Established NM2 filaments are sites of enhanced filament assembly. (A) Cartoon of protein nanocage used as a known standard for the calibration curve. (B) Cartoons of the domains of the standard proteins used with the number of subunits per polymer and representative images of the standard in cells. The intensity scale bar is on the right. (C) Intensity histograms of the known standards with Gaussian fits. (D) The calibration curve was generated from the intensity distributions of the standards and the number of subunits with linear regression. n = 15 cells for each candle, each experimental day. (E) Example cartoon of NM2 monomer, filament, and filaments tagged with EGFP with the number of EGFPs in those structures. (F) Cartoon delineating two different NM2 filament assembly mechanisms, initiation of new clusters, or adding to existing clusters. (G) Example frames of cluster appearance tracking (top row), filament growth in a single cluster (middle row), and all cluster growth (bottom row), with the number of events/filaments added since the previous frame displayed upper left corner of each panel. Scale bar = 5 μm; 300 nm. (H) Quantification of cluster appearance rates compared with NM2 filament growth rates. Data are presented as standard box plots with median, first and third quartiles, and min and max indicated. n = 26 cells from three independent experiments.