During mitosis, cells build mitotic spindles to faithfully segregate their chromosomes. 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). 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).

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. XMAP215/chTOG and TPX2 have been implicated as two major factors in nucleation (Roostalu et al., 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).

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). 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).

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.

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). CKAP2 expression and phosphorylation states are tightly regulated throughout the cell cycle. During G1 interphase, CKAP2 expression is at the detection level and increases at the onset of mitosis (Seki and Fang, 2007). CKAP2 is regulated by distinct phosphorylation events between different mitotic stages (Hong et al., 2009). 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).

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Protein purification gel.

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Knock-down of CKAP2 in cells interferes with proper spindle assembly and often results in multipolar spindles and misaligned chromosomes (Case et al., 2013). Overexpression of CKAP2 promotes cancer formation (Guo et al., 2017; Yu et al., 2015) and correlates with severity of the disease (Hayashi et al., 2014). Consequently, CKAP2 is a marker for the diagnosis and prognosis of several types of cancer (Hayashi et al., 2014). CKAP2 has been previously described as a potential microtubule stabilizer in cells (Jin et al., 2004; Tsuchihara et al., 2005), though it is not known whether CKAP2 directly binds to microtubules or how it affects microtubule dynamics. We were intrigued by the cell biology of CKAP2, the severe spindle phenotypes, the clinical relevance, and the limited molecular understanding of this protein. Therefore, we wanted to investigate how CKAP2 impacts microtubule dynamics.

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). About half the protein consists of the CKAP2_C domain (IPR029197), that defines the CKAP2 family. 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.

About 60% of the CKAP2 protein sequence is predicted to be intrinsically disordered (Figure 1A). We performed circular dichroism (CD) to confirm this prediction. Indeed, we find 45% of CKAP2 to be disordered in solution (Figure 1B). Another 17% of the protein contains turns, 30% β-sheets, and 8% α-helices. Interestingly, secondary structure prediction methods as well as AlphaFold predict α-helices for the ordered CKAP2 segments (Varadi et al., 2022). In contrast, our CD data predominantly show β-sheets as the main secondary structure element for CKAP2 in solution. 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).

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). At physiological tubulin levels (8 µM), increasing amounts of CKAP2 cause dose-dependent increase in light scattering and apparent absorbance (turbidity). At higher concentrations of CKAP2 turbidity continued to increase (Figure 1E) indicating that more microtubules are assembled faster. In addition, increased microtubule bundling likely contributes to the measured effect. Furthermore, CKAP2 increased turbidity at 4°C (Figure 1—figure supplement 1D), a temperature where tubulin does not form polymers and preformed microtubules depolymerize quickly in the absence of any stabilizers. Neither the mNeonGreen (mNG) nor the 6-His affinity tag had any impact on the ability of CKAP2 to aid microtubule formation (Figure 1—figure supplement 1B,C).

The bulk turbidity assay cannot distinguish between effects of CKAP2 on microtubule nucleation, growth, or stabilization. To test how CKAP2 enhances tubulin polymer formation, we reconstituted microtubule assembly in vitro using total internal reflection fluorescence (TIRF) microscopy (Gell et al., 2010). In this assay, we observe individual microtubules growing from guanosine-5′-[(α, β)-methyleno]triphosphate (GMPCPP)-stabilized microtubule ‘seeds’ (Figure 2A). At 8 µM tubulin, the lower end of estimated cellular tubulin concentrations and in the presence of CKAP2, observation of individual growth events was only possible at very low CKAP2 concentrations (≤50 nM) due to high levels of spontaneous nucleation beyond that point. The microtubule growth rate increased linearly with CKAP2 at these concentrations (Figure 2B and Figure 2—figure supplement 1A).

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The data and analysis for microtubule growth rates for different concentrations of Cytoskeleton-Associated Protein 2 (CKAP2).

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To measure microtubule growth across a wide range of CKAP2 concentrations and determine apparent tubulin on-and-off rates (k_a and k_d) we reduced the tubulin concentration about 100-fold (to 50–300 nM) to mitigate spontaneous nucleation and observe individual microtubule growth events. The resulting growth curves (Figure 2C and Figure 2—figure supplement 1B) illustrate microtubule elongation at substantially lower tubulin concentrations than previously observed (Wieczorek et al., 2015). Microtubules polymerized with an apparent assembly rate constant (k_a) of 2.6 ± 0.06 dimers μM⁻¹ s⁻¹ in controls and 142 ± 3.3 dimers μM⁻¹ s⁻¹ in the presence of 500 nM CKAP2, representing a 54-fold increase in k_a (Figure 2D). By comparison, members of the XMAP215/chTOG family of microtubule polymerases achieve about fivefold maximal increase in k_a (Brouhard et al., 2008). We observed a 42-fold increase in k_a for 125 nM CKAP2. Increasing the CKAP2 concentration beyond 500 nM did not further increase growth rates. We observe a smaller effect (~threefold) on the tubulin off-rate k_d (Figure 2E).

From the growth curves in Figure 2C and the spontaneous nucleation we observe at quasi-physiological concentrations of CKAP2 and tubulin, it is apparent that CKAP2 is able to shift the critical concentration (C_c) for microtubule elongation into the low nanomolar range (from 2.89 ± 0.12 to 0.02 ± 0.002 μM with 500 nM CKAP2) (Figure 2F).

To characterize the nucleation behaviour of microtubules we measured the probability of a seed to nucleate a microtubule over time (Wieczorek et al., 2015; Figure 3A). At quasi-physiological tubulin levels microtubules nucleated from seed templates faster in the presence of as ≥25 nM CKAP2 (Figure 3B). When we explored a wider range of CKAP2 and tubulin concentrations, we found a very strong promotion of templated nucleation by CKAP2 even at 100-fold lower tubulin concentrations compared to controls (Figure 3C and Figure 3—figure supplement 1A, E). The tubulin concentration for the half-maximal probability to nucleate within 1 min is reduced from 6.85 ± 0.48 µM in controls to 0.05 ± 0.01 µM for 0.5 µM CKAP2-mNG. CKAP2 facilitates templated nucleation from seeds at tubulin concentrations as low as 50 nM. Further, we quantified the spontaneous nucleation, that is in the absence of templates, in our microscopy assay by counting the number of microtubules formed after 2 min (Figure 3D; Roostalu et al., 2015). We found that CKAP2 dramatically increases spontaneous nucleation with a shift in critical tubulin concentration from 25.4 ± 1.7 to 0.25 ± 0.02 μM (Figure 3E and Figure 3—figure supplement 1B). CKAP2 therefore promotes both templated and spontaneous nucleation. In the presence of CKAP2, the C_c of spontaneous nucleation, as well as nucleation from templates, are lowered up to 100-fold.

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The data and analysis for templated and spontaneous microtubule nucleation for different concentrations of Cytoskeleton-Associated Protein 2 (CKAP2).

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A hallmark of microtubule dynamic instability is their capacity to spontaneously switch from growth to shrinkage, a behaviour termed catastrophe (Mitchison and Kirschner, 1984). When microtubules were grown in the presence of CKAP2, catastrophe levels were severely reduced (Figure 4A). We quantified this effect and determined that CKAP2 lowers catastrophe frequencies in a concentration-dependent manner, reducing them to near zero at 50 nM CKAP2 (Figure 4B). In the presence of higher concentrations of CKAP2, catastrophe frequency was at least an order of magnitude lower than in controls (Figure 4C and Figure 4—figure supplement 1A, B, C). The only other family of MAPs with a comparable effect on microtubule catastrophes are the cytoplasmic linker-associated proteins (CLASPs) (Aher et al., 2018; Lawrence et al., 2018; Majumdar et al., 2018).

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The data and analysis for catastrophe frequency measurements.

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A prediction for a potential cytoskeletal growth factor is the interaction with both the growing filament end and free monomers, as it has been shown for members of the chTOG/XMAP215 family for microtubules (Ayaz et al., 2014) and formins for actin (Kovar et al., 2006). Using size exclusion chromatography, we do not find any significant interaction of CKAP2 and tubulin in solution (Figure 5A). In contrast, CKAP2 is able to recruit soluble tubulin to microtubules. Microtubule lattice-bound CKAP2 recruits free Atto-663 tubulin to GMPCPP microtubule seeds (Figure 5b, c). CKAP2 therefore possesses a distinct microtubule lattice as well as likely one or multiple tubulin-binding sites. Recruitment of tubulin by lattice-bound CKAP2 could therefore play a role in promoting microtubule nucleation and growth.

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The data and analysis for tubulin recruitment by Cytoskeleton-Associated Protein 2 (CKAP2) and shrinkage rate for guanosine-5′-[(α,β)-methyleno]triphosphate (GMPCPP) microtubules in the presence and absence of CKAP2.

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Many of the proteins promoting microtubule nucleation and growth possess high-affinity-binding sites at the plus end of dynamic microtubules (e.g. XMAP215/chTOG, DCX, TPX2). Here, XMAP215/chTOG is thought to help the addition of tubulin dimers to the growing end (Howard and Hyman, 2007). To determine if CKAP2 interacts with the growing microtubule end, we performed our TIRF microscopy assay at subsaturating CKAP2 concentrations. Indeed, at low protein concentrations (6.5 nM), CKAP2 tracks microtubule ends (Figure 5D). At higher concentrations (12.5 nM) end tracking is obscured by lattice-binding reminiscent of end tracking behaviour observed for the microtubule nucleation factors DCX and TPX2 (Figure 5D; Bechstedt and Brouhard, 2012, Roostalu et al., 2015). CKAP2 shows higher affinity to the dynamic microtubule lattice over the GMPCPP lattice, unlike TPX2 (Roostalu et al., 2015). Similar to TPX2 (Roostalu et al., 2015) and DCX (Bechstedt and Brouhard, 2012), CKAP2 recognizes microtubule curvature (Figure 5e, f), which is thought to correlate with the high-affinity-binding site of these proteins at the microtubule end (Bechstedt et al., 2014; Roostalu et al., 2015). In contrast, members of the XMAP215/chTOG family display robust autonomous end tracking at all physiologically relevant concentrations and do not recognize lattice curvature (Brouhard et al., 2008; Roostalu et al., 2015).

We next tested if CKAP2 can promote the removal of tubulin dimers from the lattice in the absence of tubulin. Catalyzing this reverse reaction of microtubule growth is a hallmark of bona fide microtubule polymerases of the XMAP215/chTOG family (Brouhard et al., 2008). We did not observe any depolymerization over ~10 hr (Figure 5g, h and Figure 5—figure supplement 1A) when we incubated GMPCPP-stabilized microtubules with CKAP2 at concentrations as high as 1 µM, while controls without CKAP2 displayed an expected depolymerization rate of about 0.013 ± 0.003 µm/min (Figure 5g, h). CKAP2 does not catalyze the reverse reaction. On the contrary, CKAP2 strongly reduces the tubulin off-rate.

Our results show that CKAP2 has a strong effect on microtubule growth, nucleation, and stabilization in vitro.

Cellular concentrations of CKAP2 in Xenopus eggs have been measured at about 60 nM in conjunction with 8.5 µM tubulin (Wühr et al., 2014). At these quasi-physiological CKAP2 and tubulin concentrations (assuming 60 nM and 8 µM) CKAP2 has a strong effect on the microtubule growth rate (~fivefold) in our assays (Figure 1B). Further, templated nucleation is almost instantaneous (Figure 3B) and spontaneous nucleation is detectable. Catastrophe frequency is approaching zero at this concentration regime (Figure 4B). Therefore, in cells, CKAP2 could increase microtubule nucleation, growth rates, and suppress catastrophe. Interestingly, at quasi-cellular CKAP2 concentrations, the effects on microtubule dynamics in our in vitro assays are not fully saturated. Hence, cells may be susceptible to increased levels of CKAP2 as they have been observed in several types of cancer.

Notably, we purify CKAP2 from E. coli likely in an unphosphorylated state. In cells, sharp phosphorylation events are observed for CKAP2 between different mitotic stages which could modify and shift the effect of CKAP2 on different microtubule dynamics parameters (Hong et al., 2008).

At full biochemical capacity of CKAP2, we observed a 100-fold increase in spontaneous and templated nucleation as well as a 54-fold increase in apparent tubulin on rate k_a, and a complete suppression of microtubule catastrophe. Combined, these effects result in CKAP2 being the most potent microtubule assembly factor characterized to date.

Mechanistically, CKAP2 combines characteristics of microtubule nucleators (TPX2, DCX) as well as polymerases (XMAP215/chTOG) and anti-catastrophe factors (CLASPs). Like TPX2 and DCX, CKAP2 aids spontaneous nucleation. All three proteins display end tracking behaviour at low concentrations as well as lattice curvature recognition. These characteristics seem to be a hallmark of microtubule nucleation factors and might point towards the structural nature of the nascent nucleus. Similar to TPX2, CKAP2 is largely intrinsically disordered and dampens tubulin off-rates and microtubule dynamicity (Reid et al., 2016; Roostalu et al., 2015). A potential mechanism for nucleation by CKAP2 is the stabilization of the nascent microtubule nucleus (Roostalu and Surrey, 2017). Similar to the CLASP anti-catastrophe factors, CKAP2 lowers the catastrophe frequency to virtually zero (Aher et al., 2018; Lawrence et al., 2018; Majumdar et al., 2018), allowing for continued microtubule growth even at nanomolar concentrations of tubulin.

Other MAPs that stabilize the microtubule lattice like DCX (Moores et al., 2006), TPX2 (Reid et al., 2016), and CLASPs (Lawrence et al., 2018; Yu et al., 2016), as well as stabilizing drugs like paclitaxel (Zanic et al., 2013) or Guanosine-5'-triphosphate (GTP) analogues like GMPCPP (Hyman et al., 1992), do not severely impact the microtubule growth curve. Therefore, lattice stabilization alone cannot account for the severe impact of CKAP2 on the apparent tubulin on rate k_a.

Unlike TPX2, DCX, and CLASPs, CKAP2 also acts, in part, like a polymerase. Similar to known polymerases of the XMAP215/chTOG family, microtubule-bound CKAP2 recruits tubulin and increases the association rate constant (k_a) at the microtubule end (Brouhard et al., 2008; Gard and Kirschner, 1987). In contrast to XMAP215/chTOG family members, CKAP2 is able to push microtubule growth to the physical limit as defined by diffusion of the tubulin dimer to the growing microtubule end (Odde, 1997; Zanic et al., 2013). Unlike a polymerase, CKAP2 does not catalyze the reverse reaction of tubulin polymerization in the absence of tubulin substrates. CKAP2 either does not operate as a bona fide polymerase, or the depolymerase activity is masked by the lattice stabilization activity of CKAP2. In contrast to XMAP215/chTOG, CKAP2 does not interact with GDP tubulin in size exclusion chromatography experiments.

In many ways, microtubule growth in the presence of CKAP2 resembles that of microtubules grown in the presence of the slowly hydrolysable GTP analogue, GMPCPP. With GMPCPP, microtubules nucleate at nanomolar concentrations of tubulin and are relatively stable (Desai and Mitchison, 1997; Hyman et al., 1992). However, GMPCPP does not increase apparent on rate k_a, like CKAP2.

How does CKAP2 combine multiple functionalities? The protein is about one third of the size of XMAP215/chTOG, about half the size of CLASPs, and is lacking any known microtubule or tubulin-binding domains. We believe that the highly disordered nature of CKAP2 (Figure 1a, b) contributes to its ability to dynamically interact with the microtubule lattice as well as tubulin and impact microtubule assembly to an extreme extent. The advantage of disordered over folded domains is the absence of a structured, tight binding conformation to a substrate or intermediate state. In case of a microtubule growth factor, lack of a fixed structure would allow for fast molecular recognition of the tubulin dimer as well as for rapid unbinding, that is letting go of the dimer when incorporated (Uversky and Dunker, 2013). Binding of CKAP2 to microtubules likely causes the adoption of a secondary structure of part of the protein. Such structural rearrangements could explain the lack interaction with tubulin in solution in contrast to lattice-bound CKAP2. Structural as well as structure–function studies are needed to determine the interaction domains with microtubules and tubulin and provide us with further mechanistic insight.

Even small changes in microtubule assembly rates are correlated with increased levels of chromosomal instability (CIN) in cells (Ertych et al., 2014). CIN can provide the high adaptation capability necessary for tumor initiation and progression. Correspondingly, it has been shown that reducing microtubule growth rates in cancer cells by chemical or genetic manipulation suppresses CIN (Ertych et al., 2014). If the capacity of CKAP2 to promote microtubule nucleation and growth we find in vitro even partially translates into cells, we would expect significant changes in microtubule dynamics upon reduced or increased CKAP2 protein levels. Upregulation of a potent growth factor like CKAP2 as observed in many cancers could therefore be directly responsible for CIN, and subsequently for aneuploidy, and malignancies.

Cells need to tightly regulate the expression and activity of a potent microtubule growth factor like CKAP2. Known regulatory measures include rapid degradation by APC (Seki and Fang, 2007) as well as distinct phosphorylation and dephosphorylation events in between different stages of the cell cycle (Hong et al., 2008; Hong et al., 2009). Phosphorylation could alter the effects of CKAP2 on microtubule dynamics and tune CKAP2 functionality between nucleation, stabilization, and growth.

Together, our findings identify the mitotic spindle protein CKAP2 as the most potent microtubule growth factor to date. The protein displays unprecedented impact on microtubule growth, nucleation, and catastrophe. Our results suggest an explanation for the observed CKAP2 knock-down spindle phenotypes and the oncogenic potential through the misregulation of microtubule dynamics.

All data generated or analysed during this study are included in the manuscript and supporting files.

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Author details

1. Thomas S McAlear

   Department of Anatomy and Cell Biology, McGill University, Montréal, Canada

   Contribution

   Data curation, Formal analysis, Investigation, Methodology, Software, Visualization

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-6097-0103

2. Susanne Bechstedt

   Department of Anatomy and Cell Biology, McGill University, Montréal, Canada

   Contribution

   Conceptualization, Funding acquisition, Investigation, Methodology, Project administration, Supervision, Writing - original draft

   For correspondence

   susanne.bechstedt@mcgill.ca

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-4706-9975

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