Journal: bioRxiv

Article Title: UNC-45A drives ATP-independent microtubule severing via defect recognition and repair inhibition, contributing to neurite dystrophy

doi: 10.1101/2025.08.26.671351

Figure Lengend Snippet: A. Representative kymographs showing GFP-UNC-45A and GFP-katanin binding to microtubules. 100nM GFP-UNC-45A or 25nM GFP-katanin were used for the binding assay. These kymographs were used to calculate the duration of association and binding frequency. B. Quantification of binding kinetics of GFP-UNC-45A and GFP-katanin to microtubules. N= 527 and 542 binding events were quantified for GFP-UNC-45A and GFP-katanin, respectively. C. Left , representative images of biotinylated MT (visualized with DIC) treated with 100µM taxol for 1 hr and repaired with 500nM rhodamine-tubulins (red) minus/plus 100nM GFP-UNC-45A (green). Right , quantification of fluorescent intensity of rhodamine-tubulins binding to MTs minus/plus UNC-45A and expressed as normalized free tubulin AFU. n= 100 MTs per condition were quantified. Error bars represent mean ± SD. D. Experimental set-up for microtubule-repair experiments in the presence of free tubulin and UNC-45A. E . Top four panels , representative images of biotinylated MT treated with 100µM taxol for 1 hr and exposed (or not, last four panels from the bottom-minus UNC-45A) to 250nM GFP-UNC-45A and increasing concentrations (250nM-5uM) of rhodamine-tubulin. Second four panels from top , green channel (UNC-45A). Third four panels from top, red channel (rhodamine tubulin). Fourth four panels from top, merged image of green and red channels. F. Quantification of fluorescent intensity of rhodamine-tubulins binding to microtubules expressed as AFU of tubulins bound to microtubules for different input of rhodamine tubulin. n= 80 MTs per condition from two independent experiments were quantified. Error bars represent mean ± SD. G . Mean ± SD of AFU of tubulins bound to microtubules is plotted over the input free tubulin. Error bars represent mean ± SD. H . Difference in mean rhodamine-tubulin AFU between minus/plus 250nM GFP-UNC-45A is plotted over the input free tubulin concentrations. n= 80 microtubules per condition were quantified. Error bars represent mean ± SE.

Article Snippet: Porcine brain tubulin (T240), rhodamine-labeled tubulin (TL590M), and biotinylated tubulin (T333) were purchased from Cytoskeleton.

Techniques: Binding Assay