NM2 structures amplify to sub-resolution stacks before partitioning. (A) Example frames of EGFP-NM2A filament appearance, partitioning, and cluster growth. Scale bar = 300 nm. (B) Cartoon depicting two non-exclusive mechanisms of NM2 partitioning, containing either single or multiple filaments. (C) Classification of single time-point or fixed frames of NM2 fluorescent structures grouping structures based on the number of peaks/puncta and assigning them to stages of partitioning. (D) EGFP-NM2A sum projection example frame (left) with a leading edge outlined in orange and zoom inset in magenta. Scale bar = 10 μm. Zoom inset (middle). Scale bar = 1 μm. Zoom inset overlay (right) displays detected NM2 filaments color-coded into classes based on the number of detected puncta in the fluorescent structures. Scale bar = 1 μm. (E) Example calibration curve for a fixed molecular counting data set with linear regression. n = 15 cells for each candle, each experimental day. (F) Fixed NM2 partitioning results with a gray dotted line to indicate a single mature NM2 filament. n = 45 cells over three experiments. (G) Live NM2 partitioning results with a gray dotted line to indicate a single mature NM2 filament. n = 26 cells over three experiments. Data for (F and G) presented as standard box plots with median, first and third quartiles, and min and max indicated.