![]() Furthermore, CKAP2 increased turbidity at 4☌ ( Figure 1-figure supplement 1D), a temperature where tubulin does not form polymers and preformed microtubules depolymerize quickly in the absence of any stabilizers. In addition, increased microtubule bundling likely contributes to the measured effect. At higher concentrations of CKAP2 turbidity continued to increase ( Figure 1E) indicating that more microtubules are assembled faster. At physiological tubulin levels (8 µM), increasing amounts of CKAP2 cause dose-dependent increase in light scattering and apparent absorbance (turbidity). To test for the ability of CKAP2 to influence microtubule polymer formation, we used recombinantly expressed full-length protein ( Figure 1C) in a bulk light scattering assay ( Figure 1C). Notably, the measured disorder and secondary structure composition in solution for CKAP2 could change upon phosphorylation or interaction with tubulin, microtubules, or other binding partners as it has been shown for many intrinsically disordered proteins ( Uversky, 2019). In contrast, our CD data predominantly show β-sheets as the main secondary structure element for CKAP2 in solution. Interestingly, secondary structure prediction methods as well as AlphaFold predict α-helices for the ordered CKAP2 segments ( Varadi et al., 2022). Another 17% of the protein contains turns, 30% β-sheets, and 8% α-helices. Indeed, we find 45% of CKAP2 to be disordered in solution ( Figure 1B). We performed circular dichroism (CD) to confirm this prediction. Like TPX2, CKAP2 is predicted to be highly intrinsically disordered in solution, and both proteins are significantly enriched in lysines (CKAP2 ~10%, TPX2 ~13%), likely to facilitate interactions with the negatively charged C-terminal tails of tubulin.Ībout 60% of the CKAP2 protein sequence is predicted to be intrinsically disordered ( Figure 1A). About half the protein consists of the CKAP2_C domain (IPR029197), that defines the CKAP2 family. The primary amino acid sequence of CKAP2 is poorly conserved compared to other MAPs like TPX2 and chTOG and does not contain any known microtubule-binding domains ( Figure 1a, Figure 1-figure supplement 1A). The anaphase-promoting complex (APC/C) marks CKAP2 through a conserved, N-terminal KEN-box motif ( Figure 1-figure supplement 1A) for degradation at the end of mitosis to eliminate all CKAP2 from the newly formed daughter cells ( Hong et al., 2007 Seki and Fang, 2007). CKAP2 is regulated by distinct phosphorylation events between different mitotic stages ( Hong et al., 2009). ![]() During G1 interphase, CKAP2 expression is at the detection level and increases at the onset of mitosis ( Seki and Fang, 2007). CKAP2 expression and phosphorylation states are tightly regulated throughout the cell cycle. ![]() The Cytoskeleton-Associated Protein 2 (CKAP2) is an intrinsically disordered protein ( Figure 1a) that localizes to centrosomes and mitotic spindles during cell division ( Seki and Fang, 2007). Given the robustness of spindle assembly as well as the presence of microtubules in cells lacking chTOG ( Gergely et al., 2003), TPX2 ( Aguirre-Portolés et al., 2012), and γ-tubulin ( Strome et al., 2001), it seems likely that additional factors can promote microtubule formation in spindles. Notably, the γ-TURC only weakly increases microtubule nucleation and is thought to require activation of the complex itself or the help of MAPs like the microtubule polymerase XMAP215/chTOG or the nucleation factor TPX2 ( Consolati et al., 2020 Kollman et al., 2010 Moritz et al., 1995 Thawani et al., 2018). Microtubules nucleate primarily from centrosomes as well as several centrosome-independent microtubule nucleation pathways aided by γ-tubulin ring complexes (γ-TURCs) ( Petry and Vale, 2015). In addition, XMAP215/chTOG family members are the only protein shown to speed up microtubule growth by more than a factor of 3 in in vitro assays ( Brouhard et al., 2008). XMAP215/chTOG and TPX2 have been implicated as two major factors in nucleation ( Roostalu et al., 2015). How exactly microtubule nucleation and growth in mitotic spindles is controlled and if all the major assembly factors have been identified remains an open question. Microtubule turnover increases significantly in mitosis resulting from increased microtubule nucleation ( Piehl et al., 2004) in combination with shorter microtubule lifetimes ( Saxton et al., 1984). Assembling mitotic spindles requires a complete rearrangement of the microtubule cytoskeleton, which is driven by the concerted action of microtubule-associated proteins (MAPs) and motor proteins ( Kapoor, 2017). ![]() During mitosis, cells build mitotic spindles to faithfully segregate their chromosomes. ![]()
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