NMIIA mutations associated with Myh9-RDs alter stack assembly on actin stress fibers without affecting bipolar filament formation in MKs. (A) Confocal Airyscan images of NMIIA head, NMIIA tail, and F-actin in MKs spread on collagen I. Arrowheads indicate NMIIA stacks, and arrows indicate NMIIA bipolar filaments, which have been presented as zoomed images in panel B. Scale bars, 4 μm (A) and 0.5 μm (B). (C) Scatterplot showing the median and interquartile range of the distribution for bipolar filament lengths in WT and Myh9-RD mutant MKs (NMIIA^GFP/NMIIA, n = 120; NMIIA^R702C-GFP/NMIIA, n = 50; NMIIA^GFP/NMIIA^D1424N, n = 35). (D) Box and whisker plots representing the distribution of the lengths of NMIIA stacks (NMIIA^GFP/NMIIA, n = 8614; NMIIA^R702C-GFP/NMIIA; n = 5348; NMIIA^GFP/NMIIA^D1424N, n = 7360). (E) Representative confocal Airyscan images and (F) fluorescence intensity plots indicating the line scans used for graphical analysis of the arrangement of NMIIA filaments on actin stress fibers. (G) Scatterplots representing the median and interquartile range of the distribution for distance between 2 successive NMIIA filaments on actin stress fibers (NMIIA^GFP/NMIIA, n = 142; NMIIA^R702C-GFP/NMIIA, n = 142; NMIIA^GFP/NMIIA^D1424N, n = 144). Images were acquired with either a Plan-Apochromat ×63 or ×100 objective (NA, 1.46) of a Zeiss LSM 880 confocal Airyscan microscope with Zeiss AxioCam. Images were processed with ZEN software and quantifications were performed with Nikon Elements 5.1. **P ≤ .01; ***P ≤ .001; ****P ≤ .0001.