Full length KLP-6 shows diffusive motion on microtubules

(A) Schematic drawing of the domain organization of KLP-6 motor protein and the full length of KLP-6 fused with sfGFP (KLP-6FL). NC, Neck coiled-coil domain. CC1, Coiled-coil 1 domain. FHA, Forkhead-associated domain. CC2, Coiled-coil 2 domain. MBS, Membrane-associated guanylate kinase homolog (MAGUK)-binding stalk domain. MATH, Meprin and TRAF homology domain. Calculated molecular weight (M.W.) is shown at the right side.

(B) Size exclusion chromatography of KLP-6FL. The SDS-PAGE of the elution fractions is shown beneath the profile. Asterisks indicate fractions used for mass photometry and single molecule assays. The void volume of the column is indicated. Asterisks indicate fractions that are used for mass photometry and TIRF assays. Number shown at the left side indicates a molecular weight standard.

(C) Mass photometry of KLP-6FL. Histogram shows the particle count of KLP-6FL at 20 nM. The line shows a Gaussian fit (mean ± standard deviation (S.D.): 124 ± 15.5 kDa).

(D) Representative kymographs showing the motility of 5 pM KLP-6FL in the presence of 2 mM ATP. Note that KLP-6FL shows only one-dimensional diffusion on microtubules but does not show any processive runs. Horizontal and vertical bars show 10 µm and 10 seconds, respectively.

KLP-6 has a second microtubule binding domain

Schematic drawing of the domain organization of KLP-6 analyzed and representative TIRF images showing the bindings of GFP fused KLP-6 deletion mutant proteins (GFP, green) to microtubules (MT, cyan). The tail domain alone binds to microtubules. Bar, 10 µm.

KLP-6 is converted to a processive motor upon dimerization

(A) Schematic drawing of the domain organization of KLP-6(1-390) and KLP-6(1-390)LZ.

(B) Size exclusion chromatography of KLP-6(1-390) (orange) and KLP-6(1-390)LZ (cyan). The SDS-PAGE of the elution fractions is shown beneath the profiles. The number shown at the left side indicates molecular weight standard. Orange and cyan asterisks indicate fractions used for single molecule assays. Note that the fraction indicated by an orange arrow does not contain KLP-6(1-390) protein.

(C) Representative kymographs showing the motility of 5 pM KLP-6(1-390) and KLP-6(1-390)LZ in the presence of 2 mM ATP. Horizontal and vertical bars show 5 seconds and 5 µm, respectively.

(D) Dot plots showing the velocity of KLP-6(1-390) and KLP-6(1-390)LZ. Each dot shows a single datum point. Green bars represent mean ± S.D.. n = 255 for KLP-6(1-390)LZ. nd, no directional movement was detected in KLP-6(1-390).

(E) Dot plots showing the run length of KLP-6(1-390) and KLP-6(1-390)LZ. Each dot shows a single datum point. Green bars represent median value and interquartile range. n = 255 for KLP-6(1-390)LZ.

n.d., no directional movement was detected in KLP-6(1-390).

KLP-6 is a constitutive monomer

(A) Schematic drawing of the domain organization of KLP-6(1-587) and UNC-104(1-653). Calculated molecular weight is written at the right side.

(B) Size exclusion chromatography of KLP-6(1-587) (plum) and KLP-6(1-587)(D458A) (purple). 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 of KLP-6(1-587) and KLP-6(1-587)(D458A). Histograms show the normalized particle count of KLP-6(1-587) (blue) and KLP-6(1-587)(D458A) (orange) at 40 nM. Lines show Gaussian fits (mean ± S.D.: 94 ± 44 kDa and 88 ± 35 kDa for KLP-6(1-587) and KLP-6(1-587)(D458A), respectively).

(D) Representative kymographs showing the motility of 5 pM KLP-6(1-587) and KLP-6(1-587)(D458A) in the presence of 2 mM ATP. Note that no directional movement was detected in either case. Horizontal and vertical bars show 10 µm and 10 seconds, respectively.

(E) Dot plots showing the landing rate of KLP-6(1-587) and KLP-6(1-587)(D458A). Each dot shows a single datum point. Green bars represent median value. n = 33 microtubules. Mann-Whitney U test. ****, p < 0.0001.

(F) Dot plots showing the dwell time of KLP-6(1-587) and KLP-6(1-587)(D458A) on microtubules. Each dot shows a single datum point. Green bars represent median value and interquartile range. n = 338 and 351 particles for KLP-6(1-587) and KLP-6(1-587)(D458A), respectively. Mann-Whitney U test. ****, p < 0.0001.

UNC-104 is converted to a processive dimer upon autoinhibition release

(A) Schematic drawing of the domain organization of UNC-104(1-653). Calculated molecular weight is written at the right side.

(B) Size exclusion chromatography of UNC-104(1-653) (blue) and UNC-104(1-653)(E412K) (orange). The SDS-PAGE of the elution fractions is shown beneath the profiles. Blue and orange asterisks indicate fractions used for mass photometry and single molecule assays. The number shown at the left side indicates molecular weight standard. Blue arrows indicate expected dimer fraction of wild-type UNC-104(1-653).

(C) Mass photometry of UNC-104(1-653) and UNC-104(1-653)(E412K). Histograms show the normalized particle count of UNC-104(1-653) (blue) and UNC-104(1-653)(E412K) (orange) at 10 nM. Lines show Gaussian fits. UNC-104(1-653) is distributed within 100 ± 15.3 kDa range (mean ± S.D.). UNC-104(1-653)(E412K) shows two peaks consisting of 32% and 27% of particles which are distributed within 100 ± 15.3 kDa and 192 ± 31 kDa range, respectively (mean ± S.D.). Note that wild-type UNC-104(1-653) also has a very small peak around 200 kDa, but the number of datum point is too little for Gaussian fitting.

(D) Representative kymographs showing the motility of 2 nM UNC-104(1-653) and UNC-104(1-653)(E412K) in the presence of 2 mM ATP. Horizontal and vertical bars show 10 µm and 10 seconds, respectively.

(E) Dot plots showing the landing rate of UNC-104(1-653) and UNC-104(1-653)(E412K). Each dot shows a single datum point. Green bars represent median value n = 31 and 30 microtubules for UNC-104(1-653) and UNC-104(1-653)(E412K), respectively. Mann-Whitney U test. ****, p < 0.0001.

(F) Dot plots showing the velocity of UNC-104(1-653) and UNC-104(1-653)(E412K). Each dot shows a single datum point. Green bars represent mean ± S.D.. n = 603 and 624 particles for UNC-104(1-653) and UNC-104(1-653)(E412K). Student t-test, ns, p = 0.7 and statistically not significant.

(G) Dot plots showing the run length of UNC-104(1-653) and UNC-104(1-653)(E412K). Each dot shows a single datum point. Green bars represent median value and interquartile range. n = 603 and 624 particles for UNC-104(1-653) and UNC-104(1-653)(E412K). Mann-Whitney U test. ****, p < 0.0001.

CC2 domain of UNC-104, but not KLP-6, is capable to form a dimer

(A) Coiled coil prediction of KLP-6 (aa 1-587) and UNC-104 (aa 1-653). NC, CC1 and CC2 domains are indicated. Prediction was performed using the Marcoil algorism.

(B) Schematic drawing of the domain organization of mScarlet-I(mSca), KLP-6CC2-mSca and UNC-104CC2-mSca analyzed in panel C.

(C) Size exclusion chromatography of mSca (blue), KLP-6CC2-mSca (plum) and UNC-104CC2-mSca (orange). The SDS-PAGE of the elution fractions is shown beneath the profiles. The number shown at the left side indicates molecular weight standard.

The CC2 domain is essential for the stable dimer formation in UNC-104

(A) Schematic drawing of the domain organization of UNC-104(1-653) and UNC-104(1-594). Calculated molecular weight is shown at the right side.

(B) Size exclusion chromatography of UNC-104(1-594) (blue) and UNC-104(1-594)(E412K) (orange). The SDS-PAGE image of the elution fractions is shown beneath the profiles. The number shown at the left side indicates molecular weight standard. Both proteins show almost identical profile.

(C) Mass photometry of UNC-104(1-594) (blue) and UNC-104(1-594)(E412K) (orange) at 10 nM. Linges show Gaussian fits. Both UNC-104(1-594) and UNC-104(1-594)(E412K) have a single peak which is Gaussian distributed within 98 ± 14.3 kDa range and within 99 ± 13.6 kDa range, respectively (mean ± S.D.).

(D) Representative kymographs showing the motility of 2 nM UNC-104(1-594) and UNC-104(1-594)(E412K) in the presence of 2 mM ATP. Horizontal and vertical bars show 10 µm and 10 seconds, respectively.

(E) Dot plots showing the landing rate of UNC-104(1-594), UNC-104(1-594)(E412K), UNC-104(1-653) and UNC-104(1-653)(E412K). For comparison, data for UNC-104(1-653) and UNC-104(1-653)(E412K) are replotted from Figure 5E. Each dot shows a single datum point. Green bars represent median value and interquartile range. N = 31, 32, 31, 30 microtubules for UNC-104(1-594), UNC-104(1-594)(E412K), UNC-104(1-653) and UNC-104(1-653)(E412K), respectively. Kruskal-Wallis test followed by Dunn’s multiple comparison test. ***, p < 0.001. ****, p < 0.0001. ns, p > 0.05 and statistically not significant.

(F) Dot plots showing the velocity of UNC-104(1-594), UNC-104(1-594)(E412K), UNC-104(1-653) and UNC-104(1-653)(E412K). For comparison, data for UNC-104(1-653) and UNC-104(1-653)(E412K) are replotted from Figure 5F. Green bars represent mean ± S.D.. n = 467, 609, 603, 624 particles for UNC-104(1-594), UNC-104(1-594)(E412K), UNC-104(1-653) and UNC-104(1-653)(E412K), respectively. One-way ANOVA test followed by Šidák’s multiple comparison test. ns, p > 0.05 and statistically not significant.

(G) Dot plots showing the run length of UNC-104(1-594), UNC-104(1-594)(E412K), UNC-104(1-653) and UNC-104(1-653)(E412K). For comparison, data for UNC-104(1-653) and UNC-104(1-653)(E412K) are replotted from Figure 5G. Each dot shows a single datum point. Green bars represent median value and interquartile range. Kruskal-Wallis test followed by Dunn’s multiple comparison test. n = 467, 609, 603, 624 particles for UNC-104(1-594), UNC-104(1-594)(E412K), UNC-104(1-653) and UNC-104(1-653)(E412K), respectively. ****, p < 0.0001. ns, p > 0.05 and statistically not significant.