Given the similarities between the antiparallel homodimers formed by M18Aβ and NM2, as well as the conservation of charge repeats between them, we hypothesized that M18A might be capable of binding NM2 via interactions similar to those used within homo-oligomers of these myosins. We tested this hypothesis using cosedimentation. When analyzed independently in the presence of 150 mM KCl, the majority of NM2A sedimented, whereas the majority of M18Aβ did not ( Figures 1 C and 1D). In contrast, cosedimentation of a fixed concentration of M18Aβ with increasing concentrations of NM2A led to an increased proportion of M18Aβ in the pellet ( Figure 1 C), suggesting that M18Aβ interacted with NM2A filaments. Importantly, M18Aβ also cosedimented with increasing concentrations of NM2B or NM2C ( Figure S1 G), indicating the interaction occurs with all NM2 isoforms. Furthermore, preliminary cosedimentation experiments demonstrated direct interaction between the M18Aα isoform and NM2 tail fragments (data not shown; but see Figure S4 B below). Surprisingly, cosedimentation of a fixed concentration of NM2A with increasing concentrations of M18Aβ resulted in a lower proportion of NM2A in the pellet ( Figure 1 D). Therefore, although M18Aβ and NM2 directly interact, excess M18Aβ appears to interfere with NM2A filament assembly. EM performed on an equimolar mixture of M18Aβ and NM2A (in 150 mM KCl) revealed filaments with a shorter mean length (227 ± 42 nm) than filaments of NM2A alone (314 ± 28 nm) ( Figures 1 E and 1F). Additionally, the length distribution of copolymers was significantly broader than that of filaments containing only NM2A ( Figure 1 F). Together, these data are consistent with coassembly of M18A with NM2 into mixed bipolar filaments and with the ability of M18A to influence the size and organization of NM2 filaments.

Figure 2 TIRF Microscopy of M18A-NM2A Copolymers Show full caption (A) TIRF image showing copolymers of M18Aβ:NM2A (e.g., spot 1). (B) Magnified inset showing low-intensity puncta consistent with small M18A:NM2A oligomers (e.g., spot 2) and single molecules of M18A (e.g., spot 3) or NM2A (e.g., spot 4). (C) Photobleaching traces of spots highlighted in (A) and (B). Montages above each plot show images of the denoted spots over time. Each image of spot 1 shows the average of a 30 s bin, and each image of spots 2, 3, and 4 shows the average of a 4 s bin. Arrows denote photobleaching events. See also Figure S2

To further demonstrate M18A/NM2A coassembly in vitro, we performed total internal reflection fluorescence (TIRF) microscopy using myosins possessing distinct N-terminal HaloTags. Alexa Fluor 488 (AF488)-labeled HaloTag-NM2A (green) and tetramethylrhodamine (TMR)-labeled HaloTag-M18Aβ (red) were mixed in a 1:1 molar ratio and bound to a coverslip ( Figure 2 A). A variety of fluorescence intensities were observed for individual puncta, consistent with the high SD observed for copolymer length in EM. The brightest puncta ( Figure 2 A, spot 1), which likely correspond to filamentous structures, contained both proteins, although their relative amounts varied. Photobleaching traces of these bright puncta ( Figure 2 C, spot 1) revealed large numbers of photobleaching events for each myosin, consistent with these puncta corresponding to bipolar filaments assembled from many NM2A and M18Aβ molecules. Some dimmer puncta also appeared to contain both myosins ( Figure 2 B, spot 2). Photobleaching analysis of these puncta ( Figure 2 C, spot 2) showed two photobleaching steps for each color, consistent with the lower limit of hetero-oligomerization being a heterodimer containing one two-headed molecule each of NM2A and M18Aβ. Some of the dimmest puncta appeared to consist of only green or red fluorescence, most likely corresponding to individual molecules of each myosin ( Figure 2 B, spots 3 and 4). Indeed, these smaller, monochromatic species showed stepwise photobleaching ( Figure 2 C, spots 3 and 4), consistent with their being two-headed monomers or four-headed homodimers of the respective myosins. Strong colocalization and large puncta were also observed when polymerization was induced by phosphorylation of RLCs in a mixture of the myosins using MLCK ( Figure S2 A). Colocalization of the two myosins was absent in high-salt buffer, further demonstrating that copolymerization is dependent on ionic strength ( Figure S2 B). Using the overall intensity of each punctum along with the average decrease due to quantal photobleaching of single fluorophores in each field, we estimated the number of each myosin per punctum. The distributions of filaments formed in the absence of ATP and formed by RLC phosphorylation were similar, in both cases showing a wide range of ratios of the two myosins ( Figures S2 C and S2D). In contrast, puncta in high-salt conditions typically consisted of low numbers of the respective myosin ( Figure S2 E). Together, these data using purified proteins argue strongly that M18A and NM2 coassemble into hybrid filaments in vitro.