Panel A shows a Coomassie blue-stained SDS-PAGE gel with lanes labeled S and P for supernatant and pellet, respectively, and conditions with and without tubulin. Panel B shows a similar gel with different Ana1 fragments. Panel C displays a cartoon representation of the Ana1 180-497 structural model, highlighting surface-exposed positive charges and mutated residues. Panel D shows a similar cartoon for the Ana1 1404-1729 structural model. Panel E contains confocal microscopy images of in vitro Taxol-stabilized microtubules mixed with different Ana1 fragments, with a scale bar of 20 micrometers.
Ana1 binds to and bundles MTs in vitro. (A and B) Coomassie blue–stained SDS-PAGE gel of a MT cosedimentation experiment performed with 2 mg/ml polymerized tubulins and different Ana1 fragments. The experiment was repeated three times with similar results. (C and D) Cartoon representations for AF3-predicted Ana1180–497 or Ana11404–1729 structural models showing the distribution of surface-exposed positive charges. Residues subjected to mutation are highlighted in the zoomed-in view boxes. (E) Confocal images of in vitro Taxol-stabilized MTs at 1 μM (ratio of rhodamine-labeled tubulin/unlabeled tubulin is ∼10%; red) mixed with 1 μM Ana1180–497 or 2 μM Ana11404–1729 protein. Scale bar, 20 µm. Identical imaging settings were used for all groups. Note that an inset is included for tubulin-only control, showing images of rhodamine-labeled tubulin with different contrasts to the protein–tubulin mixture group for better visualization. Source data are available for this figure: SourceData F7.