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 and 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 = 71, 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 = 70, respectively.

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).

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 show 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 are 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 of human Kid. Histograms show the number of counts at 5 nM. Lines show Gaussian fits (mean ± S.D.: 110 ± 21 kDa and 211 ± 21 kDa, respectively).

(D) Mass photometry of Xenopus Kid. Histograms show the number of counts at 5 nM. Lines show Gaussian fits (mean ± S.D.: 117 ± 33 kDa and 208 ± 49 kDa, respectively).

Note that majority of hKid and XKid are dimers in the size exclusion chromatography but they are mostly dissociated to monomers in mass photometry.

Conserved coiled-coil domain is required for the processive motion

(A) Schematic representation illustrating the domain organization of XKid(1-495) and XKid(1-437).

(B) Representative SDS-PAGE analysis of purified XKid(1-495) 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 and D) Representative kymographs showing the motility of 20 pM XKid(1-495) (C) and XKid(1-437) (D) in the presence of 2 mM ATP. Note that no directional movement was detected in XKid(1-437). Scale bars: horizontal 5 µm; vertical 60 seconds.

(E) Dot plots showing the velocity of XKid(1-495) and XKid(1-437). Each dot represents an individual measurement of velocity. Green bars represent mean ± S.D.. n= 57 particles for XKid(1-495). nd, not detected.

(F) Dot plots showing the run length of XKid(1-495) and XKid(1-437). Each dot shows a single datum point. Green bars represent mean ± S.D.. n = 57 particles for XKid(1-495). nd, not detected. (G and H) Schematic drawing of XKidCC-mScarlet (G) and a representative result of size exclusion chromatography (H). XKidCC-mScarlet (magenta) and mScarlet (cyan) are shown.

Untypical neck linker of Kid can support processive movement of KIF1A

(A) Schematic representation illustrating the domain organization of KIF1A(1-393)LZ, XKid(1-495), KIF1A(1-350) and a chimera protein KIF1AMD-XKidSt. Note that KIF1A(1-393)LZ and XKid(1-495) are processive motors and KIF1A(1-350) is a non-processive 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 and E) Representative kymographs showing the motility of KIF1AMD-XKidSt and KIF1A(1-393)LZ in the presence of 2 mM ATP. Note that KIF1AMD-XKidSt exhibits diffusion-like fluctuations while they are moving. This phenomena is not observed in KIF1A(1-393)LZ. Scale bars: horizontal 10 µm; vertical 10 seconds.

(F) Dot plots showing the velocity of KIF1AMD-XKidSt and KIF1A(1-393)LZ. 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.

(G) Dot plots showing the run length of KIF1AMD-XKidSt and KIF1A(1-393)LZ. 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.

DNA movement driven by XKid

1nM of XKid fused with sfGFP and 20 nM of Cy3 labelled double-strand or single-strand DNA were mixed and observed under the TIRF microscopy.

(A-C) Representative kymographs showing the movement of full-length XKid and double-strand DNA (A), full-length XKid and single-strand DNA (B) and XKid(1-495) and double-strand DNA. Scale bars: horizontal, 10 µm; vertical, 60 seconds.

(D) A dot plot showing the frequency of DNA movement along microtubules, normalized by microtubule length (1 µm) and observation time window (1 minute). Each dot represents an individual measurement obtained from observations of different microtubules. N = 22, 10 and 35 microtubules, respectively.

Model

Kid forms dimers and transports chromosomes along prometaphase spindle microtubules by directly binding with genomic DNA.