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. 2022 Jun 22;11:e78653. doi: 10.7554/eLife.78653

Figure 2. Pathogenic mutations in KIF22 do not disrupt the localization of the motor.

(A) Immunofluorescence images of HeLa-Kyoto cells expressing KIF22-GFP constructs in prophase (top two rows) and metaphase (bottom two rows). KIF22-GFP was visualized using an anti-GFP antibody. Images are maximum intensity projections in z of five frames at the center of the spindle (metaphase cells) or maximum intensity projections in z of two frames (prophase cells). Fixed approximately 24 hr after treatment with doxycycline to induce expression. Scale bars 5 μm. (B–J) Fluorescence recovery after photobleaching (FRAP) of KIF22-GFP (B–D), KIF22-GFP R149Q (E–G), and KIF22-GFP V475G (H–J) in interphase nuclei (B, E, H) or on metaphase (C, F, I) or anaphase (D, G, J) chromosomes. Bleaching occurred at time 0. Thin lines are traces from individual cells and thick lines represent means. Intensity values are normalized to the KIF22-GFP intensity in the first imaged frame before bleaching. Interphase measurements (B, E, H) obtained from six KIF22-GFP cells from four experiments, nine KIF22-GFP R149Q cells from five experiments, and six KIF22-GFP V475G cells from four experiments. Metaphase measurements (C, F, I) obtained from 6 KIF22-GFP cells from four experiments, 14 KIF22-GFP R149Q cells from five experiments, and 12 KIF22-GFP V475G cells from four experiments. Anaphase measurements (D, G, J) obtained from eight KIF22-GFP cells from four experiments, seven KIF22-GFP R149Q cells from five experiments, and seven KIF22-GFP V475G cells from three experiments. See Figure 2—source data 1.

Figure 2—source data 1. Fluorescence recovery after photobleaching (FRAP).
KIF22-GFP intensities measured in FRAP assays.

Figure 2.

Figure 2—figure supplement 1. HeLa-Kyoto and RPE-1 stable cell lines express mutant KIF22.

Figure 2—figure supplement 1.

(A) Immunofluorescence images of HeLa-Kyoto cells expressing KIF22-GFP under the control of a doxycycline inducible promoter. Images are maximum intensity projections in z of five frames at the center of the spindle. Fixed approximately 24 hr after siRNA transfection and treatment with doxycycline to induce expression. Scale bar 5 μm. KD: knockdown. (B–E) Quantification of KIF22 fluorescence intensity in untreated HeLa-Kyoto cells transfected with control siRNA (B), cells treated with doxycycline to induce expression and transfected with control siRNA (C), untreated cells transfected with KIF22 siRNA (D), and cells treated with doxycycline and transfected with KIF22 siRNA (E) normalized to the mean intensity of uninduced, control knockdown cells (endogenous KIF22 expression level) for each cell line (B). Data in (B, D) are presented with the same y-axis scale as data in (C, E) for comparison (left), and with independently scaled y-axes to show data variability (right). Twenty-seven GFP, 24 KIF22-GFP, 27 KIF22-GFP R149Q, 28 KIF22-GFP P148L, 25 KIF22-GFP P148S, 27 KIF22-GFP R149L, and 30 KIF22-GFP V475G untreated cells transfected with control siRNA (B), 24 GFP, 24 KIF22-GFP, 31 KIF22-GFP R149Q, 30 KIF22-GFP P148L, 27 KIF22-GFP P148S, 30 KIF22-GFP R149L, and 33 KIF22-GFP V475G doxycycline-treated cells transfected with control siRNA (C), 21 GFP, 31 KIF22-GFP, 27 KIF22-GFP R149Q, 32 KIF22-GFP P148L, 22 KIF22-GFP P148S, 22 KIF22-GFP R149L, and 25 KIF22-GFP V475G untreated cells transfected with KIF22 siRNA (D), 26 GFP, 26 KIF22-GFP, 32 KIF22-GFP R149Q, 28 KIF22-GFP P148L, 28 KIF22-GFP P148S, 27 KIF22-GFP R149L, and 33 KIF22-GFP V475G doxycycline-treated cells transfected with KIF22 siRNA (E) from three experiments. (F) DAPI-stained metaphase chromosome spreads from uninduced RPE-1 cell lines with inducible expression of GFP, KIF22-GFP, KIF22-GFP R149Q, or KIF22-GFP V475G. Scale bar 10 μm. Images are representative of three experiments. (G) Numbers of chromosome pairs counted in metaphase spreads prepared from RPE-1 stable cell lines. Dashed line indicates the expected chromosome number for diploid human cells (46). The mode for each cell line is 46. Fifty-five GFP, 58 KIF22-GFP, 53 KIF22-GFP R149Q, and 57 KIF22-GFP V475G spreads from three experiments. (H–K) Quantification of KIF22 fluorescence intensity in untreated RPE-1 cells transfected with control siRNA (H), cells treated with doxycycline to induce expression and transfected with control siRNA (I), untreated cells transfected with KIF22 siRNA (J), and cells treated with doxycycline and transfected with KIF22 siRNA (K) normalized to the mean intensity of uninduced, control knockdown cells for each cell line (H). Twenty-three GFP, 27 KIF22-GFP, 25 KIF22-GFP R149Q, and 27 KIF22-GFP V475G untreated cells transfected with control siRNA (H), 24 GFP, 27 KIF22-GFP, 27 KIF22-GFP R149Q, and 28 KIF22-GFP V475G doxycycline-treated cells transfected with control siRNA (I), 21 GFP, 24 KIF22-GFP, 24 KIF22-GFP R149Q, and 21 KIF22-GFP V475G untreated cells transfected with KIF22 siRNA (J), 24 GFP, 29 KIF22-GFP, 26 KIF22-GFP R149Q, and 24 KIF22-GFP V475G doxycycline-treated cells transfected with KIF22 siRNA (K) from three experiments. For (B–E) and (H–K), bars indicate means. P values from Brown-Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparisons test. P values are greater than 0.05 for comparisons without a marked p value. See Figure 2—figure supplement 1—source data 1.
Figure 2—figure supplement 1—source data 1. HeLa-Kyoto and RPE-1 cell lines.
KIF22 expression levels in HeLa-Kyoto and RPE-1 cell lines. Counts of number of chromosomes in metaphase chromosome spreads prepared from RPE-1 acceptor cells.
Figure 2—figure supplement 2. Pathogenic mutations in KIF22 do not disrupt the localization of the motor in RPE-1 cells.

Figure 2—figure supplement 2.

(A) Immunofluorescence images of RPE-1 cells expressing KIF22-GFP constructs in prophase (top two rows) and metaphase (bottom two rows). KIF22-GFP was visualized using an anti-GFP antibody. Images are maximum intensity projections in z of five frames at the center of the spindle (metaphase cells) or maximum intensity projections in z of three frames (prophase cells). Fixed approximately 18 hr after treatment with doxycycline to induce expression. Scale bars 5 μm. (B) Time-lapse images of fluorescence recovery after photobleaching (FRAP) in HeLa-Kyoto cells expressing KIF22-GFP. Bleaching occurred at time 0, and arrowheads indicate bleached area. Scale bars 10 μm. Images are representative of three or more experiments.