FIGURE 2:

The C-terminal NLS regulates anillin’s cortical affinity during cytokinesis. (A) A cartoon schematic shows the C-terminus of anillin with binding domains and the NLS as indicated (magenta = RBD; green = C2; yellow = PH). Underneath is a ribbon structure of the RBD (magenta) and C2 domain (green). The amino acids of the NLS and those that were mutated (850KK851-DE or 887KK888-DE in the longer isoform) are shown in the box. On the right, immunoblots show the in vitro binding of GST-tagged importin-β (Imp-β) with MBP or MBP-tagged C2 domain (Ctl) vs. the C2 domain with the NLS mutations (NLS). Inputs are shown on the left, and pull downs on the right. (B) Time-lapse images show FRAP of HeLa cells expressing mCherry:tubulin (magenta) and GFP-tagged C-terminus of anillin (green), or with the NLS mutations, or after treatment with MCAK RNAi. The boxed insets show the ROIs that were photobleached in Fire LUTs. The scale bars are 10 or 2 μm for boxed insets. The blue arrow points to overextended microtubules. Indicated times are before (−) or after (+) photobleaching. (C) A graph shows the fraction of fluorescence recovery (y-axis) over time (x-axis; seconds) for control (n = 15), NLS mutant anillin (n = 12), or after MCAK RNAi (n = 8). The green lines indicate the immobile fraction, while the gray lines show the mobile fraction. Bars show SD. The table shows the maximum recovery (y_max), fluorescence recovery time constant (τ), and half-life (τ_1/2) for each condition as indicated. SEM are shown in parentheses. Below the table, cartoon schematics of cells show how mutating the NLS causes a faster recovery vs. MCAK RNAi, which recovers similar to control. However, both the NLS mutant and MCAK RNAi have reduced mobility.