A Phosphorylation Switch Governs KIF11’s Mechanical Output During Mitosis

Figure 1.. Phospho-mimetic mutations in KIF11 alter motor force production, velocity, and MT interaction dynamics. Force, velocity, and MT binding kinetics of single-molecule KIF11 pseudo-dimer constructs were measured using an optical trap. (A) Plot of force-velocity curves for the indicated constructs. N = 47, 65, 88 and 70 max force producing traces selected from 14, 9, 12 and 11 single-molecule beads of WT, 211E, 213E and 125E KIF11 motors. Error bars represent SEM for all traces at the given force bin. (B) Plot of peak forces measured for each of the indicated constructs. (C) Plot of MT bound times measured for each of the indicated constructs. Data points represent individual single-molecule measurements. (D) Plot of bead rebinding intervals measured for each of the indicated constructs. (B-C) Data points represent all single-molecule events of motor activity detected in the trap for the beads in 1 A. Mean ± SEM is indicated for each data set. Data were compared using student t-test (* p<0.05 *** p<0.001).

Figure 2.. The Y211E phospho-mimetic mutation significantly slows KIF11’s neck-linker (NL) docking. NL-docking was measured by time-resolved FRET. (A) Total fluorescence transients of MT-bound AEDANS/DDPM-labeled KIF11NL-WT (magenta) or KIF11NL-Y211E (cyan) following mixing with 5 mM MgATP. (B) Mole-fraction transients obtained by analyzing time-resolved fluorescence decays to a two-state docked/undocked model. Single exponential fits to the transients (black lines). WT k = 22.43 s−1 (22.89 to 23.99 95% CI), Y211E k = 8.1 s−1 (7.917 to 8.298 95% CI). (C) Rate constants from N = 3 biological replicates measuring ATP-driven neck-linker docking measured by total fluorescence or model dependent time-resolved FRET mole fraction analysis.

Figure 3.. The KIF11 Y211E and Y211F mutations disrupt spindle formation in RPE1 cells. Spindle morphology was assessed in fixed RPE-1 cells expressing siRNA-resistant KIF11-mEm WT, the phospho-mimetic Y211E, or the non-phosphorylatable Y211F constructs. (A) Representative immunofluorescence images taken from each cell line, treated with either control or KIF11 siRNA. Cells were stained for GFP (green), DNA (DAPI blue), tubulin (red) or KIF11. Cells were fixed 24 hr after inducing expression of KIF11-mEm constructs with addition of doxycycline, and at least 24 hr incubation with siRNAs. (B) Mitotic cells were counted and categorized as having bipolar spindles (in metaphase), disorganized spindles, multipolar, or monopolar spindles. The percentage of cells with each spindle phenotype was determined for each condition. Cell counts for each cell line: RPE1-KIF11-WT-mEM - Control KD: 83; Control KD (+KIF11-WT-mEM): 73; KIF11 KD: 161; KIF11 KD (+KIF11-WT-mEM): 84; n = 3 experiments. RPE1-KIF11-Y211E-mEm - Control KD (+KIF11-Y211E-mEm): 70; KIF11 KD (+KIF11-Y211E-mEm): 247; n = 3 experiments. KIF11-Y211F-mEm - Control KD (+KIF11-Y211F-mEm): 38; KIF11 KD (+KIF11-Y211F-mEm): 63; n = 3 experiments.…

Figure 4.. Expression of KIF11 Y211E slows spindle pole separation, while Y211F reduces maximum spindle length. Spindle polar separation was tracked in live movies of RPE1-KIF11-mEm expressing cells. Cells were induced to express KIF11-mEm constructs for 24 hr before live imaging with the addition of doxycycline and treated with control or KIF11 siRNA. Cells were also treated with monastrol overnight to inhibit KIF11 motors. This treatment collapses all spindles to monopoles and arrests cells in mitosis. Tubulin was labeled using SiR-tubulin several hours prior to imaging. Monastrol was washed out immediately before the start of imaging. Spindle pole separation was tracked using the SiR-tubulin signal after monastrol washout in individual cells at 30 sec intervals. (A) Representative stills of SiR-tubulin signal to show spindle pole separation after monastrol washout in each cell line, treated with either control or KIF11 siRNA. (B) The distance between spindle poles was tracked for cells in each line after monastrol washout, either in control siRNA (left) or KIF11 siRNA (right). (C) The average velocity of spindle pole separation was calculated for cells in each line after monastrol…

Figure 5.. KIF11 Y211E displays faster turnover in mitotic spindles. KIF11-mEm construct turnover was measured in mitotic RPE1 cells using fluorescence recovery after photobleaching (FRAP). RPE1 cells were induced to express KIF11-mEm-WT, Y211E, or Y211F constructs with 24 hr doxycycline treatment, and either control or KIF11 siRNA. Using a 405 nm laser, cells were photobleached in a small region of interest on spindle microtubules, and fluorescence intensity recovery over the following 20 sec was measured. (A) Fluorescence intensity traces after photobleaching. Traces have been corrected for photobleaching due to image acquisition. (B) Half times (t1/2) calculated for each KIF11-mEm construct. Cell counts for each cell line: RPE1-KIF11-WT-mEM - Control KD: 16; KIF11 KD: 11 bipolar, 7 monopolar; n = 3 experiments. RPE1-KIF11-Y211E-mEm – KIF11 KD: 21, n = 3 experiments. KIF11-Y211F-mEm - KIF11 KD: 16 bipolar, 16 monopolar, n = 3 experiments. Data were compared by running a one-way ANOVA with Šídák’s test for multiple comparisons: p-value style: <0.05 (*), <0.01 (**), <0.001 (***), and <0.0001 (****). If no significance is indicated, the result was not significant (>0.05).

Figure 6.. KIF11 Y211E does not impede microtubule sliding by WT KIF11. (A) Plot shows Cy-3 labeled MT gliding velocities in the presence of the indicated ratios of KIF11 WT and KIF11 Y211E pseudo-dimers. Data points represent the average velocities of individual motile MTs tracked for 200/1000 consecutive images. Number of MTs tracked, n = 32,17, 31, 33, 19 for WT:Y211E ratios of 1:0, 0:1, 1:1, 5:1, 10:1 respectively. Data were compared using a student t-test student (** p<0.01).