Figure 6.
KIF4A, but not the T799/S801 phosphorylation site mutant, promotes prometaphase chromosome congression.(A) Localization of KIF4A and KID in mitosis. CRISPR-tagged EGFP-KIF4A cells were fixed and stained for endogenous KID. Images were acquired with an Airyscan system. (B) HeLa cells were transfected for 48 h before thymidine synchronisation with control, siKIF4A, siKID, and siKIF4A + KID. After thymidine release and fixation, cells were stained for endogenous KIF4A. Arrows mark chromosomes and chromosome arms showing delayed congression to the metaphase plate. (C) mScarlet-KIF4A WT, FF1220AA, or T799A/S801A were expressed in a CRISPR-edited SMC2-EGFP cell line codepleted for KIF4A and KID. Cells were imaged undergoing mitosis, and representative maximum projections of the SMC2-EGFP signal are shown. Arrows mark chromosomes and chromosome arms showing delayed congression to the metaphase plate. (D) The plot shows the mean time and individual data points from nuclear envelope breakdown (NEBD) to anaphase onset in SMC2-EGFP cells transfected with either siControl or siKIF4A + KID and rescued with KIF4A WT and mutants (siControl n = 22, siKIF4A+KID n = 59, rescue KIF4A WT n = 16, FF1220AA n = 14, and T799A/S801A n = 20). Error bars indicate the SD. A nonparametric Kruskal-Wallis test (P < 0.0001) with a 95% confidence interval was performed, and conditions were compared after analysis by Dunn's test (**, P < 0.001; *, P < 0.1). (E) HeLa cells codepleted of KIF4A and KID and transfected with GFP-KIF4A constructs were fixed and stained for DNA and tubulin. The bar graph displays the extent of chromosome alignment for KIF4A and KID codepleted cells and for cells expressing GFP-KIF4A WT and mutants (siKIF4A+KID n = 103, rescue KIF4A WT n = 98, FF1154AA n = 73, FF1220AA n = 56, T799A/S801A n = 60, and T799A/S801A n = 66). Error bars indicate the SD (for three independent experiments).

KIF4A, but not the T799/S801 phosphorylation site mutant, promotes prometaphase chromosome congression. (A) Localization of KIF4A and KID in mitosis. CRISPR-tagged EGFP-KIF4A cells were fixed and stained for endogenous KID. Images were acquired with an Airyscan system. (B) HeLa cells were transfected for 48 h before thymidine synchronisation with control, siKIF4A, siKID, and siKIF4A + KID. After thymidine release and fixation, cells were stained for endogenous KIF4A. Arrows mark chromosomes and chromosome arms showing delayed congression to the metaphase plate. (C) mScarlet-KIF4A WT, FF1220AA, or T799A/S801A were expressed in a CRISPR-edited SMC2-EGFP cell line codepleted for KIF4A and KID. Cells were imaged undergoing mitosis, and representative maximum projections of the SMC2-EGFP signal are shown. Arrows mark chromosomes and chromosome arms showing delayed congression to the metaphase plate. (D) The plot shows the mean time and individual data points from nuclear envelope breakdown (NEBD) to anaphase onset in SMC2-EGFP cells transfected with either siControl or siKIF4A + KID and rescued with KIF4A WT and mutants (siControl n = 22, siKIF4A+KID n = 59, rescue KIF4A WT n = 16, FF1220AA n = 14, and T799A/S801A n = 20). Error bars indicate the SD. A nonparametric Kruskal-Wallis test (P < 0.0001) with a 95% confidence interval was performed, and conditions were compared after analysis by Dunn's test (**, P < 0.001; *, P < 0.1). (E) HeLa cells codepleted of KIF4A and KID and transfected with GFP-KIF4A constructs were fixed and stained for DNA and tubulin. The bar graph displays the extent of chromosome alignment for KIF4A and KID codepleted cells and for cells expressing GFP-KIF4A WT and mutants (siKIF4A+KID n = 103, rescue KIF4A WT n = 98, FF1154AA n = 73, FF1220AA n = 56, T799A/S801A n = 60, and T799A/S801A n = 66). Error bars indicate the SD (for three independent experiments).

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