Images were all taken at the same laser settings and objectives using Nikon Imaging Software Elements and the same Nikon A1R confocal microscope. Actin, β-tubulin, and nuclei channels were saved separately as TIFF files. These images were analyzed using ICY software; images were thresholded to remove background, and the nucleus signal was merged with both the actin and β-tubulin signals. Masks were produced of these merged images and overlaid, after which the difference between the two masks was calculated to determine the number of pixels representing the actin signal without a corresponding β-tubulin signal. This value was converted to square micrometers to determine the surface area per field of view with the presence of actin present and absence of β-tubulin.

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