Figure 6.
Figure 6. Live-cell imaging analysis of cells rescued with Aurora B phosphomimetic Cdt1 mutant reveals a severe delay in mitotic progression. (A) Scheme of the time course and still images obtained at the indicated time points during the imaging of live mitotic progression in different stable cell lines rescued with either the plasmid vector (analogous to Cdt1 depletion) or by WT or Aurora B mutant Cdt1 expression. Chromosomes are marked with Hoechst dye. (See also Videos 4, 5, 6, and 7.) (B) Quantification of A. Time (in min) elapsed between the establishment of a metaphase plate and the final fate of each of the imaged mitotic cells, which included delayed anaphase onset or a prolonged mitotic arrest, was plotted on the y-axis for each of the samples imaged live as indicated. Statistical significance assessed by the two-sided unpaired nonparametric Student’s t test. (C) Comparative tabulation of MT-binding parameters and phospho-regulation of relevant kinetochore-associated proteins from vertebrates. (D) Working model depicting the role of Cdt1 in providing an additional kMT-attachment site besides the CH and the N-terminal tail domain of the Hec1 subunit of the Ndc80 complex. The CH domains in Hec1 and Nuf2 along with the Hec1 unstructured tail bind MTs. The unstructured ∼40-aa loop region of Hec1 recruits Cdt1 by interacting with the N-terminal 1–320 aa of Cdt1 (Varma et al., 2012). This induces a conformational shift in the Ndc80 complex in a manner that is dependent on Cdt1 binding to MTs. In prometaphase, when kinetochores are not bi-oriented, Aurora B kinase is active to destabilize the erroneous attachments by phosphorylating several kinetochore proteins including Ndc80 (1), Ska (2), and Cdt1 (3) shown in the present study, all of which bind to MTs. Upon phosphorylation, these proteins exhibit reduced affinity for MTs. As observed for the Ska complex, which cannot interact with the KMN network effectively upon its phosphorylation by Aurora B (4); whether Cdt1~P can dock on to the Hec1 loop (5) with similar efficiency to unphosphorylated Cdt1 is still undetermined. In metaphase, when the Aurora B gradient diminishes at kinetochores, Cdt1 becomes proficient to bind to MTs (6), thus providing an additional site for kMT attachment besides the CH and the tail domains of Ndc80. Whether Cdt1 can interact with the Ska complex in its free or loop-bound state and if this interaction is dependent on their phosphorylation status (7 and 8) is yet to be determined. The model also depicts the possibility of interaction of other MAPs with the Hec1 loop, either alone or with the help of Cdt1 or Ska or both (9), as shown using dashed arrows.

Live-cell imaging analysis of cells rescued with Aurora B phosphomimetic Cdt1 mutant reveals a severe delay in mitotic progression. (A) Scheme of the time course and still images obtained at the indicated time points during the imaging of live mitotic progression in different stable cell lines rescued with either the plasmid vector (analogous to Cdt1 depletion) or by WT or Aurora B mutant Cdt1 expression. Chromosomes are marked with Hoechst dye. (See also Videos 4, 5, 6, and 7.) (B) Quantification of A. Time (in min) elapsed between the establishment of a metaphase plate and the final fate of each of the imaged mitotic cells, which included delayed anaphase onset or a prolonged mitotic arrest, was plotted on the y-axis for each of the samples imaged live as indicated. Statistical significance assessed by the two-sided unpaired nonparametric Student’s t test. (C) Comparative tabulation of MT-binding parameters and phospho-regulation of relevant kinetochore-associated proteins from vertebrates. (D) Working model depicting the role of Cdt1 in providing an additional kMT-attachment site besides the CH and the N-terminal tail domain of the Hec1 subunit of the Ndc80 complex. The CH domains in Hec1 and Nuf2 along with the Hec1 unstructured tail bind MTs. The unstructured ∼40-aa loop region of Hec1 recruits Cdt1 by interacting with the N-terminal 1–320 aa of Cdt1 (Varma et al., 2012). This induces a conformational shift in the Ndc80 complex in a manner that is dependent on Cdt1 binding to MTs. In prometaphase, when kinetochores are not bi-oriented, Aurora B kinase is active to destabilize the erroneous attachments by phosphorylating several kinetochore proteins including Ndc80 (1), Ska (2), and Cdt1 (3) shown in the present study, all of which bind to MTs. Upon phosphorylation, these proteins exhibit reduced affinity for MTs. As observed for the Ska complex, which cannot interact with the KMN network effectively upon its phosphorylation by Aurora B (4); whether Cdt1~P can dock on to the Hec1 loop (5) with similar efficiency to unphosphorylated Cdt1 is still undetermined. In metaphase, when the Aurora B gradient diminishes at kinetochores, Cdt1 becomes proficient to bind to MTs (6), thus providing an additional site for kMT attachment besides the CH and the tail domains of Ndc80. Whether Cdt1 can interact with the Ska complex in its free or loop-bound state and if this interaction is dependent on their phosphorylation status (7 and 8) is yet to be determined. The model also depicts the possibility of interaction of other MAPs with the Hec1 loop, either alone or with the help of Cdt1 or Ska or both (9), as shown using dashed arrows.

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