Role of the nonhelical tailpiece of myosin-II in regulating filament architecture and function

Figures

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  Figure 1.. Essential role of the NHT in preventing uncontrolled NM-II assembly into large filaments. (A) Electrophoretic analysis of purified recombinant NM-IIs. The first 1–8 lanes show samples run on 10% SDS-PAGE gels, while the 9–14 lanes show samples run on 4–12% gradient gels. All gels were stained with Coomassie blue. (B–E) Electron micrographs of polymerized NM-IIA and NM-IIAΔNHT (B), NM-IIB and NM-IIBΔNHT (C), NM-IIC and NM-IICΔNHT (D), and NM-IIA-IIB-NHT and NM-IIA-IIC-NHT (E). (F) Individual filaments of NM-IIA and NM-IIAΔNHT in higher magnification. Myosins were polymerized overnight on ice in a buffer containing 10 mM MOPS (pH 7.0), 2 mM MgCl2, 150 mM NaCl, and 0.1 mM EGTA. Note: The image shown in Fig. 1 C (top panel) is identical to that shown in Fig. 3 A (left panel), and the image shown in Fig. 1 D (top panel) is identical to that shown in Fig. 3 C (left panel). These images are included in multiple figures to facilitate comparison of the relevant data. Source data are available for this figure: SourceData F1.

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  Figure 2.. Quantitative analyses of filament dimensions of NM-IIA, NM-IIB, and NM-IIC and their NHT variants. Comparison of filament length, width, and bare-zone dimensions of WT NM-IIA, NM-IIB, and NM-IIC, alongside NHT chimeras and NHT deletion variants. Filament lengths, widths, and bare-zone lengths were quantified from electron micrographs similar to those in Figs. 1 and 3. Data are presented as the mean ± SD.

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  Figure 3.. NHTs of NM-IIB and NM-IIC control their filament size. (A) Electron micrographs of polymerized NM-IIB and NM-IIB-IIC-NHT. (B) Zoomed-in view of polymerized NM-IIB, NM-IIB-IIC-NHT, and NM-IIBΔNHT. (C) Electron micrographs of polymerized NM-IIC and NM-IIC-IIB-NHT. (D) Zoomed-in view of polymerized NM-IIC, NM-IIC-IIB-NHT, and NM-IICΔNHT. Myosins were polymerized under the same conditions as described in Fig. 1. Note: The image shown in Fig. 3 A (left panel) is identical to that shown in Fig. 1 C (top panel), and the image shown in Fig. 3 C (left panel) is identical to that shown in Fig. 1 D (top panel). These images are included in multiple figures to facilitate comparison of the relevant data.

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  Figure 4.. NHT length as a major determinant of NM-IIA filament size. (A–D) Electron micrographs of polymerized NM-IIA1933X (A), NM-IIA1945X (B), NM-IIA2NHT (C), and NM-IIA-AM1Ct (D). (E) Individual filaments of these NM-IIA NHT variants in higher magnification. Myosins were polymerized under the same conditions as in Fig. 1. (F) Filament dimensions of WT NM-IIA and its NHT variants plotted against the total number of aa in their NHTs. Filament dimensions were measured from electron micrographs (Figs. 1 and 4) using MetaMorph. Data are presented as the mean ± SD.

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  Figure 5.. Intermediate structures formed during NM-IIAΔNHT polymerization. (A) Representative images of folded monomers (M), antiparallel dimers (D), and tetramers (T). (B) Arrows indicate antiparallel tetramers of different lengths along growing filaments. Myosin was polymerized for 4 s and immediately fixed with glutaraldehyde to visualize polymerization intermediates (A and B). (C) Stacked filaments lacking visible bare zones. Samples were polymerized overnight.

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  Figure 6.. NHT deletion does not affect the CC for NM-II polymerization in the absence of ATP. (A and B) CCs of NM-IIs for polymerization. NM-IIs at varying concentrations were incubated overnight as described in Fig. 1, and warmed to room temperature for 30 min, and light scattering was measured at 365 nm using a PTI fluorimeter. CC was determined from the intersection of the plotted curves for polymerized and monomeric myosin. Data represent the average of two independent experiments. (C) Effect of NHT deletion on NM-II solubility. Full-length and NHT-deleted NM-IIs (150 nM) were incubated as in Fig. 1 with varying NaCl concentrations. After centrifugation at 400,000 × g for 20 min to separate polymerized (pellet) and soluble (supernatant) myosin, the amounts of polymerized and unpolymerized myosin were determined by Coomassie-stained SDS-PAGE. Data represent the average of two independent experiments.

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  Figure 7.. NHT truncations in NM-IIA lead to the formation of enlarged bipolar filaments in vivo. (A) iSIM images of NM-IIB–knockdown, NM-IIA-KO U2OS cells expressing GFP-NM-IIA, GFP-NM-IIAΔNHT, GFP-NM-IIA1933X, or GFP-NM-IIA1945X. Individual bipolar filaments in the cytosol are highlighted (white boxes), with enlarged views shown in adjacent panels. (B) Line-scan quantification of GFP intensity along myosin heads and bare zones from images as in A. Only GFP clusters lacking NM-IIB were included. Data are presented as the mean ± SD. (C) Immunogold-labeling PREM analysis of bipolar filaments from NM-IIB–knockdown, NM-IIA-KO U2OS cells expressing GFP-NM-IIA, GFP-NM-IIAΔNHT, or GFP-NM-IIA1933X. GFP was labeled with 18-nm gold particles (yellow). Filament length, bare-zone length, and bare-zone width were measured from >40 individual filaments per condition and plotted. n indicates the number of myosin filaments analyzed for each of the NM-IIA NHT variant. P values were determined using a two-sided Mann–Whitney U test. (D) FRAP analysis of GFP-NM-IIA, GFP-NM-IIAΔNHT, GFP-NM-IIA1933X, and GFP-NM-IIA1945X in NM-IIB–knockdown, NM-IIA-KO U2OS cells. 72 h after NM-IIB siRNA treatment, a region of…

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  Figure S1.. Western blot analyses of NM-IIB knockdown efficiency and GFP-tagged NM-IIA NHT variant expression levels in NM-IIA-KO U2OS cells. Related to Figs. 7 and 8. (A) Knockdown efficiency of NM-IIB and expression levels of GFP-NM-IIA NHT variants (indicated by GFP-NM-IIA*) in NM-IIA-KO U2OS cells used for iSIM analyses (related to Fig. 7, A and B). (B) Knockdown efficiency of NM-IIB in NM-IIA-KO U2OS cells used for immunogold-labeling PREM to assess filament size and for immunostaining of pRLC and ppRLC (related to Fig. 7 C and Fig. 8 D). Source data are available for this figure: SourceData FS1.

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  Figure S2.. Western blot analyses of knockdown efficiencies of NM-IIB in NM-IIA-KO U2OS cells and of NM-IIA and NM-IIB in WT U2OS cells expressing GFP-NM-IIA NHT variants used for FRAP analyses. Related to Fig. 7. (A) Knockdown efficiency of NM-IIB in NM-IIA-KO U2OS cells expressing GFP-NM-IIA NHT variants used for FRAP analyses (related to Fig. 7, D and E). (B) Knockdown efficiencies of NM-IIA and NM-IIB in WT U2OS cells expressing GFP-NM-IIA NHT variants used for FRAP analyses (related to Fig. 7, F and G). Source data are available for this figure: SourceData FS2.

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  Figure S3.. Western blot and immunofluorescence analyses of NM-IIA-KO cells with NM-IIB knocked down, expressing GFP-NM-IIA NHT variants, and treated with the ROCK inhibitor. Related to Fig. 8. (A) Knockdown efficiency of NM-IIB and expression levels of GFP-NM-IIA NHT variants (indicated by GFP-NM-IIA*) in NM-IIA-KO U2OS cells used for Y-27632 treatment analyses in live cells (related to Fig. 8 A). (B–F) Effects of Y-27632 treatment on RLC phosphorylation (ppRLC) and stress fiber organization in fixed NM-IIB–knockdown, NM-IIA-KO U2OS cells analyzed by light microscopy (related to Fig. 8 D). Representative images of DMSO-treated and Y-27632–treated cells with indicated staining (B) are shown. The percentages of cells lacking pRLC (C) or ppRLC (D), as well as cells lacking pRLC but retaining stress fibers (E) or lacking ppRLC but retaining stress fibers (F), are quantified. (G) Knockdown efficiency of NM-IIB in NM-IIA-KO U2OS cells expressing GFP-NM-IIA NHT variants used for Y-27632 treatment analyses by immunogold-labeling PREM (related to Fig. 8, E–G). Source data are available for this figure: SourceData FS3.

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  Figure 8.. NHT truncations in NM-IIA slow stress fiber disassembly and enhance assembly when RLC phosphorylation is inhibited. (A) Stress fiber dynamics in response to inhibition of RLC phosphorylation. 72 h after NM-IIB knockdown by siRNA, NM-IIA-KO U2OS cells expressing GFP-NM-IIA, GFP-NM-IIAΔNHT, GFP-NM-IIA1933X, or GFP-NM-IIA1945X were treated with 10 µM Y-27632 and imaged by time-lapse microscopy at 5-min intervals. Representative images at 0, 30, and 80 min are shown. GFP-NM-IIA* indicates NHT variants. See also Videos 1 and 2. (B) Percentage of cells in A retaining stress fibers over time. Data are presented as the mean ± SD from three independent experiments. At time 0, all groups showed 100% stress fiber retention with zero variance; statistical testing was therefore not performed. At 30 min, P values for NM-IIA compared with NM-IIAΔNHT, NM-IIA1933X, and NM-IIA1945X were 0.017, 0.010, and 0.010, respectively. At 80 min, P values for NM-IIA compared with NM-IIAΔNHT, NM-IIA1933X, and NM-IIA1945X were 0.008, 0.001, and 0.004, respectively. (C) Percentage of cells in A forming NM-IIA* aggregates over time. Data are presented as the mean ± SD from three independent experiments. At time…

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  Figure S4.. Western blot and immunofluorescence analyses of WT U2OS cells with NM-IIA/NM-IIB knocked down, expressing GFP-NM-IIA NHT variants, and treated with the ROCK inhibitor. Related to Fig. 9. (A) Knockdown efficiencies of NM-IIA and NM-IIB and expression levels of GFP-NM-IIA NHT variants (indicated by GFP-NM-IIA*) in WT U2OS cells used for Y-27632 treatment analyses in live cells (related to Fig. 9 A). (B–H) Effects of Y-27632 treatment on RLC phosphorylation (pRLC and ppRLC) and stress fiber organization in fixed NM-IIA– and NM-IIB–knockdown WT U2OS cells analyzed by light microscopy. (B) Knockdown efficiencies of NM-IIA and NM-IIB in WT U2OS cells used for Y-27632 treatment analyses by immunogold-labeling CL-PREM (related to Fig. 9, D–F), as well as by pRLC and ppRLC immunofluorescence staining (see panels C and D). (C and D) Representative images of pRLC (C) or ppRLC (D) staining in DMSO-treated and Y-27632–treated cells are shown. (E–H) The percentages of cells lacking pRLC (E) or ppRLC (F), as well as cells lacking pRLC but retaining stress fibers (G) or lacking ppRLC but retaining stress fibers (H), are quantified. Source data are available for this figure: SourceData FS4.