Fig. 1: Morphology and composition of lumenal actin filaments.

a Representative fluorescence microscopy images (n > 3) showing control (top) and kinesore-treated (bottom)) HAP1 cells stained using antibodies targeting beta-tubulin (magenta) and actin (green). Scale bar = 20 µm. b, c Tomogram slices (in longitudinal (b) (Scale bar = 100 nm) and transverse orientations (c) (Scale bar = 25-nm)) showing microtubules within a projection from a kinesore-treated HAP1 cell. Class I lumenal filaments are highlighted with magenta arrows and one example is boxed in magenta. Images here and below are representative of >3 independent freezing and imaging sessions. d Examples of Class II and Class III filaments (top) and transitions between filament morphologies (middle and bottom). Class I is shaded magenta, Class II is green and Class III is orange (Scale bar = 25-nm). e Distribution of the length of filaments found in the microtubule lumen by their class. The total filament length was 22.7 µm which is 27.4% of the total microtubule length (82.7 µm) across 144 microtubules in 26 tomograms. f Layer lines from an in vitro example (blue scale bar = 37 nm) of a canonical F-actin labelled with real space distances annotated. This is representative of >10 filaments. g Layer lines of an example class I filament (from HAP1 cells) (magenta scale bar = 27 nm) labelled with real space distances, representative of >10 filaments h An example of a segment of class I filament that has been straightened, inverted, and projected in z, which was averaged to produce a helical reconstruction map (EMD-50845). This is representative of 5 similar reconstructions. When structures of actin (PDB: 8D17, light blue) was docked in, it was insufficient to fill the map, whereas cofilin-actin (PDB: 3J0S, actin in dark blue, cofilin in magenta) is in good agreement with the model.