Endogenous NM IIA and NM IIB form heterotypic filaments in live cells. (A) Cartoon depicting the co-assembly of EGFP-NM IIA with endogenous NM IIB. When NM IIB is localized using an antibody that recognizes its C-terminus (together with a red secondary antibody; red Y), the resulting heterotypic filament will display a green-red-green pattern when imaged with TIRF-SIM. (B) Cartoon depicting the co-assembly of endogenous NM IIA and endogenous NM IIB filament. Staining with antibodies that recognize the C-termini of both NM IIA and NM IIB, together with green (green Y) and red (red Y) secondary antibodies, respectively, should result in a strong overlap between the two signals when heterotypic filaments are present. If both signals are of equal intensity and perfectly overlapping, the result will be a single yellow punctum. Any offset in the two signals will result in a punctum with a yellow center and red and green flanking regions. Signal offset could result from steric hindrance between adjacent antibodies, uneven labeling of filaments by the antibodies, and/or uneven distribution of isoforms on either side of the bipolar filament. In addition, the intensity of the yellow signal will be diminished in heterotypic filaments where the signal for one isoform is much stronger than the signal for the other isoform. (C) COS-7 cells expressing EGFP-NM IIA were fixed, immuno-stained for endogenous NM IIB (followed by a red secondary), and imaged with TIRF-SIM. The white numbered boxes correspond to the magnified insets to the right. The scale bars represent 2 μm for the larger image and 300 nm for the insets. (D-F) MDA-MB-231 cells were fixed, immuno-stained with a rabbit polyclonal antibody for endogenous NM IIA (followed by a green secondary) and a mouse monoclonal antibody for endogenous NM IIB (followed by a red secondary), and imaged with TIRF-SIM. (D and E) Shown are TIRF-SIM images of lamellar regions, transverse arcs and ventral stress fibers. The numbered boxes correspond to the row of images to the right, which present grey scale images for αNM IIA and αNM IIB and the merged image. Arrowheads indicate some of the overlapping green and red puncta indicative of heterotypic filaments. Note that yellow is not obvious in D2 because the signal for NM IIA is much stronger than the signal for NM IIB. The scale bars represent 2 μm for the larger images and 300 nm for insets. (F) Discrete individual puncta were analyzed for their relative content of endogenous NM IIA and NM IIB and the results plotted as a function of distance from the cell edge in 2 μm increments (see Experimental Procedures for details). The data, which is plotted on a log scale as geometric means with 95% confidence intervals, is a compilation of over 8, 800 puncta from 6 cells. Note that the ratios in (F) are relative and do not necessarily represent that actual ratio of NM IIA to NM IIB in individual filaments due to differences in expression and assembly levels (see Experimental Procedures). See also .