Fig. 4: SMYD2 regulates the localization of CHMP2B and the timing of abscission.

A Western blots of protein extracts from HeLa cell lines stably expressing CHMP2B_GFP and Cherry_SMYD2 treated for 24 h with DMSO or SMYD2 inhibitor (BAY-598 10 µM) were blotted for CHMP2B K6me1 or CHMP2B antibodies (N = 3). Loading control: Ponceau red. B Abscission time (from furrow onset to the microtubule cut) of HeLa cell line stably expressing CHMP2B_GFP and treated as indicated. (N = 3, n > 65 cells, median (red lines) and quartiles (dotty lines), Two-sided Mann–Whitney test). C Western blots of protein extracts from HeLa cell line stably expressing CHMP2B_GFP treated with control or SMYD2 siRNAs were blotted as indicated (N = 3). Loading control: Tubulin and Ponceau red. D Abscission time was determined as in (B). (N = 3, n > 75, median (red lines) and quartiles (dotty lines), Mann–Whitney test). E Left panels: live cell imaging of CHMP2B_GFP HeLa cell line treated as indicated. Time 00 is set prior the intercellular bridge formation. Last time point corresponds to the microtubule cut. Scale bar = 5 um. Right panel: Duration of each abscission stage (N = 3, n > 65 cells, median (red lines) and quartiles (dotty lines), Two-sided Mann–Whitney test, ns non-significant (p > 0.05)). F Left panels: live cell imaging of CHMP2B_GFP HeLa cell line treated as indicated. Time 00 is set prior the intercellular bridge formation. Last time point corresponds to the microtubule cut. Scale bar = 5 um. Right panel: Duration of each abscission stage (N = 3, n > 75 cells, median, Two-sided Mann–Whitney test, ns non-significant (p > 0.05)). G Western blots of protein extracts from CHMP2B K6A_GFP HeLa cell line treated with control or SMYD2 siRNAs were blotted as indicated (N = 3). Loading controls: Tubulin and Ponceau red staining. H Abscission time of CHMP2B K6A_GFP HeLa cell line treated with control or SMYD2 siRNAs was determined as in (B). (N = 3, n > 41 cells, median (red lines) and quartiles (dotty lines), Two-sided Mann–Whitney test, ns non-significant (p > 0.05). I Left panels: live cell imaging of CHMP2B K6A_GFP HeLa cell line treated as indicated. Time 00 is set prior the intercellular bridge formation. Last time point corresponds to the microtubule cut. Scale bar = 5 um. Right panel: Duration of each abscission stage (N = 3, n > 41 cells, median (red lines) and quartiles (dotty lines), Two-sided Mann–Whitney test, ns non-significant (p > 0.05)). J Recombinant CHMP3, CHMP2B-∆C and CHMP2B-∆C K6me1 proteins were purified and visualized using Coomassie (left) and Ponceau red staining (right). CHMP2B K6 methylation was assessed by western blot using CHMP2B K6me1 antibody (N = 2). K Left panel: Sorting coefficient plot showing preferential assembly of fluorescently labeled CHMP3 on positively curved membranes when in the presence of methylated CHMP2B. (N = 2, median (red lines) and quartiles (black lines), Two-sided t-test, (p < 0.05)). Right panel: Representative fluorescence micrograph used to calculate sorting coefficient values showing recruitment of fluorescently labeled CHMP3 on pulled lipid tubes from GUVs. Magenta is membrane signal, cyan is CHMP3 signal. Scale bar = 5 um.