Figure 1.
Schematic representations of the myosin V loop 2 constructs.
(A) Schematic diagram depicting the domain structure of myo V with its surface-exposed loop 2 shown in yellow. The C-terminal GCN4 motif ensures dimerization. (B) Schematic domain model of truncated myo V in its homodimeric form. The regions are color-coded for each structural motif. The same set of colors is used in A and B. (C) Comparison of the loop 2 sequences from myo V Wildtype, Minus4 and Minus13. Mutants Minus4 (blue) and Minus13 (green) were designed by altering positively charged amino acids (lysines and arginines) to alanine and glutamic/aspartic acid, respectively. (+) and (−) indicate positively and negatively charged amino acids, respectively. The net charge of each construct is indicated in brackets next to the respective construct.
Figure 2.
Interaction of myosin V with microtubules under increasing ionic strength conditions.
TIRFM movie sequences of single-molecule experiments with 100 nM Cy3-labeled myo V Wildtype (A), Minus4 (B) and Minus13 (C) on Atto488-labeled microtubules under increasing salt concentrations (i.e. 25, 50, 100 and 200 mM KCl), were analyzed for microtubule-association and subsequent diffusion over a 70-seconds time period. For this, the total number of microtubule-associated myo V particles (shaded) and the number of diffusing particles (solid) per unit length of the microtubules and time of measurement are plotted as a function of KCl concentration. (*) indicates that in those cases only one event in total was counted. Error bars represent mean ± confidence interval (α = 0.95).
Figure 3.
One-dimensional diffusion of myosin V loop 2 constructs on microtubules.
(A) Kymographs of sequential frames depicting diffusive movement of single Cy3-labeled myo V Wildtype, Minus4 and Minus13 molecules (top to bottom, pseudo-colored green) on Atto488-labeled microtubules in 25 mM KCl. Microtubules are not shown for this purpose. Control represents a stationary, non-diffusing motor molecule on the microtubule. (B) TIRFM movie sequences of single myo V molecules on microtubules in 25 mM KCl were analyzed and then plotted as a displacement histogram. A single Gaussian (solid color-coded lines) was fitted to the data using equation 1 (Methods). From the obtained fit, the variance σ was used to calculate the diffusion coefficient D according to the equation, D = σ/2t, where t is the time interval between images, resulting in DWt = 0.113 µm2/s (n = 464), DMinus4 = 0.089 µm2/s (n = 801) and DMinus13 = 0.081 µm2/s (n = 425). Black, blue and green color-coded fit-lines depict the Gaussian fit for the individual displacement distribution of myo V Wildtype, Minus4 and Minus13, respectively.
Table 1.
Key parameters of diffusion of myosin V (Wildtype) and two myosin V loop 2 mutants.
Figure 4.
Interaction of myosin V with S-microtubules.
(A) Removal of the Carboxy-terminal E-hook from microtubules. (Left panel) SDS/12% PAGE gel of untreated microtubules (lane 1), and after subtilisin-treatment (lane 2). (Middle and right panel) Western blots of these two lanes with anti-α or anti-β tubulin antibodies. Subtilisin-treatment resulted in the complete loss of epitope reactivity, hence complete E-hook removal can be assumed. (B) TIRFM movie sequences of single-molecule experiments with 100 nM Cy3-labeled myo V Wildtype, Minus4 and Minus13 on Atto488-labeled S-microtubules, in 25 mM KCl were analyzed for microtubule-association and subsequent diffusion over a 70-seconds time period. The total number of S-microtubule-associated and diffusing myo V particles per unit length of the microtubules and time of measurement is plotted as category plot for the respective myo V constructs. Error bars represent mean ± confidence interval (α = 0.95).
Figure 5.
One-dimensional diffusive motion of myosin V on microtubules lacking the E-hook.
(A) The displacement between successive image frames of diffusive movements for myo V Wildtype on S-microtubules was determined. TIRFM movie sequences of 100 nM Cy3-labeled myo V Wildtype on Atto488-labeled microtubules were analyzed and plotted as a displacement histogram. The diffusion coefficient D was calculated according to the equation, D = σ/2t, where t represents the time interval between images and σ the variance. σ was obtained from the Gaussian fit (solid line), resulting in D = 0.226 µm2/s (n = 327). (B) Kymograph of sequential frames depicting the diffusive motion of a single Cy3-labeled myo V Wildtype molecule (pseudo-colored green) on Atto488-labeled S-mmicrotubules (for this purpose not visualized). Control represents a stationary, non-diffusing motor molecule on the microtubule.
Figure 6.
The balance between attraction forces determines the diffusive state of myosin V on microtubules.
(Left part) Strong attraction forces prevent microtubule-bound myo V molecules from advancing to the diffusive state. This Trapped State is achieved, if in addition to electrostatic also non-ionic attraction forces become increasingly dominant (Minus4 and Minus13 on S-microtubules). (Middle part) Diffusion takes place if for myo V the attraction toward the microtubule is of moderate strength. This Diffusive State in general is achieved when attraction and repulsion outweigh each other. Two different possibilities might account for that behavior. First, electrostatic and non-electrostatic interaction forces at the myo V binding-interface are well-balanced (Wildtype on untreated and S-microtubules); second, strong loop 2-derived ionic attraction is dominated by ionic repulsion elements (E-hooks) on the microtubule binding-interface (Minus4 on untreated microtubules). (Right part) Weak attraction toward microtubules prevents myo V from binding effectively to the filament, and hence diffusion becomes unlikely. This Dissociative State is given, if electrostatic repulsion via hydrophilic surface structures (E-hooks) becomes predominant (Minus13 on untreated microtubules). Red and blue colors indicate strong and weak attraction forces toward the microtubule surface, respectively.