Multimodal regulation of myosin VI ensemble transport by cargo adaptor protein GIPC

Figures

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  Figure 1. GIPC MIR increases the actin gliding speed of myosin VI.A, schematic illustrating the cargo adaptors with MIR and myosin VI constructs used in this study. B, schematic representation of the construct and assay design for the bimolecular FRET assay. C, dose–response curve for the change in the FRET ratio (ΔFRET) versus the adaptor protein concentration for GIPC MIR. The ΔFRET is calculated as the difference between the FRET ratios for the adaptor present and no adaptor conditions. Each dose–response curve value was derived from three independent protein preparations. The dose–response curve was fit to a hyperbolic equation, and the dissociation constant (Kd) was calculated as the protein concentration for the half-maximal response. Error bars are SD. D, schematic design of the myosin VI surface gliding assay. E, actin gliding speed with respect to myosin VI surface concentration in the presence and absence of GIPC. Error bars are SD. F, schematic design of myosin VI DNA nanotube actin gliding assay. G, actin gliding speed observed in the DNA nanotube actin gliding assay. For each condition, speed was computed from n ≥ 300 actin gliding traces. Error bars are SD. Significance was…

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  Figure 2. Multimotor DNA origami motility on single actin filaments.A, schematic illustrating multiple motor DNA origami motility assays on single actin filaments. B–D, quantification of the average cargo speed (B), motile events per field of view (C), and cargo run length (D) of DNA origami motility for the different conditions listed. Mean value of all motile parameters for each experimental repeat was estimated from ≥50 DNA origami motility events. ∗∗∗p < 0.001, comparisons of nonsignificant values (p > 0.05) are not shown. All experimental conditions in the figure were performed across three independent protein preparations, and data shown represent the mean ± SD across preparations.

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  Figure 3. Full-length (FL) myosin VI functions as a monomeric complex with GIPC MIR.A, schematic representation of myosin VI constructs and configuration used in the actin landing rate assay. B, log–log plot of actin landing rate with respect to motor density. A slope ≤1 indicates processive motility. R2 values for the linear fits were 0.913 for the HMM dimer, 0.884 for FL myosin VI, and 0.823 for FL myosin VI in the presence of GIPC. C, schematic indicating the myosin VI constructs used in the single-molecule TIRF assay. D, representative kymographs for the motility of the indicated constructs from C. E, cumulative distribution function (CDF) of the run lengths observed for each of the indicated constructs from C and D. F, Cy3 spot intensity for each of the constructs used in the single-molecule TIRF motility assay. G, schematic representation of the myosin VI cargo-binding domain (CBD) constructs and FRET assay used to probe GIPC MIR influence on myosin VI dimerization. H, FRET ratios of the constructs depicted in G in the presence or the absence of GIPC MIR. Experiments were performed using N ≥ 3 independent protein preparations. Significance was calculated using Student’s t test. GIPC,…

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  Figure 4. GIPC MIR binding releases myosin VI autoinhibition.A, schematic illustrating the construct design and assay for the myosin VI conformational FRET sensor. B, representative emission spectra (normalized to emission at 475 nm) illustrating the occurrence of FRET between mCitrine and mCerulean in the myosin VI conformational sensor. Samples were excited with 430 nm light. C, FRET ratios calculated for the myosin VI FRET sensor under the indicated conditions for both the WT and RRL to AAA mutation in myosin VI. Significance was computed using one-way ANOVA with a post hoc Tukey’s test. D, gliding velocity of actin in a surface motility assay for the RRL to AAA mutation in myosin VI. Experiments were performed using N ≥ 3 independent protein preparations. ∗∗p < 0.01; ∗∗∗p < 0.001. GIPC, GAIP-interacting protein, C terminus; MIR, myosin-interacting region; ns, not significant.

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  Figure 5. GIPC MIR increases the stroke distance of the myosin VI lever arm.A, depiction of myosin VI labeled beads in a single-molecule optical trap experiment. B and C, example of optical trap traces for full-length myosin VI alone (B) and in the presence of GIPC MIR (C). Black solid lines indicate the baseline with filled in arrowheads indicating the interarrival time and hollow arrowheads indicating the dwell time. D, trap displacement data for myosin VI-labeled beads. E and F, cumulative distribution function (CDF) of the dwell (E) and interarrival (F) times for myosin VI in the presence and absence of GIPC MIR. Detailed explanations of the fits are presented in the Experimental procedures section. Experiments were performed using N ≥ 3 independent protein preparations. GIPC, GAIP-interacting protein, C terminus; MIR, myosin-interacting region.

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  Figure 6. GIPC MIR decreases actomyosin crossbridge stiffness, leading to enhanced motor speed.A, schematic of the design of the proximal tail conformation sensor for myosin VI. B, representative anisotropy decay traces for the proximal tail conformation sensor in the presence and absence of GIPC MIR. C, decay times for the proximal tail conformation sensor for either the WT or RRL to AAA mutant in both the presence and absence of GIPC MIR. D, schematic depiction of the surface gliding nanotube assay with stiff and flexible linkages. E, actin gliding velocities of myosin VI in the presence or absence of GIPC MIR, when attached to nanotubes via either a stiff or a flexible oligo. Experiments were performed using N ≥ 3 independent protein preparations. Significance was assessed using either Student’s t test or a two-way ANOVA with post hoc Tukey’s test. ∗∗∗p < 0.001. GIPC, GAIP-interacting protein, C terminus; MIR, myosin-interacting region; ns, not significant.

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  Figure 7. GIPC MIR drives smooth and processive ensemble cargo motility.A, schematic illustrating the configuration of myosin VI on the DNA origami scaffold. B, example of merged image of the actin cytoskeleton extracted from a fish keratocyte (red) with DNA origami (green). The scale bar represents 10 μm. C, representative trajectories of myosin VI-labeled DNA origami on a keratocyte actin network. A sampling of trajectories is shown to aid in visualization of behavior. Inset, representative trajectories depicting pausing behavior (cyan circles). Circles represent one or more pauses in close proximity. D, average velocity of DNA origami scaffold with full-length myosin VI, in the presence or the absence of GIPC MIR, on the keratocyte actin network. E, individual cumulative distribution functions (CDFs) of run lengths for six measured keratocytes. F, trajectory run length in the absence or the presence of GIPC MIR. F, run lengths were calculated as the average run length of trajectories greater than 320 nm for each keratocyte. The data shown are the mean value for six keratocytes from each condition. G–J, analysis of parameters involved in pausing behavior of DNA origami scaffolds on the…

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  Figure 8. Model for multimodal regulation of the dual transporter-anchor function of myosin VI by GIPC. Myosin VI binding to GIPC MIR releases motor autoinhibition, extends the lever arm, and increases the flexibility of the motor–cargo linkage. This results in increased speed of single motors and enhances the processivity in ensembles. GIPC-mediated regulation leads to fast and smooth cargo transport across dense actin networks. GIPC, GAIP-interacting protein, C terminus; MIR, myosin-interacting region.