Polymerization pathway of mammalian nonmuscle myosin 2s

Significance

Nonmuscle myosin 2 (NM2) filaments are highly dynamic, polymerizing and depolymerizing in situ at different cellular sites where they are essential for multiple functions including cell division, cell motility, endocytosis, and exocytosis. Therefore, the mechanism of myosin filament assembly and disassembly has high importance. NM2 monomers can be either folded or unfolded. It has been thought that unfolded monomers and antiparallel bipolar dimers are the fundamental polymerization units. We now show that antiparallel, bipolar filaments are formed in vitro by the conversion of folded monomers to folded antiparallel dimers and then antiparallel tetramers that unfold, forming bipolar tetramers. Mature filaments consist of multiple unfolded tetramers with entwined bare zones. These results should assist our understanding of myosin filament dynamics in vivo.

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.

Like all class 2 myosins, the three mammalian nonmuscle myosin 2s (NM2A, NM2B, and NM2C) are hexameric monomers consisting of two long heavy chains (HCs), two short regulatory light chains (RLCs), and two short essential light chains (ELCs). The HCs comprise an N-terminal globular motor domain (head) with actin-activated ATPase activity, a lever arm (neck) that binds an RLC and an ELC, and a long helical tail terminating with a short C-terminal nonhelical tailpiece (1–3). The hexameric monomers are formed by homodimerization of the helical tails of two HCs into a coiled-coil. Under physiological conditions in vitro, the NM2 monomers polymerize into short, bipolar filaments (4–6) by antiparallel association of their helical tails (7).

NM2 filaments are highly dynamic in situ, frequently disassembling and reassembling at different locations within the cell (8–12) where, with actin filaments, they perform multiple diverse functions including cell division, cell adhesion, cell migration, endocytosis and exocytosis, and cell and tissue morphogenesis (13). Therefore, the mechanisms of polymerization and depolymerization of NM2s are of substantial interest.

NM2 monomers (14), like smooth muscle myosin 2 (SM2) monomers (15, 16), can exist in either of two conformations (16), folded (10S) and unfolded (6S), both of which have been shown to be present in motile fibroblasts (17). SM2 monomers and filaments have been extensively characterized (18). The addition of ATP to RLC-unphosphorylated SM2 monomers favors the 10S-folded conformation in which the motor domains of the two HCs associate and the coil-coiled helical tail bends at two sites, forming three approximately equal, closely packed segments; the two N-terminal segments of the folded tail bind to the necks of the associated heads (19). Phosphorylation of Ser19 (20) of the RLCs inhibits the ATP-induced head–head and neck–tail interactions, shifting the monomer equilibrium from the 10S-folded to the 6S-unfolded conformation. Under physiological ionic conditions, the addition of ATP to filaments of RLC-unphosphorylated SM2 causes the filaments to disassemble into folded monomers. Therefore, it was concluded that folded SM2 monomers are unable to polymerize, whereas unfolded SM2 monomers are polymerization competent (18).

The polymerization and depolymerization of NM2 filaments have been studied in vitro with NM2s extracted from cells (4, 14) and, more recently, with purified recombinant NM2s (5, 6). Based on their similarity to SM2, it has been proposed, and generally accepted, that the disassembly of filaments of RLC-unphosphorylated NM2s by the addition of ATP and filament reassembly after RLC-phosphorylation, as measured by light scattering (5, 21, 22), results from a shift of the monomer conformation from unfolded to folded upon the addition of ATP and back to the unfolded conformation after RLC-phosphorylation.

However, we recently reported (6) that, after overnight polymerization of 300 nM RLC-unphosphorylated NM2B under physiological ionic conditions (150 mM NaCl) in the presence of ATP, all the nonfilamentous NM2 was in folded conformation: ∼10% of the myosin was folded monomers, ∼45% was antiparallel folded dimers and antiparallel folded tetramers, ∼25% was higher oligomers, and ∼20% was filaments. This led us to speculate that NM2s may polymerize from folded monomers, not from unfolded monomers [a possibility once suggested by Trybus and Lowey (23) for SM2 and by Kendrick-Jones et al. (14) for NM2s], but the polymerization pathway from folded monomers to unfolded filaments was not apparent in our earlier experiments.

In the present study, we diluted monomeric RLC-phosphorylated NM2B (pNM2B) and RLC-unphosphorylated NM2s from 600 mM NaCl into 150 mM NaCl with or without 1 mM ATP and immediately (within 4 s or less) fixed the myosin with glutaraldehyde to capture intermediate structures during polymerization and to inhibit their depolymerization when diluted for electron microscopy. Similar experiments were performed with the addition of 1 mM ATP to filamentous RLC-unphosphorylated NM2s polymerized overnight in 150 mM NaCl in the absence of ATP. The results described below led to the conclusion that folded antiparallel tetramers are the principal filament assembly units and that depolymerization of RLC-unphosphorylated filaments by the addition of ATP is essentially the reverse of the polymerization pathway.

Results

Formation of Folded Monomers, Dimers, and Tetramers.

Recombinant NM2s were expressed in SF-9 cells and purified by anti-FLAG antibody affinity chromatography. The RLC of purified NM2B was phosphorylated by recombinant smooth muscle myosin light-chain kinase. As documented by electrophoresis (Fig. 1), the NM2s were highly purified, and the NM2B-RLCs were fully phosphorylated. Aliquots of ∼2 µM NM2s in 600 mM NaCl, 10 mM Mops, and 1 mM DTT were stored in liquid N₂.

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.

We diluted 1.48 µM pNM2B in 600 mM NaCl to 370 nM in 150 mM NaCl, 10 mM Mops (pH 7.0), 2 mM MgCl₂, 0.1 mM EGTA, 1 mM DTT, and 1 mM ATP. As measured by light scattering (Fig. 2), polymerization of pNM2B was rapid and almost complete by 4 s. As the apparent critical concentration of pNM2B+ATP is ∼33 nM (6), only ∼10% of the myosin would not have been polymerized.

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.

Aliquots of the polymerizing myosin were fixed with 0.1% glutaraldehyde at 4 s. As expected from the light-scattering data, electron microscopy field images of samples fixed at 4 s showed mostly filaments (Fig. 3). Also, as expected, 100% of pNM2B in 600 mM NaCl was unfolded monomers (Fig. 4). However, 4 s after dilution in polymerization buffer, images of 504 nonfilamentous pNM2B structures in field images from two experiments, such as that shown in Fig. 3, consisted of 52% folded monomers, 43% folded antiparallel dimers, and 4% folded antiparallel tetramers; about 2% of total dimers were folded parallel dimers. Typical structures are shown in Fig. 4. No unfolded monomers remained after dilution to 150 mM NaCl, and no unfolded dimers were observed.

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.)

Folded Structures in Growing Filaments.

Notably, 10 of the 13 filaments in Fig. 3 have folded structures within a bare zone between the two ends of mature filaments (5, 6). (More extensive images of filaments with folded structures are shown in Figs. 5–7.) About 67% of 258 pNM2B filaments and 92% of 102 NM2B filaments polymerized for 4 s in the presence of ATP had folded or partially folded structures in what would become a bare zone, whereas only 39% of 454 NM2A filaments, 9% of 420 NM2B filaments, and 31% of 472 NM2C filaments polymerized for 4 s in the absence of ATP had similar folded structures. The remaining filaments had clean bare zones. These data suggest that the presence of ATP in the polymerization buffer inhibits the unfolding of NM2B and that RLC phosphorylation promotes the unfolding of NM2B and probably also of NM2A and NM2C.

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. 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.)

Folded Antiparallel Tetramers Appear to Be the Principal Assembly Unit.

Folded antiparallel tetramers (orange arrows in Figs. 5–7), identified by the number of heads at one end, the head area, and the thickness of the region between the two ends, were associated with growing filaments (Figs. 5 and 7). The filament-associated tetramers have various lengths (Fig. 5, 8–13) consistent with different extents of unfolding. In addition, folded antiparallel dimers, although much fewer than folded tetramers, were also associated with growing pNM2B filaments in the presence of ATP (Fig. 5, 5–7, blue arrows). Multiple clusters of four heads (Fig. 6, red arrows) and three heads (Fig. 6, white arrows) are clearly identified in filaments of NM2A, NM2B, and NM2C polymerized without ATP and pNM2B filaments polymerized with ATP, indicating that the head clusters are one end of unfolded tetramers. Even the larger polymerizing filaments (Fig. 7) had multiple partially unfolded structures broadly distributed along the filament length; folded tetramers (Fig. 7, orange arrows) and clusters of four heads (Fig. 7, red arrows) and three heads (Fig. 7, white arrows) are identified.

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.)

Mature Filaments Consist of Multiple Unfolded Tetramers with Entwined Bare Zones.

The filaments that had fully polymerized in 4 s had clear bare zones consisting of multiple entwined unfolded tetramers (Fig. 8) with clusters of four heads (Fig. 8, red arrows) at the bipolar ends. The widths of the entwined tetramers in mature filaments (Fig. 8) are similar to the widths of unfolded tetramers (Figs. 5, 16 and 17 and 6, NM2C). On average, the mature filaments polymerized for 4 s were similar in total length, bare zone length, and bare zone width (Table 1) to previous data for filaments polymerized overnight (6).

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.)

Table 1.

Dimensions of NM2s filaments polymerized for 4 s

	NM2A	NM2B	NM2C	pNM2B+ATP
Filament length	341.5 ± 49.5 (n = 60)	310.8 ± 28.4 (n = 65)	285.3 ± 15.4 (n = 55)	310.1 ± 43.7 (n = 55)
Bare zone length	169.3 ± 19.2 (n = 35)	198.0 ± 21.6 (n = 60)	223.3 ± 18.3 (n = 35)	165.2 ± 18.2 (n = 40)
Bare zone width	11.5 ± 2.3 (n = 40)	10.5 ± 1.8 (n = 80)	6.0 ± 0.9 (n = 40)	10.3 ± 1.8 (n = 70)

Data are from electron microscopy images such as those shown in Fig. 8. Dimensions are in nanometers.

Depolymerization of RLC-Unphosphorylated NM2A and NM2B Filaments by Addition of ATP.

It has long been known that the addition of ATP induces depolymerization of filaments of RLC-unphosphorylated SM2 (15) and NM2 (21), but the pathway of depolymerization had not been studied. We added ATP to filaments of RLC-unphosphorylated NM2A and NM2B that had been polymerized overnight in the absence of ATP (Fig. 9, 1). Within 4 s after the addition of ATP, folded structures and broadly distributed heads appeared in what had been a bare zone (Fig. 9, 2), and bundles of folded antiparallel dimers also were present (Fig. 9, 3). Depolymerizing filaments (Fig. 9, 4–6) are shorter than filaments before depolymerization (Fig. 9, 1) with folded structures within bare zones of varied thickness. Antiparallel folded tetramers were formed during depolymerization (Fig. 9, 7–9). These results suggest that depolymerization of NM2 filaments is the reverse of polymerization.

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.)

Discussion

We conclude that the present data, together with the data in our prior publication (6), provide strong evidence that the polymerization in vitro of both RLC-unphosphorylated NM2s and RLC-phosphorylated NM2s proceeds from folded monomers to folded antiparallel dimers and folded antiparallel tetramers that unfold into bipolar antiparallel tetramers (Fig. 10). This is strongly suggested by the fact that 39% of growing filaments of NNM2A polymerized in the absence of ATP, and 67% of growing filaments of pNM2B in the presence of ATP had folded structures in the middle region. The lower number of nonpolymerized folded tetramers relative to folded monomers and folded dimers at the early stages of polymerization suggests that folded tetramers may unfold more rapidly than folded monomers and that dimers associate to form folded tetramers. Folded antiparallel tetramers may also be formed by the association of a folded dimer with a folded dimer already associated with a filament (Fig. 5, 5–7).

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.)

The absence of a bare zone during polymerization is inconsistent with the prior model based on the initial association of unfolded monomers with unfolded bipolar dimers that associate into filaments, nor have we seen any unfolded antiparallel dimers in any of our images. Additionally, the number of folded tetramers associated with the bare zones of polymerizing filaments greatly exceeded the number of folded dimers associated with the bare zones.

Mature filaments consist of multiple bipolar tetramers with entwined bare zones (Fig. 10). Although we and others did not report it, reexamination of previously published data (4–6) on the polymerization in vitro of purified NM2s reveals entwined structures in the filament bare zones (e.g., in figures 1 and 4 of ref. 4, figure 4 of ref. 5, and figure 9 of ref. 6). Entwined bare zones might increase the stability of NM2 filaments in situ.

Depolymerization of NM2 filaments in vitro appears to be the reverse of polymerization (Fig. 9), as similar intermediate structures were formed during polymerization and depolymerization; at the onset of depolymerization, filaments formed folded structures that then disassociated. A similar depolymerization process was reported in blebbistatin-treated rat embryo fibroblasts by Shutova et al. (figure S1 in ref. 17); the authors state: “The head domains of bipolar filaments in blebbistatin-treated cells were often splayed apart or unusually broadly distributed along the filament length whereas their bare zone was frequently bent.”

The assembly pathway described in this paper will contribute to our understanding of the processes of the multiple cell biological functions of nonmuscle myosin 2s. Folded monomers and dimers might better serve for storage and transportation in situ than extended monomers, and a polymerization pathway in which multiple folded tetramers open and assemble into mature filaments could be more efficient than the sequential addition of unfolded dimers or tetramers. Of course, the polymerization pathway in vivo might be modified by the phosphorylation status of the several phosphorylation sites on the HCs or by the interaction of NM2s with other proteins at any stage of the polymerization process (2, 3, 24).

Materials and Methods

Expression and Purification of Myosins.

The cDNAs of NM2A HC (Homo sapiens myosin heavy chain 9), NM2B HC (H. sapiens myosin heavy chain 10 transcript variant 2), and mouse NM2C HC (Mus musculus myosin heavy chain 14 transcript variant 1) were cloned onto pFastbac 1, the Bac-to-Bac plasmid, for expression in Sf-9 cells (Invitrogen). A FLAG tag (DYKDDDDK) was added to the N termini of the HCs to facilitate purification of the recombinant myosins. The cDNAs of NM2 RLC (H. sapiens nonmuscle myosin II, sequence ID NP_291024.1) and ELC (M. musculus nonmuscle myosin II, sequence ID NP_034990.1) were cloned onto pFastbac 1 for expression in Sf-9 cells. The baculoviruses for expressing HCs and LCs were constructed according to the company’s product manual (Invitrogen). Recombinant full-length myosins were produced by coexpression of the HC and two light-chain baculoviruses in Sf-9 cells. The recombinant NM2s were purified as described (6) using anti-FLAG resin (Sigma-Aldrich) affinity chromatography. After dialysis against 10 mM Mops (pH 7.0), 600 mM NaCl, and 1 mM DTT to remove the FLAG peptides, the purified myosins (2 µM), were aliquoted and stored in liquid nitrogen.

Preparation of Calmodulin and Myosin II RLC Kinase.

Rat calmodulin was expressed and purified from Escherichia coli. FLAG-tagged rabbit smooth muscle kinase (NP_001075775) was expressed using Baculovirus and was purified from Sf-9 cells.

Phosphorylation of NM2B.

Phosphorylation of the RLC was performed as described previously (6). pNM2B was aliquoted and stored in liquid N₂.

Protein Concentration Assay and Electrophoresis.

Protein concentrations were determined using the Bradford reagent (Bio-Rad) with purified myosin as the standard; its protein concentration determined by UV absorbance: myosin (mg/mL) = (A280 – A260)/0.5 (5). Urea-glycerol PAGE was performed according to a modified method as described previously (25). SDS/PAGE was performed by standard procedures on 10% NuPAGE gels (Invitrogen).

Polymerization and Depolymerization.

Myosin samples were cleared before use by centrifugation at 300,000 × g for 15 min at 4 °C in a Beckman TL-100 centrifuge. Myosins were polymerized at a concentration of 370 nM in 10 mM Mops (pH 7.0), 150 mM NaCl, 2 mM MgCl₂, 1 mM DTT, and 0.1 mM EGTA without or with 1 mM ATP and were fixed immediately (∼4 s) by incubation for 1 min with 0.1% glutaraldehyde in assembly buffer at room temperature. The cross-linking reaction was stopped by adding a 10% volume of 1 M Tris (pH 8.0) buffer. NM2A and NM2B were also polymerized overnight and then were depolymerized by the addition of 10 mM ATP in assembly buffer to a final concentration of 1 mM ATP and were fixed quickly (within 4 s) by incubation for 1 min with 0.1% glutaraldehyde in assembly buffer at room temperature.

Electron Microscopy.

After glutaraldehyde fixation, samples were diluted to about 200 nM, and 4 μL was applied to a UV light-pretreated carbon-coated copper grid and stained with 1% uranyl acetate. Micrographs were recorded on a JEOL 1200EX II microscope at room temperature. Filament lengths and widths were determined with MetaMorph software (MetaMorph, Inc.).

Light-Scattering Assay of pNM2B Polymerization.

Polymerization of pNM2B at the same concentration and with ATP was monitored by light scattering for 80 min at 20 °C in a PTI fluorometer with excitation at 365 nm (slit width 0.5 nm) and detection at 365 nm (slit width 0.5 nm).