Molecular mechanisms of cytokinesis defects by the RACGAP1 variants in the GAP domain. (A) Positions of the variants in a crystal structure of the CYK4 GAP domain in a complex with Cdc42 (PDB:5C2J). The arginine finger (R385) and the 2 affected residues (L396 and P432) are marked in blue, green, and magenta, respectively. The region between R385 and P432 (cyan) is located close to the 3 subtype-specific residues on the GAP-interacting surface of the GTPases (S88, E95, and N132 on CDC42). The residues of the GTPases are color coded for the difference between the subtypes of Rho GTPases (canonical Rho, Rac, and Cdc42): pink, conserved among the 3 subtypes; orange, Rho specific (ie, identical between Rac and Cdc42); green, Rac specific; khaki, Cdc42 specific; and purple, different in all 3. A closer look from a different angle and a similar mapping of the key residues on the complex with RhoA (PDB:5C2K) are found in supplemental Figure 4. (B) GAP activity (ie, the stimulation of GTPase of human RhoA, Cdc42, and Rac1 by the CYK4 GAP domain with or without mutations [WT, P432S, L396Q, and R385A]). Kinetic efficiency (k_cat/K_M) was estimated with a linear model between the rate of GAP-stimulated GTPase activity vs the concentration of the GTPase (supplemental Figure 5) and shown with standard error. P values of the statistical test for the difference from the control (WT GAP) were corrected for multiple comparisons by Dunnett’s method. (C) Cell division of the HeLa cell lines stably expressing GFP-tagged CYK4 WT or variants as well as the control cell line expressing GFP alone (supplemental Figure 6 shows further characterization of the cell lines) was monitored by live microscopy after depletion of the endogenous CYK4. Frequency of cytokinesis failure calculated from the indicated numbers of failed cytokinesis in all the cell divisions in at least 3 independent experiments per line is shown with the 95% confidence interval (CI). P values of the statistical test for the difference from the WT cell line (w6) were corrected for multiple comparisons by Dunnett’s method. (D) Stills from live microscopy of the cells stably expressing a GFP-tagged WT or variant CYK4 after depletion of the endogenous protein. GFP fluorescence (green) is merged on the brightfield images (grayscale; unmerged images are found in supplemental Figure 7). The transition from metaphase to anaphase was monitored by chromosomes (yellow arrowheads). Postmitotic nuclei are marked with yellow dotted curves. White arrows indicate the localization of CYK4 GFP to the spindle midzone and midbody. Magenta arrows indicate the abnormal recruitment of the P432S variant to the equatorial cortex, which failed to form a cleavage furrow. Numbers are time (minutes) after the last timeframe with aligned metaphase chromosomes (scale bar 10 μm). (E) Proposed model for the significance of target preference of the GAP domain of CYK4 in cytokinesis. Elevated GAP activity of the P432S variant on RhoA in combination with the reduced activity of the L396Q variant on Rac/Cdc42 disturbs the proper balance between these Rho GTPases, leading to the cytokinesis failure of erythroblasts and CDA III. *P < .05, **P < .01, ***P < .001.