Bipolar filaments of human nonmuscle myosin 2-A and 2-B have distinct motile and mechanical properties

Figure 1.. Nonmuscle myosin 2-B filaments move processively along actin filaments. (A) Movie frames showing HaloTag-NM2-B filaments (individual filaments marked by colored arrows), moving along Alexa Fluor 647 phalloidin labeled actin filaments (blue). (B) Kymograph for NM2-B filaments showing clear processive movement (diagonal lines) and long run lengths. (C) Frequency distribution histogram of NM2-B filament velocity. Black line is the Gaussian fit to the data yielding a velocity of 43 ± 1 nm · s-1(mean ± SEM; SD = 27 nm · s−1, R2 = 0.98). (D) Frequency distribution histogram of NM2-B filament run length. Black line is the single exponential fit to the data. The characteristic velocity and run length with their standard errors obtained from the fits are indicated in the graphs (n = 1463, R2 = 0.99). 10.7554/eLife.32871.007Figure 1—source data 1.Data for the velocity (panel C) and run length (panel D).

Figure 1—figure supplement 1.. GFP and HaloTags do not significantly interfere with myosin filament structure or motile function. (A) SDS-polyacrylamide gel of the constructs used in the experiments. The position of the RLC and the GFP-RLC are indicated. Lane 1, Molecular weight markers; lane 2, GFP-RLC-NM2-A; lane 3, HaloTag-NM2-A; lane 4, GFP-RLC-NM2-B; lane 5, HaloTag-NM2-B, lane 6 HaloTag-NM2-B Tail. (B) Negatively stained EM images of the bipolar filaments formed by GFP-RLC- and HaloTag-NM2-A and -NM2-B. For all constructs, the filaments have similar appearances and dimensions as unlabeled NM2-B filaments (Billington et al., 2013). Scale bars 100 nm. (C) Frequency distribution histograms of actin filaments velocity in the actin gliding assays. Black lines are the Gaussian fits to the data. The velocity and standard error obtained from the fit are indicated in each panel. The n value, SD and R2 values are 190, 9 nm · s−1, 0.99; 1617, 11 nm · s−1, 0.99; 471, 18 nm · s−1, 0.98; and 221, 15 nm · s−1, 0.98 for GFP-RLC-NM2-A, HaloTag NM2-A, GFP-RLC-NM2-B and HaloTag-NM2-B, respectively. 10.7554/eLife.32871.004Figure 1—figure supplement 1—source data 1.Data for the velocity distributions shown in panel C.

Figure 1—figure supplement 2.. GFP-RLC-NM2-B filaments move processively along actin filaments. (A) Movie frames showing GFP-RLC-NM2-B filaments (green and arrows), moving along rhodamine phalloidin-labeled actin filaments (red). (B) Kymograph for NM2-B filaments showing clear processive movement (diagonal lines) and long run lengths. (C) Frequency distribution histogram of NM2-B filaments run length. Black line is the single exponential fit to the data. The reported error is the S.E.M. (R2 = 0.94). 10.7554/eLife.32871.006Figure 1—figure supplement 2—source data 2.Run length data for GFP-RLC NM2-B.

Figure 2.. Determination of the number of NM2-B motor domains required for filament processivity. (A) Negative stain EM images of bipolar filaments formed by 100% HaloTag-NM2-B (left panel), 100% Halotag-NM2-B tail fragments (middle panel) and bipolar co-filament at 1:5 mixing ratio (NM2-B:Tail) (right panel). Arrows indicate a myosin motor domain. Arrowheads indicate a HaloTag moiety which can be seen as discrete globular domain smaller in size that the motor domain. (B) Frequency distribution histograms of NM2 co-filament run length. Black lines are the single exponential fit to the data. The mixing ratio and the characteristic run length obtained from the fit are indicated in each panel (n = 957, 378 and 113 for mixing ratios of 1:1, 1:2 and 1:5 NM2-B:Tail, respectively R2 = 0.99 for each fitting). (C) Characteristic run length is plotted as a function of number of motors per half filament. The black line is the linear fit to the data (R2 = 0.99). (D) Frequency distribution of velocity for all mixing ratios of co-filaments. The experimentally determined average number of motors per half filament are given for each mixing experiment in the inset. Lines are the Gaussian fits to the data.…

Figure 2—figure supplement 1.. HaloTag-NM2-B and HaloTag-NM2-B myosin tail co-polymerize. (A) Frequency distributions of the total fluorescence intensity of Alexa Fluor 488 (AF488) labeled molecules from AF488-HaloTag-NM2-B filaments and co-filaments formed by mixing AF488-HaloTag-NM2-B full-length with tetramethylrhodamine (TMR)-labeled HaloTag-NM2-B myosin tail for the three mixing ratio. Only filaments moving along actin were chosen for this analysis. (B) Dependency of the number of NM2-B molecules that formed the co-filaments, calculated according to Equation (2), on the mixing ratio (black symbols). Black line is the linear fit to the data. The error bars are S.E.M. The red line is the calculated relation assuming that the co-polymerization of NM2-B molecules and tails and the subsequent association of these filaments with actin is unbiased. The error bars represent the S.E.M. The deviation from this line suggests that filaments with a higher percentage of NM2-B have a greater probability of binding to and moving along the actin filament. 10.7554/eLife.32871.017Figure 2—figure supplement 1—source data 1.Fluorescent intensity values of hybrid filaments of NM2-B and NM2-B trail fragment shown in Panel A.

Figure 2—figure supplement 2.. Rate of movement of hybrid filaments of full length NM2-B and NM2-B Tail. Frequency distribution histograms at three mixing ratios. The velocity and S.E.M. are given above the histograms. The standard deviations and R2 values for the various mixing experiments are 17 nm.s−1, 0.99; 20 nm.s−1, 0.99; and 18 nm.s−1,, 0.99, respectively for the 2:1, 1:2 and 1:5 ratios of NM2-B full length:tail.

Figure 3.. NM2-A filaments move processively in methylcellulose. (A) Movie frames showing HaloTag-NM2-A filaments (red, selected ones marked by blue and yellow arrows), moving along actin filaments (blue) in presence of 0.5% methylcellulose. Under these conditions, NM2-A filaments are able to move on actin for several microns without detaching. (B) Kymograph for NM2-A filaments in presence of 0.5% methylcellulose. (C) Frequency distribution histograms of NM2-A filament velocity (n = 143). The black line is the Gaussian fit to the data. The velocity is 133 ± 10 nm.s−1 (mean ± SEM; SD = 75 nm.s−1, R2 = 0.81, ). (D) Movie frames showing HaloTag-NM2-B filaments (individual filaments marked by arrows), moving along actin filaments (green) in presence of 0.5% methylcellulose. In these conditions, the processivity of the filaments is increased dramatically relative to experiments in the absence of methylcellulose. (E) Kymograph for NM2-B filaments in presence of 0.5% methylcellulose. Most of the NM2-B filaments reached and accumulated at the end of the actin filaments as shown by the increasing in fluorescence intensity at the end of the actin filament and the vertical line at the end of the…

Figure 4.. Motile properties of NM2-A and NM2-B co-filaments. (A) Frequency distribution histograms of the run length for NM2-A:NM2-B co-filaments. Black lines are the single exponential fit to the data. The mixing ratio and the characteristic run length obtained from the fit are indicated in each panel (n, R2 = 1454, 0.98, 381, 0.99 and 1121, 0.99 for mixing ratios of 2:1, 1:1 and 1:2 NM2-B:NM2-A, respectively. The reported errors are the S.E.M. (B) Frequency distribution histograms of the velocity of mixed filaments for all mixing ratios. The FNM2-A (fraction of NM2-A in a filament) determined by quantification of the average NM2-A content per filament at each of the mixing ratios described in panel A is given in the insert The n values are 143, 1121, 381, 1454 and 1463 for FNM2-A of 1, 0.71, 0.36, 0.23, and 0, respectively. Lines are the Gaussian fit to the data. The distributions of these data are given in Figure 4—figure supplement 2. (C) The average velocity of co-filament movement as a function of FNM2-A. The velocity for 100% NM2-A is depicted in red and comes from experiments conducted in the presence of methycellulose. The error bars are S.E.M. (D) Dependence of the characteristic…

Figure 4—figure supplement 1.. NM2-A and NM2-B co-polymerize into filaments. (A) Frequency distributions of the fluorescence intensity of Halotag-NM2-A/NM2-B co-filaments at three mixing ratios and for NM2-A filaments alone. (B) Frequency distributions of the fluorescence intensity of AF488-HaloTag-NM2-B at three mixing ratio and for NM2-B filaments alone. (C) Measured versus expected fraction of NM2-A molecules in the co-filaments. FNM2-A, calculated according to Equation (4) (black symbols). Red line shows the expected values assuming random co-polymerisation of the two myosins. The error bars represent the S.E.M. 10.7554/eLife.32871.029Figure 4—figure supplement 1—source data 1.Fluorescent intensity values of mixed filaments of NM2-A and NM2-B.

Figure 4—figure supplement 2.. Velocities of co-filaments of HaloTag-NM2-B myosin and HaloTag NM2-A moving on actin filaments. Frequency distribution histograms of HaloTag-NM2-B:HaloTag-NM2A tail fragment co-filaments velocity at the three mixing ratios used in the experiments. Lines are the Gaussian fit to the data. The mixing ratio and the characteristic velocity (±standard error) obtained from the fit are reported in each panel. The n, S.D., and R2 values are 1454, 20 nm.s−1; 0.98) -, 381; 27 nm.s−1; 0.99; and 1121, 26 nm.s−1, 0.99 for mixing ratios of 2:1, 1:1 and 1:2 NM2-B:NM2A, respectively).

Author response image 1.. Each color shows the proportion of NM2-A in a given filament during a processive run.