1. Volume 37, Issue 4, Pages 350-361 (May 2016)
Separase Promotes Microtubule Polymerization by Activating CENP-E-Related Kinesin Kin7 
Panagiotis N. Moschou, Emilio Gutierrez-Beltran, Peter V. Bozhkov, Andrei Smertenko 
Developmental Cell 
Volume 37, Issue 4, Pages (May 2016)
DOI: /j.devcel
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2. Developmental Cell 2016 37, 350-361DOI: (10.1016/j.devcel.2016.04.015)
Copyright © 2016 Elsevier Inc. Terms and Conditions

3. Figure 1 AtESP Interacts with Kin7.3
(A) Yeast two-hybrid analysis of interaction between Kin7.3 fused to the activation domain and three AtESP fragments fused to the binding domain (numbers indicate first and last residues of the corresponding fragment). 2Fe-2S, iron sulfur motif; C50, caspase-like clan C50 domain; DDO, double dropout; QDO, quadruple dropout; LacZ, β-galactosidase. The experiment was repeated ten times with similar results.
(B) AT3G12020 belongs to group 7 of kinesins. Unrooted phylodendrogram is based on the alignment of motor domains of representatives of major groups of kinesins. Each descriptor consists of the kinesin group name, corresponding protein name, and GenBank accession number in parentheses. Numbers indicate bootstrap values.
(C) Mapping the region of Kin7.3 that interacts with AtESP by Y2H analysis. Numbers indicate first and last amino acids of Kin7.3 fragments. DDO, double dropout; QDO, quadruple dropout. The experiment was repeated three times with similar results. Low C, low complexity; CC, coiled coil.
(D) Representative BiFC images of nYFP-AtESPl−791 or full-length nYFP-AtESP and Kin7.3-cYFP in N. benthamiana leaves (3 days post infiltration). Reconstituted YFP labels microtubules. nYFP-AtESPl621−2178 and Kin7.3-cYFP, or Kin7.3-cYFP alone show no reconstituted signal. Insets show higher magnification of the regions in white box. The experiment was repeated three times with similar results. Scale bars, 20 μm.
(E) Quantification of BiFC YFP signal in N. benthamiana leaves (3 days post infiltration) with Kin7.3-cYFP and three fragments of AtESP. Data are mean ± SE of five independent experiments each containing five measurements. Number above bars represents the p value (one-sided Dunnett's test).
(F and G). Co-immunoprecipitation (IP) of tagged (HA- or myc-) versions of AtESP with Kin7.3 expressed in N. benthamiana leaves (3 days post infiltration). Membranes were probed simultaneously with anti-HA and anti-myc. The experiment was repeated four times with similar results.
See also Figure S1.
Developmental Cell  , DOI: ( /j.devcel )
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4. Figure 2
KISC Binds Microtubules and Promotes Microtubule Polymerization
(A) GFP-Kin7.3 binds to cortical microtubules (1), anaphase spindle (2), and phragmoplast (3) in Arabidopsis root cells. Images are representative of more than ten independent T2 lines. Scale bars, 10 μm (1) and 2 μm (2, 3).
(B) GFP-Kin7.3 binds both phragmoplast microtubules (arrow) and the expanding cell plate in Arabidopsis root cells. FM4-64 labels only cell plate (arrow). The experiment was repeated twice with similar results. Scale bar, 10 μm.
(C) Immunostaining of AtESP (green) and Kin7.3 (red) in Arabidopsis root cells. Scale bar, 10 μm.
(D) Co-localization of GFP-Kin7.3 (green) with microtubules (magenta) in double transgenic plants WT;proKin7.3:GFP-Kin7.3/pro35S:tagRFP-MAP4MBD. Yellow arrow indicates a growing microtubule end. Numbers in the upper right corner indicate time in seconds. Images are representative of five individual lines examined.
(E) Kymograph of GFP-Kin7.3 and tagRFP-MAP4MBD shows simultaneous recruitment of both proteins to the growing microtubule end. Horizontal scale bar, 1 μm; vertical scale bar, 29.9 s.
(F) Full-length Kin7.3, but not the motor fragment, can target AtESP to microtubules. AtESP promotes decoration of microtubules by Kin7.3. Images are representative of transient transfection experiments of N. benthamiana leaf pavement cells that were replicated three times with similar results. Scale bars, 20 μm.
(G and H) Parameters of microtubule dynamics in N. benthamiana leaf pavement cells expressing ECFP-Kin7.3m, ECFP-Kin7.3, or mCherry-β-tubulin in the presence or absence of GFP-AtESP (ESP). ECFP signal of Kin7.3 or Kin7.3m was used to trace microtubules. Effect of Kin7.3 on the microtubule dynamics was measured in cells expressing mCherry-β-tubulin, as Kin7.3 does not bind dynamic microtubules in the absence of AtESP. Data are mean ± SE of six independent experiments each containing ten microtubules. Means with different letters are significantly different at p < 0.05 (Student's t test).
(I) Turbidimetric analysis of tubulin polymerization in the presence of Kin7.3m and 1 mM ATP at Kin7.3m/tubulin molar ratios of 1:200, 1:100, and 1:50 or in the absence of Kin7.3m (control). The experiment was repeated three times with similar results.
(J) SDS-PAGE analysis of microtubule pellet polymerized in the absence or presence of Kin7.3m (Kin7.3m/tubulin ratio 1:50) and quantification of corresponding bands. Data are mean ± SD of four independent experiments. Means are significantly different at p < (one-sided Dunnett's test).
(K) Turbidimetric analysis of microtubule polymerization with Kin7.3m and 1 mM ATP or with Kin7.3m and 1 mM AMP-PNP (adenyl imidodiphosphate). Control sample contains tubulin only. The experiment was repeated three times with similar results.
(L) ATPase activity of Kin7.3m depends on microtubules. Data are from a representative assay repeated three times with similar results.
See also Figure S2.
Developmental Cell  , DOI: ( /j.devcel )
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5. Figure 3 Tail Domain of Kin7.3 Inhibits Activity of the Motor Domain
(A) Kin7.3t inhibits microtubule polymerization induced by Kin7.3m. Control reaction contained tubulin only. Data are from a representative turbidimetric assay repeated three times with similar results.
(B) Representative micrographs of microtubules as in (A) after 10 min of polymerization. Scale bar, 10 μm.
(C) Length of microtubules in samples shown in (B). Data are mean ± SD of three independent experiments each containing 111 microtubules. Means with different letters are significantly different at p < 0.05 (Student's t test).
(D) Kin7.3t inhibits ATPase activity of Kin7.3m at molar ratios Kin7.3m:Kin7.3t 1:1 and 1:3. Data are means ± SE of five independent experiments each containing five replicate reactions. Means with different letters are significantly different at p < 0.05 (Student's t test).
(E) ATPase activity of Kin7.3m or Kin7.3 in the presence of immunopurified full-length AtESP or in the presence of both AtESP and Kin7.3t. Data are means ± SD of three independent experiments each containing three replicate reactions. Means with different letters are significantly different at p < 0.05 (Student's t test).
(F) FRET assay of the indicated protein combinations in N. benthamiana leaves (72 hr post infiltration). ECFP-YFP-Kin7.3 was used as a positive control for the most proximal location of the fluorophores (efficiency 21%). Simultaneous co-expression of ECFP-YFP-Kin7.3 or of ECFP-Kin7.3t-YFP with tagRFP-AtESP did not affect FRET efficiency of the ECFP-YFP pair. All proteins were expressed under control of CaMV 35S promoter. Data are means ± SE of seven independent experiments each containing ten replicates. p Value is for one-sided Dunnett's test.
See also Figure S3.
Developmental Cell  , DOI: ( /j.devcel )
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6. Figure 4
AtESPrsw4 Interacts with Kin7.3 but Degrades at Restrictive Temperature
(A) In vitro pull-down assay of GST-tagged AtESP1−1000 (G-AtESP1−1000) or rsw4 mutant (G-AtESP1−1000;rsw4) with h-Kin7.3 at permissive or restrictive temperature. Free GST was used as a negative control. Input corresponds to 5% of the total reaction volume. The experiment was repeated four times with similar results.
(B) Immunoblot analysis of AtESP in WT and rsw4 after 24 hr at permissive (p) or restrictive (r) temperature. Anti-actin was used as a loading control. Five-day-old seedlings were collected and pulverized. Images are from a single experiment that was repeated three times with similar results.
(C) Immunoblot analysis of GFP-AtESPrsw4 degradation in N. benthamiana leaf pavement cells at the indicated time points at permissive (p) or restrictive (r) temperature. Time 0 corresponds to 48 hr post infiltration (minimum time required for AtESP detection). Images are from a single experiment repeated twice with similar results.
(D) Co-IP of GFP-Kin7.3 with AtESP in WT and rsw4 after 24 hr at permissive or restrictive temperature. Note that for the detection of Kin7.3, anti-Kin7.3 antibody was used. As a negative control, we performed co-IP from WT;2xpro35s:GFP-Kin7.3m plants, where we could detect signal in input samples using anti-GFP antibody. Images are from a single experiment that was repeated twice with similar results.
(E) AtESPrsw4 degrades under restrictive temperature in N. benthamiana leaf pavement cells. Time 0 corresponds to 48 hr post infiltration. GFP-AtESPrsw4 signal under restrictive temperature (6 hr) becomes cytoplasmic and partially associated with microtubules. Note the reduced binding of ECFP-Kin7.3 to microtubules and their lower density. Arrows point to the AtESPrsw4 foci on the microtubules. Scale bars, 10 μm.
See also Figure S4.
Developmental Cell  , DOI: ( /j.devcel )
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7. Figure 5 AtESP Interacts with Kin7.1 and Kin7.5
(A) The N terminus of AtESP (AA 1–791) interacts with Kin7.1, Kin7.3, and Kin7.5 in a Y2H assay, but not with Kin7.2 and Kin7.4. Kin7.x indicates an individual member of Kin7 group, where x is 1–5. DDO, double dropout; QDO, quadruple dropout; BD, binding domain; AD, activation domain. Images are from a single representative experiment repeated five times.
(B) Quantification of reconstituted YFP signal in N. benthamiana leaf pavement cells 3 days post infiltration with nYFP-AtESP and Kin7.1-cYFP, Kin7.2-cYFP, Kin7.3-cYFP, Kin7.4-cYFP, or Kin7.5-cYFP. AU, arbitrary units. Data are means ± SE of BiFC signal level measured in five independent experiments each containing ten individual measurements. Means with different letters are significantly different at p < 0.05 (Student's t test).
(C) Domain architecture of Kin7.3 homologs. Low C, low complexity; CC, coiled coil.
(D) Representative images for the localization in root meristem cells of Kin7.1, Kin7.3, and Kin7.5 GFP fusions expressed in WT plants under XVE promoter. Plants were grown on plates containing 2 μM estradiol for 48 hr. The experiment was repeated five times with similar results. Scale bars, 10 μm.
(E) Representative image of N. benthamiana leaf pavement cells transfected with GFP fusions of Kin7.1m or Kin7.5m. Scale bars, 25 μm.
(F) Localization of tail domains GFP-Kin7.1t and GFP-Kin7.5t in N. benthamiana leaf pavement cells (48 hr post infiltration). The GFP-Kin7.1t signal was patchy, suggesting partial protein aggregation. The experiment was repeated twice with similar results. Scale bars, 10 μm.
(G) Immunostaining of AtESP, Kin7.3, or β-tubulin in kin7.1kin7.3kin7.5 mutant root epidermal cells. The lack of staining with anti-Kin7.3 demonstrates specificity of the antibody. Note the reduction of AtESP labeling on phragmoplast microtubules. Data are from a single representative experiment that was repeated three times with similar results.
(H) Representative images showing the labeling of tubulin (green) and myc-AtESP (red) in WT or kin7.1kin7.3kin7.5 (3KO kin) root tips expressing myc-AtESP at similar levels. Scale bars, 10 μm.
(I) Ratio of myc-AtESP signal on the microtubules to the cytoplasmic signal in WT, kin7.1kin7.3kin7.5 (3KO kin), and XVE>Kin7.3t. Data are means ± SE of five independent experiments each containing ten phragmoplasts. Numbers above bars represent the p values of one-sided Dunnett's test.
(J) Expression of myc-AtESP under the control of 35S promoter in WT, kin7.1kin7.3kin7.5, and XVE>Kin7.3t backgrounds. Anti-actin was used to show equal protein loading. The experiment was repeated twice with similar results.
See also Figure S5.
Developmental Cell  , DOI: ( /j.devcel )
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8. Figure 6
KISC Functions in the Establishment of Cell Polarity and in Karyokinesis
(A) Effect of microtubule-destabilizing drug APM (10 nM; 6 days) on the root length of WT and mutant seedlings (ratio of values of treated to untreated samples). Data are means ± SE of five independent experiments each containing 20 treated or untreated seedlings. Means with different letters are significantly different at p < 0.05 (Student's t test).
(B) Root curvature (deviation from the vertical vector, anticlockwise) of WT and mutant seedlings treated with 10 nM APM. Data are means ± SE of five independent experiments each containing 20 seedlings. Means with different letters are significantly different at p < 0.05 (Student's t test).
(C) Root length of WT and mutant seedlings treated with 10 nM APM (3 days permissive, 1 day restrictive, 2 days permissive). Data are means ± SE of five independent experiments each containing 20 seedlings. Note that the restrictive temperature did not have a significant effect on kin7.3 root growth. Means with different letters are significantly different at p < 0.05 (Student's t test).
(D) PIN2 localization in root cortex cells of WT, rsw4, kin7.1kin7.3kin7.5, and rsw4;Kin7.3m. Micrographs are representative of an experiment that was replicated three times. Scale bars, 10 μm.
(E) Frequency of root cortex cells with inverted polarity in WT, WT;Kin7.3m, rsw4, rsw4;Kin7.3m, XVE>Kin7.3t, and kin7.1kin7.3kin7.5. Data are means ± SD of three independent experiments each containing three replicates. Means with different letters are significantly different at p < 0.05 (Student's t test).
(F) Root tip curvature in the gravitropism assay in WT, XVE>Kin7.3t, and kin7.1kin7.3kin7.5. Data are means ± SD of three independent experiments each containing three replicates. Asterisk indicates significant difference from WT at p < 0.05 (Student's t test).
(G) In vitro pull-down assay of G-AtESP1−1000 with h-Kin7.3 or h-Kin7.31−717. 1, h-Kin7.31−717; 2, h-Kin7.3. The experiment was repeated three times with similar results.
(H) Complementation of the root-length phenotype on APM of kin7.1kin7.3kin7.5 with proKin7.3:GFP-Kin7.3. Numbers indicate independent lines. Data are means ± SE of five independent experiments each containing ten seedlings. Means with different letters are significantly different at p < 0.05 (Student's t test).
(I) Effect of microtubule-destabilizing drug APM (10 nM; 6 days) on the root length of WT and XVE>Kin7.3t (ratio of values of treated to untreated samples). Data are means ± SE of five independent experiments each containing 20 treated or untreated seedlings. p Value is for one-sided Dunnett's test.
(J) Representative images of root curvature (deviation from the vertical vector, anticlockwise) of WT and XVE>Kin7.3t seedlings treated with 2 μM estradiol. The experiment was repeated three times with similar results.
(K) Root curvature (deviation from the vertical vector, anticlockwise) of WT and mutant seedlings treated with 10 nM APM. Data are means ± SE of five independent experiments each containing 20 seedlings. p Value is for one-sided Dunnett's test.
(L) Root-length ratio (treated to untreated) of WT, kin7.1kin7.3kin7.5, and XVE>Kin7.3t seedlings after simultaneous application of 2 μM estradiol and 10 nM APM for 5 days. Data are means ± SE of five independent experiments each containing 20 seedlings. Means with different letters are significantly different at p < 0.05 (Student's t test).
(M) Lagging chromosomes in XVE>Kin7.3t line. DNA is shown in blue and microtubules are in red. Images are from a single experiment that was repeated twice with similar results. Scale bars, 50 μm.
(N) Frequency of roots with chromosomal aberrations (lagging chromosomes and chromosome bridges) in WT, rsw4, and XVE>Kin7.3t seedlings 72 hr after induction with 2 μM estradiol at the restrictive temperature. Data are means of two replicates. nd, not detected.
See also Figure S6.
Developmental Cell  , DOI: ( /j.devcel )
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9. Figure 7
Functions of KISC Partially Depend on the Catalytic Activity of AtESP
(A) Microtubule dynamics in WT;pro35S:β-tubulin, WT;2xpro35S:GFP-Kin7.3m, rsw4;pro35S:β-tubulin, rsw4;pro35S:HA-AtESP/pro35S:β-tubulin, rsw4;pro35S:HA-AtESPPD/pro35S:β-tubulin, and rsw4;2xpro35S:GFP-Kin7.3m plants (T2 generation). Data are means ± SE of five independent experiments each containing 20 microtubules. ∗p < 0.05; ∗∗p < 0.01, ∗∗∗p < (two-sided Dunnett's test).
(B) Root-tip curvature in the gravitropism assay in WT, rsw4, and rsw4;Kin7.3m. Data are means ± SD of three independent experiments each containing three replicate reactions. Asterisks indicate significant difference from WT at p < 0.05 (Student's t test).
(C) Co-IP of HA-AtESP or HA-AtESPPD with GFP-Kin7.3. Input was 5% of the total volume. 1, GFP + HA-AtESP; 2, HA-AtESP + GFP-Kin7.3; 3, HA-AtESPPD + GFP-Kin7.3. The experiment was repeated three times with similar results. Numbers on the left-hand side of the blots denote the size of molecular markers in kDa.
(D) GFP-AtESP or GFP-AtESPPD promote even labeling of microtubules by ECFP-Kin7.3. Images are representatives of an experiment that was replicated three times with similar results. Scale bar, 20 μm.
(E) Root meristem width of WT, WT;pro35S:HA-AtESP, WT;pro35S:HA-AtESPPD, and rsw4 plants grown at the restrictive temperature for 72 hr. Data are means ± SE of five independent experiments each containing ten seedlings. Means with different letters are significantly different at p < 0.05 (Student's t test).
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2016 Elsevier Inc. Terms and Conditions