Myosin-Va binds to and mechanochemically couples microtubules to actin filaments

Abstract

Myosin-Va was identified as a microtubule binding protein by cosedimentation analysis in the presence of microtubules. Native myosin-Va purified from chick brain, as well as the expressed globular tail domain of this myosin, but not head domain bound to microtubule-associated protein-free microtubules. Binding of myosin-Va to microtubules was saturable and of moderately high affinity (approximately 1:24 Myosin-Va:tubulin; Kd = 70 nM). Myosin-Va may bind to microtubules via its tail domain because microtubule-bound myosin-Va retained the ability to bind actin filaments resulting in the formation of cross-linked gels of microtubules and actin, as assessed by fluorescence and electron microscopy. In low Ca2+, ATP addition induced dissolution of these gels, but not release of myosin-Va from MTs. However, in 10 microM Ca2+, ATP addition resulted in the contraction of the gels into aster-like arrays. These results demonstrate that myosin-Va is a microtubule binding protein that cross-links and mechanochemically couples microtubules to actin filaments.

Figure 1.

Myo5a binds to MTs. Sedimentation of Myo5a (0.06 μM), G-tail (1.7 μM), and Head (0.4 μM) domains in the absence (–MTs) or presence (+MTs) of taxol-stabilized MTs (3.6 μM) was performed. Content of Myo5a, G-Tail, and Head domains (top) and tubulin (bottom) in the pellet (P) and supernatant (S) fractions was assessed by immunoblot by using anti-Myo5a head or tail antibodies and Coomassie blue staining, respectively. The cosedimentation of Head domain with F-actin (4.8 μM; Head+Actin panels on right) in the absence and presence of ATP is also shown. Pellet and supernatant content of Myo5a head domain was determined by immunoblot with anti-Myo5a head antibodies and that of actin by Coomassie Blue staining.

Figure 2.

Sedimentation analysis of Myo5a-MT binding. Mixtures of taxol-stabilized, MAP-free MTs (1.3 μM polymer) and 0–0.45 μM Myo5a were sedimented and the concentration of Myo5a in the supernatant and pellet fractions determined by SDS-PAGE densitometry. The Myo5a binding saturates at ∼1:23 M ratio with tubulin polymer and binds with an apparent K_d of ∼70 nM.

Figure 3.

Myo5a-dependent cross-linking of MTs to F-actin. (a–d) Fluorescence images of fluorescein-labeled MTs (1.5 μM; a and c) and Texas Red-phalloidin–labeled F-actin (0.25 μM; b and d) in the absence (a and b) and presence (c and d) of Myo5a (0.5 μM). (e–j) Mixtures of MTs and F-actin in the absence (e–g) and presence (h–j) of Myo5a. k–m: Mixture of myosin-II-cross–linked arrays of F-actin and MTs. (n–p) Mixture of MTs, F-actin, and Myo5a in the presence of 2 mM ATP, under low Ca²⁺ conditions. The merged images show the MT signal in green and F-actin in red. Bar, 5 μm.

Figure 4.

Sedimentation of Myo5a with MTs and F-actin in the absence and presence of ATP. (A) Anti-Myo5a immunoblot analysis of pellet and supernatant fractions after sedimentation of Myo5a (0.06 μM) alone (M5a), Myo5a and F-actin (0.4 μM) in the absence (M5a+A) and presence (M5a+A+ATP) of ATP, Myo5a and MTs (3 μM) in the absence (M5a+MT) and presence (M5a+MT+ATP) of ATP, and Myo5a, F-actin, and MTs in the absence (M5a+A+MT) and presence (M5a+A+MTs+ATP) of ATP. (B) Quantification of the amount of total Myo5a present in the pellet fraction by densitometric scans of immunoblots.

Figure 5.

EM of thin sections of a pellet formed by cosedimentation of MTs (1.4 μM) and F-actin (0.3 μM). Regions near the top (A) and bottom (C) as well as the interface (B) between the layers of F-actin and MTs are shown. Note the alignment of both actin and MTs by the centrifugal field, which was roughly parallel to the vertical axis of the figure. Bar, 0.2 μm.

Figure 6.

Thin section EM of the pellet after sedimentation of the cross-linked arrays formed by addition of F-actin (0.3 μM) to MTs (1.4 μM) with bound Myo5a (0.1 μM). As in Figure 5, images from top through the bottom (A–C) of the pellet are shown. Arrows in A and B highlight interdigitating actin filaments that are free of Myo5a along their length. Arrows in C point out actin filaments decorated along their length with Myo5a. The vertical axis of the Figure 6C is roughly aligned with the centrifugal field. Bar, 0.2 μm.

Figure 7.

Myo5a-dependent contraction of MT-actin networks in the presence of Ca²⁺ and ATP. (A) Fluorescence images of actin and Myo5a (a) and mixtures of MTs, Myo5a and actin (b–d) after addition of ATP in the presence of 11 μM free Ca²⁺. Merge image shows MT fluorescence in green and actin in red. (B and C) Immunolocalization of Myo5a with either actin (B, e–j) or MTs (C, k–p) in cross-linked arrays of MTs-Myo5a and actin in the presence of Ca²⁺ and the absence (e–g, k–m) and presence (h–j, n–p) of ATP. Bar, 5 μm.

Figure 8.

Time-lapse microscopy of the Ca²⁺ and ATP-dependent contraction of Myo5a cross-linked networks of MTs and actin. Visualization of either actin (A, C, and D) or MT (B) fluorescence after addition of either ATP (A and B), buffer (C) or ATPγS (D) is shown. Time in seconds after perfusion is indicated. Panels on the right show the distribution of both actin and MTs at the start and end of the time-lapse series (C). Bar, 5 μm.

Figure 9.

EM of pellet formed by sedimentation of the aster-like MT-Myo5a-actin networks after addition of Ca²⁺ and ATP. Low (A, bar, 1 μm) and high (B; bar, 0.2 μm) magnification images are shown.