Polymerization pathway of mammalian nonmuscle myosin 2s

Abstract

The three mammalian nonmuscle myosin 2 (NM2) monomers, like all class 2 myosin monomers, are hexamers of two identical heavy (long) chains and two pairs of light (short) chains bound to the heavy chains. The heavy chains have an N-terminal globular motor domain (head) with actin-activated ATPase activity, a lever arm (neck) to which the two light chains bind, and a coiled-coil helical tail. Monomers polymerize into bipolar filaments, with globular heads at each end separated by a bare zone, by antiparallel association of their coiled-coil tails. NM2 filaments are highly dynamic in situ, frequently disassembling and reassembling at different locations within the cell where they are essential for multiple biological functions. Therefore, it is important to understand the mechanisms of filament polymerization and depolymerization. Monomers can exist in two states: folded and unfolded. It has been thought that unfolded monomers form antiparallel dimers that assemble into bipolar filaments. We now show that polymerization in vitro proceeds from folded monomers to folded antiparallel dimers to folded antiparallel tetramers that unfold forming antiparallel bipolar tetramers. Folded dimers and tetramers then associate with the unfolded tetramer and unfold, forming a mature bipolar filament consisting of multiple unfolded tetramers with an entwined bare zone. We also demonstrate that depolymerization is essentially the reverse of the polymerization process. These results will advance our understanding of NM2 filament dynamics in situ.

Keywords: filaments; nonmuscle myosin 2; polymerization.

Fig. 1.

Electrophoretic analysis of purified recombinant NM2A (2A), NM2B (2B), NM2C (2C), and pNM2B (p2B). (Left) SDS/PAGE of NM2A, NM2B, and NM2C. (Right) Urea-glycerol electrophoresis of unphosphorylated and RLC-phosphorylated NM2B; ∼10% of pNM2B was diphosphorylated (band below pRLC). pRLC, phosphorylated RLC.

Fig. 2.

Polymerization of pNM2B in the presence of ATP measured by the light-scattering assay. The myosin concentration was 370 nM, and buffer composition was 10 mM Mops (pH 7.0), 2 mM MgCl₂, 150 mM NaCl, 0.1 mM EGTA, 1 mM DTT, and 1 mM ATP. Inset shows 0–30 s polymerization.

Fig. 3.

Field electron micrograph of pNM2B after polymerization for 4 s in the presence of ATP. Myosin (370 nM) was polymerized in the buffer described in the legend of Fig. 2. Unpolymerized myosins seen in the background include folded monomers (black arrows), a folded antiparallel dimer (yellow arrow), a folded parallel dimer (white arrow), and a folded tetramer (red arrow). See Fig. 4 for more images.

Fig. 4.

Electron micrographs of nonfilamentous pNM2B in 600 mM KCl (Top) and in 150 mM NaCl without (Middle) and with (Bottom) ATP. (Top) Unfolded monomers in 600 mM NaCl. The tail length is ∼150 nm. (Middle) Immediately after dilution into 150 mM NaCl, pNM2B formed folded monomers (M), folded antiparallel dimers (D), and folded antiparallel tetramers (T). (Bottom) Immediately after dilution in 150 mM NaCl+1 mM ATP, pNM2B formed folded monomers (M), folded antiparallel dimers (D), folded parallel dimers (PD), and folded antiparallel tetramers (T). There were no folded myosins in 600 mM NaCl or residual unfolded monomers in 150 mM NaCl with or without ATP, and only ∼2% of dimers were parallel dimers in 150 mM NaCl and ATP. Red arrows identify four heads in the tetramers. Folded monomers have two heads and a thick tail that is one-third the length of the tail of a fully extended monomer. Folded antiparallel dimers have two heads at both ends, and folded parallel dimers have four heads at one end only. The tails of the folded monomers almost fully overlapped in the folded dimers. Folded antiparallel tetramers have four heads at each end and are formed via association of two folded antiparallel dimers without staggering. (The scale bar applies to all figure panels.)

Fig. 5.

Electron micrographs of intermediate structures during polymerization showing the association of folded antiparallel tetramers and dimers with growing filaments and unfolding of folded tetramers. In all panels red arrows indicate clusters of four heads, orange arrows indicate folded and unfolding antiparallel tetramers, and blue arrows indicate folded antiparallel dimers. (1–4) Polymerization of NM2A and NM2C in the absence of ATP showing filament-associated tetramers. (5–7) Association of folded antiparallel dimers with pNM2B filaments polymerizing in the presence of ATP. (8–14) Association of folded antiparallel tetramers with growing pNM2B filaments in the presence of ATP. The folded tetramers are of various lengths indicative of different extents of unfolding. In 10, two folded antiparallel tetramers associate with one growing filament. (15–17) Opening of folded pNM2B tetramers in the presence of ATP with folded segments (green arrows) in the bare zones. (The scale bar applies to all figure panels.)

Fig. 6.

Electron micrographs of intermediate structures during polymerization showing clusters of four (red arrows) or three (white arrows) myosin heads in filaments of NM2A, NM2B, and NM2C in the absence of ATP and pNM2B filaments in the presence of ATP. (The scale bar applies to all figure panels.)

Fig. 7.

Electron micrographs of intermediate structures during the formation of large filaments of NM2A polymerizing without ATP (Upper Row) and pNM2B polymerizing with ATP (Lower Row). Clusters of four heads (red arrows) and three heads (white arrows) and associated folded antiparallel tetramers (orange arrows) are identified. Myosin heads are broadly distributed along the growing filaments, as is consistent with the unfolding of multiple associated folded structures. (The scale bar applies to all figure panels.)

Fig. 8.

Electron micrographs of mature filaments of NM2A, NM2B, and NM2C polymerized without ATP and pNM2B polymerized with ATP. All filaments have entwined bare zones. The red arrows (panels 1, 2, 6, and 8) indicate clusters of four heads, and white arrows (panels 1 and 8) indicate clusters of three heads, as is consistent with tetramers being the filament assembly unit. (The scale bar applies to all figure panels.)

Fig. 9.

Depolymerization of RLC-unphosphorylated NM2A (NM2A 1–9) and NM2B (NM2B 1–9) filaments after the addition of ATP. (1) Bipolar antiparallel filaments after overnight polymerization without ATP showing head clusters at both ends separated by a bare zone. (2) Filaments immediately after the addition of 1 mM ATP showing partial depolymerization with heads of folded structures in the bare zones. (3) Complete depolymerization of filaments into bundles of folded dimers. (4–6) Images of intermediates between the images in 2 and 3. In 6, a folded tetramer is associated with a depolymerizing filament. (7–9) Images of folded antiparallel tetramers formed during depolymerization. Red arrows identify clusters of four heads; orange arrows indicate folded tetramers associated with depolymerizing filaments; blue letters identify folded monomers (M), folded dimers (D), and folded tetramers (T). (The scale bar applies to all figure panels.)

Fig. 10.

Illustration of the proposed NM2 assembly pathway in 150 mM NaCl. Folded monomers form folded antiparallel dimers and folded antiparallel tetramers irrespective of RLC phosphorylation status or the presence of ATP (Top Row). Folded tetramers open to become extended, unfolded tetramers to which folded tetramers bind and unfold (Rows 2–4), forming mature filaments of multiple unfolded tetramers with entwined bare zones (Rows 5 and 6). (The scale bar applies to all figure panels.)