The straight lines through the data points are the best fits to Eq. 1. Error bars of the fitting are within data points. (C) [ATP]-dependence of the slow observed rate constants of ATP binding to Acto·Myo19-3IQ measured by light scattering. The straight lines through the data points are the best fits to hyperbola. Error bars of the fitting are within data points. (D) The ratio of the fast (Afast) to slow (Aslow) amplitudes of the fluorescence transients as a function of [ATP]. (E) The kinetic reaction mechanism for ATP induced dissociation of Acto·Myo19-3IQ.

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  Figure 3. ADP binding to Acto·Myo19-3IQ as measured by kinetic competition with ATP. (A) Normalized time courses of light scattering decrease after mixing 0.2 µM Acto·Myo19-3IQ with 125 µM ATP in the absence or presence of different [ADP] ranging from 0.125 to 25 µM. Data are averaged transients (n = 3-5). Smooth lines through the data represent best fits to double or single exponential functions. (B) [ADP]-dependence of the slow observed rate constants for Acto·Myo19-3IQ as measured by kinetic competition with 125 µM ATP. (C) Normalized amplitudes of the slow phase obtained by fitting transients to double or single exponential functions as a function of [ADP]. The solid lines are fits of the data to Eq. 2. (D) [ATP]-dependence of the observed rate constants of Acto·Myo19-3IQ·D dissociation as measured by light scattering at saturated [ADP] of 25 µM. (E) The temperature dependence of the observed lag preceding ATP (500 µM)-induced Acto·Myo19-3IQ·D (0.2 µM) dissociation as measured by light scattering at saturated [ADP] of 25 µM. The solid line represents the best fit to the sum of exponential representing lag and the observed process as described previously64, error bars of the fitting are…

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  Figure 4. Binding kinetics of mT to Myo19-3IQ and Acto·Myo19-3IQ by monitoring FRET upon tryptophan excitation to mant-fluorophore. (A) Time courses of mT fluorescence change after mixing 200 nM myosin 19-3IQ with 2 (a), 4, (b), 5 (c), 10 µM (d) mT (final concentrations). The smooth lines are the best fit to a single exponential. (B) The dependence of k obs on [mT]. The solid line through the data points is the best fits to linear equation. Error bars represent standard deviation from three independent experiments. (C) The dependence of the amplitudes on [mT]. The solid lines through the data points are the best fits to linear equation. Error bars of the fitting are within the data points. (D) The dependence of signal to noise ratio (SNR) of the experimental data on [mT]. Note the fast decrease in observable signals. (F) The kinetic reaction mechanism for mT binding to Myo19-3IQ and for Acto·Myo19-3IQ. We included in rectangular parenthesis a rapid collision complex that most likely occurs without fluorescence change.

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  Figure 5. Binding kinetics of mD to Myo19-3IQ and Acto·Myo19-3IQ by monitoring FRET upon tryptophan excitation to mant-fluorophore. (A) Time courses of 2′/3′-mD fluorescence change after mixing 200 nM Myo19-3IQ with 2 (a), 5, (b) and 10 µM (d) 2′/3′-mD. The smooth lines are the best fit to a double exponential. (B) The dependence of k obs, fast on [2′/3′-mD]. The solid line through the data points is the best fit linear function. Error bars represent standard deviation from two independent experiments. (C) The dependence of k obs, slow on [2′/3′-mD]. The solid line through the data points is the best fit to hyperbola. Error bars represent standard deviation from two independent experiments. (D) The dependence of the amplitudes of the slow phase (A slow) on [2′/3′-mD]. The solid line through the data points is the best fit to hyperbola. Error bars represent standard deviation from two independent experiments. (E) The dissociation of the 2′/3′-mD from Myo19-3IQ and from Acto·Myo19-3IQ. The time courses of the 2′/3′-mD fluorescence change after mixing 250 nM Myo19-3IQ∙mD with 1000 µM ATP in the absence or presence of 275 nM phalloidin stabilized actin filaments. The smooth line is the best…

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  Figure 6. Time course of ATP hydrolysis and ADP·Pi burst of Myo19-3IQ. Time course of ADP-Pi formation by mixing 4 µM Myo19-3IQ construct with 250 µM. ATP (initial concentrations) followed by quenching with equal volume of 2 M HCl (premixing concentration). The solid line is the best fit of the data to the Eq. 10 with k ss being slow and thus was ignored. Error bars represent standard deviation of 2-3 independent experiments.

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  Figure 7. Actin-activated Pi release from Myo19-3IQ·ADP·Pi. The time course of transient Pi release. Myo19-3IQ·ADP·Pi (0.5 µM) after mixing with 0 (a) or 80 µM (b) F-actin in the presence of 8 µM PiBiP. Myo19-3IQ·ATP were aged for 300 ms before mixing with F-actin (λex = 436 nm, with 455-nm long-pass emission filter). The solid line represents the best fit to the sum of single exponential (initial burst) and linear (steady state turnover) function. Time course in the absence of the actin appears flat over the same timescale, as Pi release from myosin alone is very slow (0.05 s−1).

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  Figure 8. Simulation of the ATPase activity and duty ratio of Myo19-3IQ (A). Simulated steady state [actin]-dependence of the ATPase activity of the Myo19-3IQ. The solid lines through the data points are the best fit to a rectangular hyperbola (\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$v={v}_{0+}({k}_{cat}[actin])/({K}_{m}+[actin]))$$\end{document}v=v0+(kcat[actin])/(Km+[actin])). (B) Duty ratio as a function of [actin]. The duty ratio was calculated from the summation of all the biochemical intermediates distribution at each [actin] at the steady-state time regime according to equation: duty ratio = (strongly bound states)/(strongly bound states + weakly bound states). The solid lines through the data points are the best fit to a rectangular hyperbola (\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$duty\,ratio=1\,\ast…

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  Figure 9. Kinetic reaction mechanism of the key transitions in Myo19 ATPase cycle contributing to the rate limiting step.

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  Figure 10. Working model for Myo19 active translocation within the filopodia. Fraction of unbound Acto·Myo19-3IQ·D (AMD) complexes as a function of ATP/ADP ratio. Fractions of unbound AMD complexes were calculated as the ratio between amplitudes observed with ATP induced AMD dissociation at saturated [ADP] = 25 µM and the total amplitude observed with ATP induced AM dissociation at no ADP present. Insert. Graphic representation of working model in the filopodia. Myo19 utilizes its ADP high affinity strong binding state to regulate its movement along the filopodia. Myo19 display ‘catch up’ movements with filopodia elongation and may fluctuate between stall and burst type movements as a function of the [ATP]/[ADP]. Actin polymerization and other cytoskeletal motors are dependent on the available ATP pool for their activity across the filopodia. ATP (and other small and large molecules) free diffusion is relatively slower due to fluid-phase viscosity in membrane-adjacent cytosol65. Hence, diffusion of ATP from the interior cytoplasm may not meet its local high demand inside filopodia. Motors are depicted as black circle (attached state) and red circles (detached states), mitochondria is…