1. Biochemistry and Chemical Biology

The chromokinesin Kid (KIF22) forms a homodimer, moves processively along microtubules, and transports double-stranded DNA

  1. Shinsuke Niwa  Is a corresponding author
  2. Natsuki Furusaki
  3. Tomoki Kita
  4. Yuki Suzuki
  5. Kyoko Chiba  Is a corresponding author
  1. Frontier Research Institute for Interdisciplinary Sciences (FRIS), Tohoku University, Japan
  2. Graduate School of Life Sciences, Tohoku University, Japan
  3. Mie University, Graduate School of Engineering, Japan
https://doi.org/10.7554/eLife.102828.3
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  1. Shinsuke Niwa
  2. Natsuki Furusaki
  3. Tomoki Kita
  4. Yuki Suzuki
  5. Kyoko Chiba
(2026)
The chromokinesin Kid (KIF22) forms a homodimer, moves processively along microtubules, and transports double-stranded DNA
eLife 13:RP102828.
https://doi.org/10.7554/eLife.102828.3
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eLife Assessment

This important study clarifies the mechanism by which the kinesin-10 motor protein, chromosome-associated kinesin, Kid (KIF22), enables chromosome movement during mitosis, demonstrating that human and Xenopus Kid proteins function as processive, homodimeric kinesins capable of processive microtubule plus-end motility. The convincing work highlights that Kid can recruit and transport duplex DNA along microtubules via its conserved C-terminal DNA-binding domain, revising our understanding of chromokinesins' role in chromosome motility during mitosis. It will be of interest to those in the molecular motor community working at the molecular, cellular, and organismal levels.

https://doi.org/10.7554/eLife.102828.3.sa0

Abstract

During prometaphase in mitosis, chromosomes are pushed toward the spindle equator. The chromokinesin Kid, also known as KIF22, moves chromosomes along spindle microtubules during prometaphase. Kid has long been considered a monomeric and nonprocessive motor, different from typical kinesins. In this study, we demonstrate that the full-length Kid forms a homodimer and moves processively along microtubules. A conserved coiled-coil domain within the stalk region of Kid is sufficient for homodimer formation and is required for the processivity of Kid. Furthermore, the neck linker and coiled-coil domains of Kid could add processive activity to the motor domain of KIF1A, suggesting that Kid contains a functional neck linker and dimerization capability, a prerequisite for the processivity of kinesin motor domains. The full-length Kid, containing a helix–hairpin–helix domain, can transport double-stranded DNA along microtubules in vitro. AlphaFold3 prediction suggests that the dimerization of Kid stabilizes the association with DNA. These findings collectively suggest the reclassification of Kid as a processive and dimeric motor that transports DNA along microtubules.

Introduction

Microtubules are reorganized to form a bipolar spindle as cells enter mitosis. In the prometaphase of mitosis, chromosomes are transported along the microtubules toward the spindle equator (Rieder et al., 1986). This process, known as chromosome congression, requires kinesin-4, kinesin-10, and kinesin-12 class of motor proteins (Bieling et al., 2010; Iemura and Tanaka, 2015; Wordeman, 2010). The mechanical forces that move chromosomes toward the spindle equator are called polar ejection forces (Brouhard and Hunt, 2005; Levesque and Compton, 2001; Rieder et al., 1986). The Kinesin-like DNA-binding protein (Kid), belonging to the kinesin-10 family and also known as KIF22, serves as a main molecular motor that transports chromosomes and generates polar ejection forces (Brouhard and Hunt, 2005; Levesque and Compton, 2001; Ohsugi et al., 2003; Thompson et al., 2022; Tokai et al., 1996; Wandke et al., 2012). Structurally, Kid contains a kinesin motor domain (Yajima et al., 2003), a coiled-coil domain (Shiroguchi et al., 2003), and a DNA-binding domain (Tokai et al., 1996; Figure 1A). In mitosis, the DNA-binding domain of Kid binds along chromosome arms (Antonio et al., 2000; Funabiki and Murray, 2000; Levesque and Compton, 2001). Using the motor domain, Kid transports chromosomes (Bieling et al., 2010; Brouhard and Hunt, 2005).

Figure 1 with 3 supplements see all
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Kid is a processive motor.

(A) Schematic illustration of the domain organization in Xenopus Kid tagged with a fluorescent protein mScarlet (XKidFL) and human Kid tagged with mNeonGreen (hKidFL). The calculated molecular weights of the fusion proteins are indicated on the right. (B) Representative SDS-PAGE analysis of purified XKidFL and hKidFL fusion proteins. The proteins are visualized using a Stain-Free gel. The molecular weight standards are indicated on the left side of the SDS-PAGE images. (C, D) Representative kymographs showing the motility of XKidFL at 20 pM (C) and hKidFL at 20 pM (D) both in the presence of 2 mM ATP. Scale bars: horizontal, 10 µm; vertical, 60 seconds. (E) Dot plots showing the velocity of XKidFL and hKidFL. Each dot shows a single datum point. Green bars represent mean ± S.D. n=51 and 52, respectively. (F) Dot plots showing the run length of XKidFL and hKidFL. Each dot shows a single datum point. Green bars represent mean ± S.D. n=51 and 52, respectively.

Figure 1—source data 1

Velocity and run length data for Figure 1E and F.

https://cdn.elifesciences.org/articles/102828/elife-102828-fig1-data1-v1.xlsx
Figure 1—source data 2

Original file for SDS-PAGE data for Figure 1B.

https://cdn.elifesciences.org/articles/102828/elife-102828-fig1-data2-v1.zip
Figure 1—source data 3

Original file for SDS-PAGE data for Figure 1B indicating the relevant bands.

https://cdn.elifesciences.org/articles/102828/elife-102828-fig1-data3-v1.zip

A single kinesin molecule can move for hundreds of steps along a microtubule without dissociating (Hackney, 1995; Hancock and Howard, 1998). This property, called processivity, requires dimerization of kinesins (Hancock and Howard, 1998). However, Kid has long been regarded as a monomeric and nonprocessive motor, which can move along microtubules in only a single step (Shiroguchi et al., 2003; Yajima et al., 2003). Consistent with these findings, Kid is unique within the kinesin superfamily due to the lack of the conventional neck coiled-coil domain (Shiroguchi et al., 2003), an element that determines the length of the neck linker essential for the coordinated movement of the two motor domains (Case et al., 2000; Isojima et al., 2010; Yildiz et al., 2008). On the other hand, a previous study showed that the coiled-coil domain of Kid interacts with full-length Kid in GST pull-down assays, suggesting this domain may mediate dimerization of Kid (Pike et al., 2018). In addition, full-length human Kid was reported to move processively along microtubules (Stumpff et al., 2012) and, Kid was proposed to be a chemically processive motor (Walker et al., 2019). However, these studies did not show the oligomeric state of Kid, nor did they provide detailed information on its motility characteristics and parameters. A Drosophila kinesin-10 motor NOD, an orthologue of Kid, is also characterized as a monomeric and nonprocessive motor (Matthies et al., 2001). Similar to Kid, NOD has a DNA-binding domain at the tail domain (Afshar et al., 1995). NOD shows a processive movement only when the protein is forcedly dimerized by the addition of an artificial coiled-coil domain (Ye et al., 2018). Nevertheless, Kid acts as an active motor in microtubule gliding assays and cargo transport assays, both of which detect movement generated by multiple Kid motors (Bieling et al., 2010; Li et al., 2016; Shiroguchi et al., 2003; Takagi et al., 2013). Collectively, it has been widely assumed that Kid, unlike other kinesins, transports chromosomes by their cooperative action of many nonprocessive monomers (Brouhard and Hunt, 2005; Iemura and Tanaka, 2015; Takagi et al., 2013; Thompson et al., 2022).

Recently, we and others have analyzed the properties of full-length kinesins (Chiba et al., 2022; Chiba et al., 2019; Fan and McKenney, 2022; Wang et al., 2022). Notably, these studies have found that monomeric kinesins, including KIF1A, UNC-104, and KIF13B, are converted to a dimer when activated (Chiba et al., 2023; Fan and McKenney, 2022; Kita et al., 2024; Tomishige et al., 2002). For instance, in the autoinhibited and inactive state, KIF1A and UNC-104 are monomeric (Kita et al., 2024; Tomishige et al., 2002). The monomeric motor domain of UNC-104 and KIF1A has a plus-end directed motor activity but exhibits one-dimensional diffusion, meaning that the efficiency is low (Okada et al., 2003; Tomishige et al., 2002). Upon the release of autoinhibition, KIF1A and UNC-104 form dimers, which exhibit efficient directional movement on microtubules (Kita et al., 2024). These studies prompted us to reanalyze the oligomeric state and the motile properties of Kid, mainly in the full length. In this study, we show that the full-length Kid protein moves processively along microtubules. Kid proteins can form dimers, but Kid proteins are dissociated to monomers at low concentrations. In the reconstitution assays in vitro, full-length Kid transport double-stranded DNA along microtubules.

Results

Kid exhibits processive movement along microtubules

To study biochemical and biophysical properties of full-length Kid, we firstly purified the full-length human Kid (hKid) and Xenopus Kid (XKid) using the baculovirus system and Sf9 cells because a previous study has succeeded in purifying functional XKid from Sf9 cells (Bieling et al., 2010; Funabiki and Murray, 2000; Takagi et al., 2013). hKid as well as XKid possesses an N-terminal motor domain, a short coiled-coil domain, and a DNA-binding tail domain (Figure 1A). Previous studies have shown that hKid and XKid, which are fused with fluorescent proteins at the C-terminal, can complement the function of hKid-depleted cells and XKid-depleted Xenopus egg extracts (Bieling et al., 2010; Soeda et al., 2016). Thus, we fused a fluorescent protein at the C-terminal of XKid and hKid. Notably, while EGFP-fused and sfGFP-fused hKid were insoluble, mNeonGreen-fused hKid was recovered from soluble fractions. As a result, we succeeded in purifying both XKid-mScarlet and hKid-mNeonGreen proteins (Figure 1B, Figure 1—figure supplement 1). Next, the motility of purified proteins was analyzed by single molecule motility assays using total internal reflection fluorescent microscopy (TIRF). We found that both XKid and hKid moved on microtubules processively (Figure 1C and D, Figure 1—videos 1 and 2). The motility of single molecules along microtubules could be observed at 20 pM of Xkid and hKid, respectively. The average velocity of XKid and hKid was approximately 110 nm/second (Figure 1E and Table 1), within the same range as the movement of chromosomes (Brouhard and Hunt, 2005). The run length of XKid and hKid was 1.8±0.7 and 1.3±0.5 µm, respectively (Figure 1F and Table 1). These data collectively suggest that full-length Kid is a processive motor protein.

Table 1
Motile properties of constructs used in this study.

Motor protein constructs underwent purification through affinity chromatography followed by size-exclusion chromatography, as detailed in the ‘Materials and methods’ section. The reported velocities and run lengths represent mean values ± standard deviation (SD). Notably, XKid(1-437) failed to demonstrate consistent processive motion across three separate protein preparations, indicating a lack of detectable activity (ND: not detected).

ProcessiveOligomeric state in SECVelocity (µm/second)Run length (µm)
Full-length hKidYesDimer0.11±0.041.8±0.7
Full-length XKidYesDimer0.11±0.041.3±0.5
XKid(1-496)YesDimer0.12±0.033.1±2.4
XKid(1-437)NoMonomerNDND
KIF1A(1-393)LZYesDimer1.4±0.411±11
KIF1AMD-XKidStYesDimer0.67±0.186.7±6.0

Full-length Kid form dimers

It has been proposed that Kid is a monomeric protein (Shiroguchi et al., 2003; Yajima et al., 2003). Even full-length human Kid protein obtained from 293T cells is indicated to be a monomer (Shiroguchi et al., 2003). However, typical kinesins show processive movement along microtubules only when they form dimers (Hancock and Howard, 1998; Kita et al., 2024; Tomishige et al., 2002). As both hKid and XKid showed processive movement on microtubules (Figure 1), we next analyzed the oligomeric state of these motors using size-exclusion chromatography (Figure 2A and B, Figure 2—figure supplement 1). We utilized the previously well-characterized kinesin UNC-104(1-653)-sfGFP as a size marker. UNC-104(1-653)-sfGFP exhibits a dimer peak at 200 kDa and a monomer peak at 100 kDa in size-exclusion chromatography (Kita et al., 2024). This characteristic makes it a suitable marker for determining the oligomeric state of hKid and XKid, given that the predicted molecular weights of hKid-mNeonGreen and XKid-mScarlet monomers are approximately 100 kDa (Figure 1A). As a result, we found that the peak fraction of full-length hKid and XKid is almost equivalent to that of UNC-104(1-653)-sfGFP dimer (Figure 2A and B, Figure 2—figure supplement 1). Notably, unlike UNC-104(1-653)-sfGFP, we did not detect any monomer peaks for hKid and XKid under these conditions. We next analyzed purified proteins recovered from the peak fractions using mass photometry (Sonn-Segev et al., 2020). Mass photometry is generally performed at nanomolar concentrations (Sonn-Segev et al., 2020). Peak fractions obtained from size-exclusion chromatography were diluted 100-fold and analyzed by mass photometry. Mass photometry analysis revealed a predominant monomer population, with a smaller fraction corresponding to dimers and trimers (Figure 2C and D). The behavior is similar to that of UNC-104(1-653)-sfGFP, which shows a dimer peak by size-exclusion chromatography at micromolar concentrations but dissociates into monomers at the nanomolar concentrations used for mass photometry (Kita et al., 2024). In addition, we consistently detected a minor trimer population under this condition (Figure 2C and D), although this species may represent an artifact because it was not detected by size-exclusion chromatography. These findings indicate that full-length hKid and XKid are capable of forming dimers, but the dimer formation is dependent on the protein concentration.

Figure 2 with 1 supplement see all
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Kid forms a weak dimer.

(A) Size-exclusion chromatography profiles of hKidFL (black) and UNC-104(1-653)-sfGFP (cyan). Below the chromatography, an SDS-PAGE image shows the elution fractions. Asterisks indicate fractions used for mass photometry and single molecule assays. The molecular weight standards are indicated on the left side of the SDS-PAGE images. (B) Size-exclusion chromatography of XKidFL (black) and UNC-104(1-653) (cyan). The SDS-PAGE of the elution fractions is shown beneath the profiles. Asterisks indicate fractions used for mass photometry and single molecule assays. The number shown at the left side indicates molecular weight standard. (C) Mass photometry analysis of human Kid at 10 nM. Histograms show particle counts, and lines indicate Gaussian fits. The mean ± SD and percentage of each peak are shown. (D) Mass photometry analysis of human Kid at 10 nM. Histograms show particle counts, and lines indicate Gaussian fits. The mean ± SD and percentage of each peak are shown. Note that the majority of hKid and XKid are dimers in the size-exclusion chromatography, but they are mostly dissociated to monomers in mass photometry.

Figure 2—source data 1

Original file for SDS-PAGE data for Figure 2A.

https://cdn.elifesciences.org/articles/102828/elife-102828-fig2-data1-v1.zip
Figure 2—source data 2

Original file for SDS-PAGE data for Figure 2B.

https://cdn.elifesciences.org/articles/102828/elife-102828-fig2-data2-v1.zip
Figure 2—source data 3

Original file for SDS-PAGE data for Figure 2A indicating the relevant bands and elution volume of each fraction.

https://cdn.elifesciences.org/articles/102828/elife-102828-fig2-data3-v1.zip
Figure 2—source data 4

Original file for SDS-PAGE data for Figure 2B indicating the relevant bands and elution volume of each fraction.

https://cdn.elifesciences.org/articles/102828/elife-102828-fig2-data4-v1.zip

A conserved coiled-coil domain is essential for the processivity

To determine the domain essential for the processive movement of Kid, we generated a series of deletion mutants. Unfortunately, we failed to purify adequate quantities and qualities of hKid deletion mutants. Thus, the following experiments were performed using XKid. We purified XKid(1-496), which lacks the DNA-binding tail domain, and XKid(1-437), which lacks the DNA-binding domain and the coiled-coil domain (Figure 3A and B, Figure 3—source data 2 and 3). Mass photometry showed that XKid(1-496) was predominantly monomeric (Figure 3C), with minor dimer and trimer populations, resembling the behavior of full-length XKid (Figure 2). In contrast, XKid(1-437) was exclusively monomeric (Figure 3D).

Figure 3
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Conserved coiled-coil domain is required for the processive motion.

(A) Schematic representation illustrating the domain organization of XKid(1-496) and XKid(1-437). The calculated molecular weights of the fusion proteins are indicated on the right. (B) Representative SDS-PAGE analysis of purified XKid(1-496) and XKid(1-437) proteins. The proteins are visualized using a Stain-Free gel. The molecular weight standards are indicated on the right side of the SDS-PAGE images. (C, D) Mass photometry analysis of XKid(1-496)-mSca and XKid(1-437)-mSca. The expected molecular masses are 86 and 79 kDa, respectively. Histograms show particle counts, and lines indicate Gaussian fits. The mean ± SD and percentage of total counts for each peak are shown. (E, F) Representative kymographs showing the motility of 10 pM XKid(1-496) (E) and XKid(1-437) (F) in the presence of 2 mM ATP. Note that no directional movement was detected in XKid(1-437). Scale bars: horizontal 10 µm; vertical 10 seconds. (G, H) Mean-square displacement (MSD) analysis of XKid(1-496) and XKid(1-437) trajectories. (G) Representative MSD curves fitted to the power-law relationship MSD = AΔtα, where α is the anomalous diffusion exponent. XKid(1-496) showed superlinear MSD scaling with α=1.68, consistent with persistent or directionally biased motion, whereas XKid(1-437) showed sublinear MSD scaling with α=0.77. (H) Distribution of α values obtained from individual trajectory fits. Each dot represents one trajectory; bars indicate mean ± SD. n=10 trajectories per construct. (I, J) Schematic drawing of XKidCC-mScarlet (I) and a representative result of size-exclusion chromatography (J). XKidCC-mScarlet (magenta) and mScarlet (cyan) are shown.

Figure 3—source data 1

MSD analysis data for Figure 3G and H.

https://cdn.elifesciences.org/articles/102828/elife-102828-fig3-data1-v1.xlsx
Figure 3—source data 2

Original file for SDS-PAGE data for Figure 3B.

https://cdn.elifesciences.org/articles/102828/elife-102828-fig3-data2-v1.zip
Figure 3—source data 3

Original file for SDS-PAGE data for Figure 3B indicating the relevant bands.

https://cdn.elifesciences.org/articles/102828/elife-102828-fig3-data3-v1.zip

Using these purified proteins, we performed single-molecule motility assays (Figure 3E–H). We found XKid(1-496) could move processively on microtubules (Figure 3E). The velocity of XKid(1-496) was approximately 120 nm/second and the run length was 3.1±2.4 µm, which are comparable to those of full-length XKid (Table 1). Thus, the DNA-binding domain of Kid is not required for processive runs. In contrast, XKid(1-437), which lacks the coiled-coil domain, did not show any processive runs (Figure 3F). To analyze the motile properties, we performed the mean square displacement (MSD) analysis (Figure 3G and H). The MSD curves were fitted to the power-law relationship MSD = A(Δt)α, where α describes how the displacement scales with time. α=1 indicates simple diffusion, α>1 indicates superlinear MSD scaling indicating persistent or directionally biased motion, and α<1 indicates sublinear MSD scaling indicating constrained motion. XKid(1-496) had an α value of approximately 1.6, consistent with directionally biased movement, whereas XKid(1-437) had an α value of approximately 0.8, indicating sublinear MSD scaling consistent with hindered motion (Figure 3G and H). This would be because XKid(1-437) did not form a homodimer, considering that coiled-coil domains are generally required for the dimerization of kinesins (Hancock and Howard, 1998; Kita et al., 2024).

To confirm that the coiled-coil domain of XKid induces dimerization, the domain was fused with mScarlet and analyzed by the size-exclusion chromatography (Figure 3I and J). As a result, the peak of XKidCC-mScarlet shifted to the larger size compared with that of mScarlet. The calculated molecular weight of XKidCC-mScarlet was 42 kDa, which is almost equivalent to the size of dimerized mScarlet. To test whether monomeric Kid molecules in solution form dimers on microtubules, hKid-mScarlet3 and hKid-mStayGold were purified separately, mixed at final concentrations of 1 pM each, and analyzed using single-molecule motility assays (Figure 4). Under these assay conditions, hKid is expected to be monomeric in solution because mass photometry showed that hKid was predominantly monomeric even at a much higher concentration of 10 nM (Figure 2). As a result, approximately 20% of motile hKid-mScarlet3 particles showed comigration with hKid-mStayGold particles along microtubules, suggesting microtubule-dependent association between hKid molecules.

Figure 4
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Human Kid-mStayGold and Kid-mScarlet3 co-migrate on microtubules.

Purified hKid-mStayGold and hKid-mScarlet3 were mixed at final concentrations of 20 pM each and analyzed by single-molecule motility assays using TIRF microscopy. Representative kymographs show hKid-mStayGold, hKid-mScarlet3, and merged signals. The schematic drawing illustrates examples of co-migrating particles on microtubules, defined as overlapping mStayGold and mScarlet3 signals moving together along the same microtubule. Co-migration was defined as overlapping mStayGold and mScarlet3 signals moving together along the same microtubule over the same time interval. Scale bars: horizontal, 10 µm; vertical, 100 seconds.

The stalk domain of XKid adds processivity to the motor domain of KIF1A

In the processive movement along the microtubules, the coordination between two motor domains is essential (Hancock and Howard, 1998). The neck linker domain, immediately following the kinesin motor domain, regulates the ATPase activity within the motor domain (Case et al., 2000). It has been shown that the optimal length of the neck linker domain is crucial for achieving coordination of the two motor domains (Isojima et al., 2010; Yildiz et al., 2008). The length of the neck linker domain is typically determined by the presence of the neck coiled-coil domain (Case et al., 2000; Shastry and Hancock, 2010). However, Kid is an exception, as it does not have the conventional neck coiled-coil domain (Figure 5A; Shiroguchi et al., 2003; Tokai et al., 1996). This unique feature of Kid supports the idea that Kid functions as a nonprocessive monomer (Yajima et al., 2003), which does not require the coordination between two motor domains. However, coiled-coil prediction tools might not identify hidden coiled-coil domains, or Kid could have a motif that induces dimerization. In the case of kinesin-1, AlphaFold2, but not coiled-coil prediction tools, can more accurately find the location of coiled-coil domains (Tan et al., 2023; Weijman et al., 2022). Therefore, we used AlphaFold2 to analyze the neck-linker and the first coiled-coil domain of XKid and hKid. The regions corresponding to residues 359–495 of XKid and 369–506 of hKid were modeled using AlphaFold2 (Figure 1A). The result suggested that the region does not have hidden coiled-coil domains nor a motif that induces dimerization, and the region is flexible (Figure 5—figure supplement 1). If the entire flexible region functions as a neck linker, its length is four times longer than that of kinesin-1 (Figure 5—figure supplement 1). To investigate if this extended neck linker of Kid can support the coordination of two motor domains, we fused the coiled-coil domain of XKid to the motor domain of KIF1A (Figure 5A). We included the possible neck linker domain of XKid in this chimera protein to test whether the neck linker of XKid is functional or not (Figure 5B). We could purify the chimeric protein, named KIF1AMD-XKidSt (Figure 5—source data 3). Similar to full-length XKid or XKid(1-497) (Figures 2D and 3C), KIF1AMD-XKidSt showed monomeric populations with minor dimer and trimer populations on mass photometry (Figure 5D). We found that KIF1AMD-XKidSt exhibited processive movement along microtubules in the single molecule motility assay (Figure 5F and Figure 5—video 1), as is the case of KIF1A(1-393)LZ (Figure 5E and Figure 5—video 2). The velocity of KIF1AMD-XKidSt was much faster than original XKid but slightly slower than KIF1A(1-393)LZ (Figure 5G and Table 1). The run length of KIF1AMD-XKidSt was shorter than KIF1A(1-393)LZ (Figure 5H and Table 1). Previous studies have shown that the motor domain of KIF1A does not exhibit processive motion when it is monomeric, but exhibits processive motion when an artificial dimer is generated using a stalk domain of kinesin-1 or a leucine zipper domain (Soppina et al., 2014; Tomishige et al., 2002). Therefore, these results suggest that the coiled-coil domain of XKid can induce dimerization of KIF1A motor domains on microtubules, and the longer neck linker domain of XKid can support the processive movement of the kinesin motor domain.

Figure 5 with 3 supplements see all
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Untypical neck linker of Kid can support processive movement of KIF1A.

(A) Schematic representation illustrating the domain organization of KIF1A(1-393)LZ, XKid(1-496), KIF1A(1-350), and a chimera protein KIF1AMD-XKidSt. Note that KIF1A(1-393)LZ and XKid(1-496) are processive motors, and KIF1A(1-350) is a nonprocessive motor. Cyan, motor domain of KIF1A; orange, motor domain of Kid; magenta, neck linker. (B) Amino acid sequences of the neck linker region. KIF1A, XKid, hKid, KIF5C, and KIF1AMD-XKidSt are shown. Cyan, motor domain of KIF1A and KIF5C; orange, motor domain of Kid; magenta, neck linker; green, neck coiled-coil domain. (C) Representative SDS-PAGE analysis of purified KIF1AMD-XKidSt fusion protein. The protein is visualized using a Stain-Free gel. The molecular weight standards are indicated on the right side of the SDS-PAGE image. (D) Mass photometry analysis of KIF1AMD-XKidSt at 10 nM. The expected molecular mass is 103 kDa. Histogram shows particle counts, and lines indicate Gaussian fits. The mean ± SD and percentage of each peak are shown. (E, F) Representative kymographs showing the motility of KIF1A(1-393)LZ and KIF1AMD-XKidSt in the presence of 2 mM ATP. Note that KIF1AMD-XKidSt exhibits diffusion-like fluctuations while they are moving. This phenomenon is not observed in KIF1A(1-393)LZ. Scale bars: horizontal 10 µm; vertical 10 seconds. (G) Dot plots showing the velocity of KIF1A(1-393)LZ and KIF1AMD-XKidSt. Each dot shows a single datum point. Green bars represent mean ± S.D. ****p<0.0001, Unpaired t-test. n=273 and 434 particles for KIF1AMD-XKidSt and KIF1A(1-393)LZ, respectively. (H) Dot plots showing the run length of KIF1A(1-393)LZ and KIF1AMD-XKidSt. Each dot shows a single datum point. Green bars represent median value and interquartile range. ****p<0.0001, Mann–Whitney test. n=273 and 434 particles for KIF1AMD-XKidSt and KIF1A(1-393)LZ, respectively.

Figure 5—source data 1

Velocity and run length data for Figure 5G and H.

https://cdn.elifesciences.org/articles/102828/elife-102828-fig5-data1-v1.xlsx
Figure 5—source data 2

Original file for SDS-PAGE data for Figure 5C.

https://cdn.elifesciences.org/articles/102828/elife-102828-fig5-data2-v1.zip
Figure 5—source data 3

Original file for SDS-PAGE data for Figure 5C indicating the relevant bands.

https://cdn.elifesciences.org/articles/102828/elife-102828-fig5-data3-v1.zip

Reconstitution of DNA transport in vitro

Kid was originally identified as a DNA-binding protein (Tokai et al., 1996). The tail domain of Kid has two helix–hairpin–helix motifs that are supposed to be a DNA-binding domain (Doherty et al., 1996). To test that Kid has an activity to transport DNA along microtubules, we mixed fluorescently labeled DNA in TIRF assays and directly observed the motility (Figure 6A–D). To study the domains essential for the transport of DNA, we used full-length XKid and a deletion mutant of Xkid for these assays. We found that full-length XKid can drive the movement of double-stranded DNA along microtubules (Figure 6A). Double-stranded DNA signals that co-migrated with XKid were 93.4 ± 10.5% (n=31 microtubules). The velocity of double-stranded DNA moving along microtubules was approximately 100 nm/second, which is similar to the velocity of XKid alone (Figure 1). In contrast, single-strand DNA movement was not driven by XKid (Figure 6B and D). Deletion of the tail domain of XKid, containing helix–hairpin–helix motifs, abolished the DNA transport activity (Figure 6C and D). We confirmed that full-length hKid can also induce the movement of double-stranded DNA along microtubules (Figure 6—figure supplement 1 and Figure 6—video 1).

Figure 6 with 2 supplements see all
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DNA movement driven by Xkid.

(A–D) sfGFP-tagged XKid at 1 nM was mixed with 20 nM Cy3-labeled double-stranded or single-stranded DNA and observed by TIRF microscopy. (A–C) Representative kymographs showing the movement of full-length XKid with double-stranded 100 bp DNA (A), full-length XKid with single-stranded 100-base DNA (B), and XKid(1-496) with double-stranded 100 bp DNA (C). Scale bars: horizontal, 10 µm; vertical, 100 seconds. (D) Frequency of DNA movement along microtubules, normalized by microtubule length and observation time. Each dot represents an individual measurement from a different microtubule. Bars indicate mean ± SD. n=29 microtubules per condition. (E–I) Single-molecule analysis of XKid–sfGFP on Cy3-labeled DNA. Purified XKid–sfGFP was mixed with Cy3-labeled DNA at final concentrations of 1 nM XKid–sfGFP and 20 nM DNA and observed by TIRF microscopy. (E–G) Representative kymographs showing the movement of full-length XKid–sfGFP on 100 bp DNA (E), 1000 bp DNA (13 ng/µl; F), and 2000 bp DNA (26 ng/µl; G). Scale bars: horizontal, 10 µm; vertical, 100 seconds. (H) Velocity of DNA movement along microtubules. Each dot represents one DNA molecule. Bars indicate mean ± SD. n=48, 53, and 53 DNA molecules for 100, 1000, and 2000 bp DNA, respectively. (I) Run length of DNA movement along microtubules. Each dot represents one DNA molecule. Bars indicate mean ± SD. n=48, 53, and 53 DNA molecules for 100, 1000, and 2000 bp DNA, respectively.

Figure 6—source data 1

Frequency data for Figure 6D.

https://cdn.elifesciences.org/articles/102828/elife-102828-fig6-data1-v1.xlsx

Next, we analyzed dsDNA fragments of different lengths. Full-length Kid transported not only 100 bp dsDNA but also longer 1000 bp and 2000 bp dsDNA fragments along microtubules (Figure 6E–G). However, the velocity and run length of DNA transport were comparable among these substrates (Figure 6H and I), suggesting that increasing DNA length does not substantially alter Kid-driven DNA transport under our reconstituted assay conditions.

Dimerization would be required for DNA transport

The structure of the XKid–DNA complex was modeled using AlphaFold3 (Figure 7A–D). We first modeled one copy of the DNA-binding domain of XKid with a 15 bp DNA fragment (Figure 7A). This prediction yielded low ipTM and pTM values of 0.12 and 0.64, respectively, and did not produce a clear XKid–DNA complex model. We then modeled two copies of the DNA-binding domain of XKid with a double-stranded DNA fragment (Figure 7B–D). In this case, AlphaFold3 generated a model in which two DNA-binding domains of XKid associate with dsDNA. The ipTM and pTM values were 0.79 and 0.84, respectively, suggesting a higher-confidence prediction of the complex. To further examine whether the DNA-binding domain of XKid intrinsically forms a stable dimer, we performed AlphaFold3 prediction in the absence of DNA. This prediction yielded only modest confidence scores (ipTM = 0.34, pTM = 0.57). These results suggest that the dimeric configuration of the DNA-binding domain of XKid is preferentially stabilized in the presence of double-stranded DNA. These results suggest that the DNA-binding domain of XKid adopts a DNA-binding architecture more readily in a dimeric configuration. To experimentally test the predicted model, we introduced the K605A mutation into XKid, as K605 was predicted to be located at the XKid–DNA interface (Figure 7C), and performed dsDNA motility assays (Figure 7D and E). The XKid(K605A) mutant failed to drive DNA movement in the reconstitution assay (Figure 7E).

Figure 7
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AlphaFold3 prediction and functional validation.

(A) AlphaFold3-predicted structure of the XKid DNA-binding domain modeled as a monomer with double-stranded DNA (dsDNA). The prediction showed a low interface confidence score between XKid and dsDNA (ipTM = 0.12, pTM = 0.64). (B–D) AlphaFold3-predicted structure of the XKid DNA-binding domain modeled as a dimer with dsDNA. The two XKid molecules are shown in green and gray, and the two DNA strands are shown in red and blue. This dimeric model showed higher confidence for the XKid–DNA complex (ipTM = 0.79, pTM = 0.84). (B) Side view of the predicted XKid dimer–dsDNA complex. (C) Magnified view of the predicted XKid–DNA interface. K605 and K606, highlighted in yellow, are positioned near dsDNA. A cyan dashed line indicates the predicted hydrogen bond between K605 and DNA. (D) View of the same model shown in (B) after a 90° rotation. (E, F) Representative kymographs showing the movement of dsDNA along microtubules. Wild-type XKid supported movement of 1000 bp dsDNA (E), whereas the K605A mutant abolished detectable dsDNA movement (F). Scale bars: horizontal, 10 µm; vertical, 100 s.

Discussion

Kid is a processive dimer on microtubules

Prometaphase chromosomes are transported along microtubules by the activity of kinesins (Iemura and Tanaka, 2015; Wordeman, 2010). Kid is the primary kinesin that transports chromosomes along spindle microtubules in prometaphase (Brouhard and Hunt, 2005; Iemura and Tanaka, 2015). Previous biochemical studies reported that purified Kid is monomeric, leading to the concept that Kid is a monomeric and nonprocessive chromokinesin (Yajima et al., 2003; Shiroguchi et al., 2003). Under this model, sustained chromosome movement would require many Kid monomers distributed along chromosome arms to act collectively. Our findings revise this view. Our data show that full-length Kid is capable of forming a dimer and exhibits a processive motion along microtubules. In addition, Kid dimers directly bind to dsDNA. Thus, the elementary force-generating unit of Kid is a single Kid dimer that functions as a processive DNA-bound motor. In the context of mitotic chromosomes, multiple processive Kid dimers bound along chromosome arms could cooperate to generate chromosome-scale polar ejection forces (Figure 8). This model preserves the likely importance of motor teams on large chromatin. However, the team is likely composed of multiple processive dimers rather than many nonprocessive monomers.

Figure 8
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Model.

Model for Kid-dependent DNA and chromosome transport. Kid forms processive dimers that can directly bind and transport double-stranded DNA along microtubules. On mitotic chromosomes, multiple Kid dimers may bind along chromosome arms and cooperate to generate polar ejection forces. This model is not drawn to scale and does not fully represent the structural complexity of condensed chromatin.

Biochemical properties of Kid

A notable difference between this study and prior studies is the protein concentration used. Previous analysis of full-length human Kid via density gradient and size-exclusion chromatography identified peak fractions through western blot due to the low concentration of recombinant Kid expressed in 293T cells (Shiroguchi et al., 2003). We show that most hKid and XKid exist as dimers in size-exclusion chromatography performed at micromolar concentrations, yet mostly dissociate into monomers in mass photometry, which is performed at nanomolar concentrations (Figure 2). Thus, the inability to detect Kid dimers in earlier studies may be attributed to the low concentration. In mass photometry, we also detected a small population with an apparent trimeric mass (Figures 2, 3, and 5). Because this species was not detected by size-exclusion chromatography, we interpret this population cautiously and cannot exclude the possibility that it represents a measurement or sample-preparation artifact rather than a stable oligomeric state of Kid. However, transient higher-order oligomerization of Kid in cells, where Kid may be locally concentrated on microtubules or chromatin, remains possible and should be examined in future studies.

Another difference is the methods to detect the processivity of motors. We observed the processivity of Kid by TIRF whereas previous studies observed the motility by optical trap. In optical trapping assays, Kid is diluted prior to being adsorbed onto beads. This dilution likely promotes the dissociation of preformed Kid dimers, consistent with our mass photometry analysis showing that Kid is predominantly monomeric at nanomolar concentrations (Figure 2), and thus optical trapping assays would primarily monitor monomeric Kid motility. By contrast, in TIRF-based motility assays, Kid in solution binds directly to microtubules. Although Kid is predominantly monomeric in solution, its accumulation on the microtubule surface may increase the local concentration of Kid and promote dimer formation on microtubules. This microtubule-dependent dimerization model could account for the processive motility observed in the TIRF assay. Consistent with this model, when separately purified Kid-mScarlet3 and Kid-mStayGold were mixed, they colocalized and moved processively together along microtubules (Figure 5). However, because of the limited temporal resolution of our two-color imaging system, we could not directly resolve the transition from monomers to dimers on microtubules. These properties, such as ‘the equilibrium between monomers and dimers’ and ‘the capability to exhibit processive movement on microtubules, even when characterized as monomers through mass photometry’ have been observed in kinesin-3 motors (Chiba et al., 2023; Fan and McKenney, 2022; Kita et al., 2024).

Neck linker of Kid

We show that Kid has an exceptionally long neck linker, approximately four times longer than that of kinesin-1. Neck-linker length strongly influences kinesin processivity, and changes in neck-linker length alter the run length and motility properties of kinesin-1, kinesin-2, and other N-terminal kinesins (Shastry and Hancock, 2010; Shastry and Hancock, 2011). However, longer or nonconventional neck linker regions can also support processive motility and may provide additional functions, such as navigation around microtubule-bound obstacles. Kinesin-2 bypasses Tau and other microtubule-bound obstacles by protofilament switching (Hoeprich et al., 2014). The neck linker of the mitotic kinesin KIF18A contributes to obstacle navigation within the mitotic spindle (Malaby et al., 2019). Thus, the exceptionally long and flexible neck linker of Kid may represent an adaptation that allows this chromokinesin to move processively along crowded spindle microtubules while remaining attached to DNA or chromatin. This possibility remains to be tested directly.

Reconstitution of chromosome congression

A previous in vitro study, using an elegant assay termed chromatin gliding assay, has shown that XKid and XKLP1/KIF4A can crosslink between DNA and microtubules (Bieling et al., 2010). However, it remains to be elusive whether the chromosome transport is mediated by direct binding between chromosomal DNA and motor proteins. In organelle transport, cargo adaptor proteins are generally required for efficient transport (Chiba and Niwa, 2024; Chiba et al., 2022). Our in vitro reconstitution suggests that Kid-dependent chromosome transport does not require cargo adaptor proteins. Rather, our results suggest that dimerization of Kid facilitates DNA binding, allowing Kid to directly couple DNA to microtubule-based motility (Figure 7). Although hKid transported 1000 bp and 2000 bp DNA fragments in vitro, their motile parameters were comparable to those of 100 bp DNA. Thus, increasing DNA length did not substantially enhance DNA transport under our reconstituted assay conditions. One possible explanation is that the interaction between Kid and naked DNA is relatively weak or dynamic, and thus only one or a small number of Kid molecules productively engage each DNA molecule during transport. Alternatively, additional Kid molecules bound to longer DNA may not strongly affect the measured motility parameters under these assay conditions. However, naked DNA does not fully recapitulate the structural and mechanical properties of condensed chromatin or mitotic chromosomes. Thus, although multiple Kid dimers may engage chromatin to generate chromosome-scale polar ejection forces, this model remains to be directly tested. Future experiments using chromatinized DNA or reconstituted chromosome-like substrates will be required to determine how Kid interacts with condensed chromatin and how multiple Kid molecules cooperate to move chromosomes during prometaphase.

It has been shown that Kid is regulated by phosphorylation by CDK1 (Ohsugi et al., 2003). It would be interesting to study the effects of phosphorylation and dephosphorylation on the Kid-dependent DNA transport using this reconstitution system. Moreover, by extending this system, it may be possible to fully reconstitute the chromosome congression in vitro by including other kinesins and microtubule-associated proteins, such as XKLP1/KIF4A, CENP-E, and NuSAP1, that are required for proper chromosome congression (Bieling et al., 2010; Iemura and Tanaka, 2015; Li et al., 2016).

Materials and methods

Plasmids

PCR was performed using a KOD FX neo DNA polymerase (TOYOBO, Tokyo, Japan). Human Kid cDNA (corresponding to DQ895829.2) was described previously (Iemura and Tanaka, 2015). Xenopus Kid (Kif22.S, corresponding to BC070549.1) was purchased from Horizon Discovery. To generate hKidFL-mNeonGreen, DNA fragments encoding human Kid and mNeonGreen were amplified by PCR and assembled into pFastbac1 (Novagen) by Gibson assembly as described (Gibson et al., 2009). To generate XKidFL-mScarlet, DNA fragments encoding XKid and mScarlet were amplified by PCR and assembled into pAcebac1 (Geneva Biotech). ORF sequences are shown in Supplementary file 1. Deletion mutants of XKid were generated by PCR-based mutagenesis. For this purpose, primers were designed through the QuickChange Primer Design tool, a web-based application (Agilent). PCR-based mutagenesis was performed using KOD plus neo DNA polymerase (TOYOBO).

Expression of XKid and hKid in Sf9 cells

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Sf9 cells were obtained from Thermo Fisher Scientific (Gibco, Cat. No. 12659017; RRID:CVCL_0549). The identity of the cell line was confirmed by the supplier by isozyme and karyotype analyses. The Master Seed Bank was tested by the supplier and confirmed to be negative for mycoplasma contamination. Sf9 cells are not listed in the ICLAC Register of Misidentified Cell Lines. Sf9 cells were maintained in Sf900 II SFM (Thermo Fisher Scientific) at 27°C. DH10Bac (Thermo Fisher Scientific) were transformed to generate bacmid. To prepare baculovirus, 1×106 cells of Sf9 cells were transferred to each well of a tissue-culture treated 6-well plate. After the cells attached to the bottom of the dishes, about ∼5 μg of bacmid were transfected using 5 μl of TransIT-Insect transfection reagent (Takara Bio Inc). 5 days after initial transfection, the culture media were collected and spun at 3000×g for 3 minutes to obtain the supernatant (P1). For protein expression, 400 ml of Sf9 cells (2×106 cells/ml) were infected with 200 µl of P1 virus and cultured for 65 hours at 27°C. Cells were harvested and stocked at –80°C.

Purification of proteins

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We failed to purify hKid-EGFP and hKid-superfolder GFP due to the insolubility. In contrast, mNeonGreen fusion and mStayGold fusion stabilized hKid and enabled purification.

Sf9 cells were resuspended in 40 ml of Kid lysis buffer (50 mM HEPES-KOH, pH 7.5, 500 mM KCH3COO, 2 mM MgSO4, 1 mM EGTA, 10% glycerol) along with 1 mM DTT, 1 mM PMSF, 0.1 mM ATP, and 0.5% Triton X-100. After incubating on ice for 10 minutes, lysates were cleared by centrifugation (100,000×g, 20 minutes, 4°C) and subjected to affinity chromatography. Lysate was loaded on Streptactin-XT resin (IBA Lifesciences, Göttingen, Germany) (bead volume: 2 ml). The resin was washed with 40 ml Kid wash buffer (50 mM HEPES-KOH, pH 8.0, 500 mM KCH3COO, 2 mM MgSO4, 1 mM EGTA, 10% glycerol). Protein was eluted with 40 ml Kid elution buffer (50 mM HEPES-KOH, pH 8.0, 500 mM KCH3COO, 2 mM MgSO4, 1 mM EGTA, 10% glycerol, 200 mM biotin). Eluted solution was concentrated using an Amicon Ultra 15 (Merck) and then separated on an NGC chromatography system (Bio-Rad) equipped with a Superdex 200 Increase 10/300 GL column (Cytiva). Peak fractions were collected and concentrated using an Amicon Ultra 4 (Merck). Proteins were analyzed by SDS-PAGE using TGX Stain-Free gel (Bio-Rad). Concentrated proteins were aliquoted and snap-frozen in liquid nitrogen.

Mass photometry

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Purified hKid and XKid obtained from the peak fractions in the SEC analysis were pooled, snap-frozen, and stored until measurement. Prior to measurement, the proteins were thawed and diluted to a final concentration of 5–10 nM in GF150 buffer (25 mM HEPES, 150 mM KCl, 2 mM MgCl2, pH 7.2). Mass photometry was performed using a Refeyn OneMP mass photometer (Refeyn) and Refeyn AcquireMP version 2.3 software, with default parameters set by Refeyn AcquireMP. Bovine serum albumin (BSA) was used as a control to determine the molecular weight. The results were subsequently analyzed, and graphs were prepared to visualize the data using Refeyn DiscoverMP version 2.3.

Preparation of microtubules

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Tubulin was purified from porcine brain as described (Castoldi and Popov, 2003). Tubulin was labeled with Biotin-PEG2-NHS ester (Tokyo Chemical Industry, Tokyo, Japan) and AZDye647 NHS ester (Fluoroprobes, Scottsdale, Arizona, USA) as described (Al-Bassam, 2014). To polymerize Taxol-stabilized microtubules labeled with biotin and AZDye647, 30 μM unlabeled tubulin, 1.5 μM biotin-labeled tubulin, and 1.5 μM AZDye647-labeled tubulin were mixed in BRB80 buffer supplemented with 1 mM GTP and incubated for 15 minutes at 37°C. Then, an equal amount of BRB80 supplemented with 40 μM taxol was added and further incubated for more than 15 minutes. The solution was loaded on BRB80 supplemented with 300 mM sucrose and 20 μM taxol and ultracentrifuged at 100,000 × g for 5 minutes at 30°C. The pellet was resuspended in BRB80 supplemented with 20 μM taxol.

Preparation of fluorescent-labeled DNA for the TIRF assay

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To prepare a 100 bp DNA fragment, oligonucleotides labeled with Cy3 were purchased from Integrated DNA Technologies, Inc (Coralville, Iowa, USA). The following oligonucleotides were synthesized:

  • Cy3-5'-GAGAATCGCCGGTTGATAATCTTCCTAGTAGGTAGTATTGGTGTTGAGTCGCTCA-3' (Oligonucleotide #1)

  • Cy3-5'-GAGAATCGCCGGTTGATAATCTTTGAGCGACTCAACACCAATACTACCTACTAGG-3' (Oligonucleotide #2)

Underlines indicate sequences that form double-stranded DNA.

Double-stranded DNA was prepared using either a VeritiPro Thermal Cycler (Applied Biosystems) or a C1000 Touch Thermal Cycler (Bio-Rad). 1 µM of Oligonucleotides #1 and #2 were mixed and subjected to the following protocol: an initial incubation at 96°C for 2 minutes, followed by incubation at 25°C for 1 minute, and subsequently to 4°C. For single-stranded DNA preparation, Oligonucleotide #1 was incubated at 96°C and then cooled to 4°C.

To prepare 1000 bp and 2000 bp DNA fragments, Cy3-labeled oligonucleotides were purchased from Eurofins Genomics K.K. (Tokyo, Japan). The following oligonucleotides were synthesized:

  • Cy3-5'-TGATGACGGTGAAAACCTCTGACAC-3' (pUC19_F)

  • 5'-TATGAGAAAGCGCCACGCTTCCCG-3' (pUC19_R_1000bp)

  • 5'-GATCGGAGGACCGAAGGAGCTAACC-3'(pUC19_R_2000bp)

PCR was performed using pUC19 as the template and KOD FX Neo DNA polymerase. After agarose gel electrophoresis, the 1000 bp and 2,000 bp DNA fragments were purified.

Double-stranded and single-strand DNA were prepared on the day of the experiment, immediately before the TIRF single-molecule motility assays. Old DNA can potentially cause high background.

TIRF single-molecule motility assays

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Purified Kid proteins described above were thawed and analyzed. KIF1A (1-393)LZ, which was described in our previous work (Anazawa et al., 2022), was also thawed and reanalyzed. TIRF assays using porcine microtubules were performed as described (Chiba et al., 2019). Glass chambers were prepared by acid washing as previously described (Chiba et al., 2022). Glass chambers were coated with PLL-PEG-biotin (50% labeled, SuSoS, Dübendorf, Switzerland) and streptavidin (Wako). Polymerized microtubules were flowed into flow chambers and allowed to adhere for 5–10 minutes. Unbound microtubules were washed away using assay buffer (90 mM HEPES-KOH pH 7.4, 50 mM KCH3COO, 2 mM Mg(CH3COO)2, 1 mM EGTA, 10% glycerol, 0.1 mg/ml biotin–BSA, 0.2 mg/ml kappa-casein, 0.5% Pluronic F127, 2 mM ATP, and an oxygen scavenging system composed of PCA/PCD/Trolox). Purified Kid was diluted to indicated concentrations in the assay buffer. Then, the solution was flowed into the glass chamber. An ECLIPSE Ti2-E microscope equipped with a CFI Apochromat TIRF 100XC Oil objective lens (1.49 NA), an Andor iXion life 897 camera, and a Ti2-LAPP illumination system (Nikon, Tokyo, Japan) was used to observe the motility. NIS-Elements AR software version 5.2 (Nikon) was used to control the system. At least three independent experiments were conducted for each measurement.

MSD analysis

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Single-particle trajectories of fluorescent XKid(1-496) and XKid(1-437) molecules were obtained by manually tracking fluorescent puncta in kymographs. For each trajectory, the MSD was calculated for each lag time, Δt, as

MSD(Δt)=⟨[x(t+Δt)−x(t)]2⟩,

where x(t) is the one-dimensional position of the particle along the microtubule or DNA axis, and the brackets denote averaging over all displacement pairs with the same lag time. When the time intervals between tracked points were not strictly uniform, Δt was defined as the mean time difference of all displacement pairs contributing to that lag. The standard error of the MSD for each lag time was calculated as SD/√N, where N is the number of displacement pairs. To minimize the influence of reduced sampling at longer lag times and end-point effects, curve fitting was restricted to the initial region of each MSD curve. Unless otherwise indicated, lag times within approximately the first 60% of the total trajectory duration were used. Lag times were included in the fitting only when they were calculated from at least 10 displacement pairs. Each MSD curve was fitted to the power–law relationship:

MSD=A(Δt)α,

where A is a scaling coefficient and α is the anomalous diffusion exponent.

AlphaFold2 analysis

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AlphaFold2 analysis was conducted on Google Colaboratory (Jumper et al., 2021; Mirdita et al., 2022). We analyzed the amino acid sequences of KIF5C, XKid, and hKid, ranging from the neck linker to the end of coiled-coil 1. The sequences are detailed in Supplementary file 2. The analysis was based on the assumption that these fragments form dimers and XKid and hKid fold in a similar manner.

AlphaFold3 analysis

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AlphaFold3 analysis was conducted on AlphaFold Server (https://alphafoldserver.com/). We analyzed the amino acid sequences of the DNA-binding domain of XKid and 15 bp dsDNA. The sequences are detailed in Supplementary file 3.

Statistical analyses and graph preparation

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Statistical analyses and graph preparation were conducted using GraphPad Prism version 10. Details on the statistical methods are provided in the figure legends. Graphs were created with GraphPad Prism version 10, exported in PDF format, and aligned using Adobe Illustrator 2023.

Data availability

Figure 1—source data 1, Figure 3—source data 1, Figure 5—source data 1 and Figure 6—source data 1 contain the numerical data used to generate the figures. Figure 1—source data 2, Figure 2—source data 1 and 2, Figure 3—source data 2 and Figure 5—source data 2 contain the original files of the full raw uncropped, unedited gels. Figure 1—source data 3, Figure 2—source data 3 and 4, Figure 3—source data 3 and Figure 5—source data 3 contain the labeled uncropped gels.

References

    1. Tokai N
    2. Fujimoto-Nishiyama A
    3. Toyoshima Y
    4. Yonemura S
    5. Tsukita S
    6. Inoue J
    7. Yamamota T
    (1996)
    Kid, a novel kinesin-like DNA binding protein, is localized to chromosomes and the mitotic spindle
    The EMBO Journal 15:457–467.

Article and author information

Author details

  1. Shinsuke Niwa

    1. Frontier Research Institute for Interdisciplinary Sciences (FRIS), Tohoku University, Sendai, Japan
    2. Graduate School of Life Sciences, Tohoku University, Sendai, Japan
    Contribution
    Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing – original draft, Project administration, Writing – review and editing
    Contributed equally with
    Natsuki Furusaki
    For correspondence
    shinsuke.niwa.c8@tohoku.ac.jp
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8367-9228

  2. Natsuki Furusaki

    Graduate School of Life Sciences, Tohoku University, Sendai, Japan
    Contribution
    Resources, Data curation, Formal analysis, Validation, Investigation
    Contributed equally with
    Shinsuke Niwa
    Competing interests
    No competing interests declared

  3. Tomoki Kita

    Graduate School of Life Sciences, Tohoku University, Sendai, Japan
    Contribution
    Resources, Data curation, Formal analysis, Validation, Investigation
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1531-565X

  4. Yuki Suzuki

    Mie University, Graduate School of Engineering, Tsu, Japan
    Contribution
    Resources, Methodology
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1848-0105

  5. Kyoko Chiba

    Frontier Research Institute for Interdisciplinary Sciences (FRIS), Tohoku University, Sendai, Japan
    Contribution
    Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Project administration, Writing – review and editing
    For correspondence
    kyoko.chiba.e7@tohoku.ac.jp
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6919-2782

Funding

Japan Society for the Promotion of Science (JP23H02472)

  • Shinsuke Niwa

Japan Society for the Promotion of Science (JP23KJ0168)

  • Tomoki Kita

Japan Society for the Promotion of Science (JP22K15053)

  • Kyoko Chiba

Uehara Memorial Foundation

  • Kyoko Chiba

Naito Foundation

  • Kyoko Chiba

Takeda Science Foundation (2024036450)

  • Kyoko Chiba

Astellas Foundation for Research on Metabolic Disorders (2024a1048)

  • Kyoko Chiba

Brain Science Foundation

  • Kyoko Chiba

MEXT Leading Initiative for Excellent Researchers (JPMXS0320200156)

  • Kyoko Chiba

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We would like to thank the members of Niwa lab for useful discussions. We also would like to thank Dr. Atsushi Nakagawa and Mr. Jiye Wang (Osaka University) for technical assistance. SN was supported by JSPS KAKENHI (grant no. JP23H02472). TK was supported by JSPS KAKENHI (grant no. JP23KJ0168). KC was supported by JSPS KAKENHI (grant no. JP22K15053), Uehara Memorial Foundation, Naito Foundation, Takeda Science Foundation (grant no. 2024036450), Astellas Foundation for Research on Metabolic Disorders (grant no. 2024a1048), Brain Science Foundation, and MEXT Leading Initiative for Excellent Researchers (grant no. JPMXS0320200156). This work was performed under the Collaborative Research Program of Institute for Protein Research, Osaka University, CR-24-02.

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  1. Shinsuke Niwa
  2. Natsuki Furusaki
  3. Tomoki Kita
  4. Yuki Suzuki
  5. Kyoko Chiba
(2026)
The chromokinesin Kid (KIF22) forms a homodimer, moves processively along microtubules, and transports double-stranded DNA
eLife 13:RP102828.
https://doi.org/10.7554/eLife.102828.3

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