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A second tubulin binding site on the kinesin-13 motor head domain is important during mitosis.

Zhang D, Asenjo AB, Greenbaum M, Xie L, Sharp DJ, Sosa H - PLoS ONE (2013)

Bottom Line: To address this issue, we investigated the in-vitro and in-vivo effects of mutating Kin-Tub-2 family conserved residues on the Drosophila melanogaster kinesin-13, KLP10A.Disruption of the Kin-Tub-2 site, despite not having a deleterious effect on MT depolymerization, results in abnormal mitotic spindles and lagging chromosomes during mitosis in Drosophila S2 cells.The results suggest that the additional Kin-Tub-2 tubulin biding site plays a direct MT attachment role in-vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, New York, United States of America ; State Key Lab of Reproductive Medicine, College of Basic Medicine, Nanjing Medical University, Nanjing, Jiangsu, China.

ABSTRACT
Kinesin-13s are microtubule (MT) depolymerases different from most other kinesins that move along MTs. Like other kinesins, they have a motor or head domain (HD) containing a tubulin and an ATP binding site. Interestingly, kinesin-13s have an additional binding site (Kin-Tub-2) on the opposite side of the HD that contains several family conserved positively charged residues. The role of this site in kinesin-13 function is not clear. To address this issue, we investigated the in-vitro and in-vivo effects of mutating Kin-Tub-2 family conserved residues on the Drosophila melanogaster kinesin-13, KLP10A. We show that the Kin-Tub-2 site enhances tubulin cross-linking and MT bundling properties of KLP10A in-vitro. Disruption of the Kin-Tub-2 site, despite not having a deleterious effect on MT depolymerization, results in abnormal mitotic spindles and lagging chromosomes during mitosis in Drosophila S2 cells. The results suggest that the additional Kin-Tub-2 tubulin biding site plays a direct MT attachment role in-vivo.

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Effect of KLP10A Kin-Tub-2 mutations on S2 cells during mitosis.(A) Western blot of cell line transfected with mRFP-WT-KLP10A (WT cell line). Lanes: WT-rescue: Cells after dsRNAi treatment against endogenous KLP10A untranslated RNA region and induction of exogenous mRFP-WT-KLP10A expression; KLP10A KD: dsRNAi treatment against KLP10A coding sequence; Control: mock dsRNAi treatment; No-label: molecular weight markers ladder. (B) Relative amount of KLP10A proteins in cell lines containing plasmids for mRFP-WT-KLP10A (top) and mRFP-KT2M-KLP10A (bottom) (N=3). (C) Fluorescent images (GFP-α-tubulin) of representative mitotic spindles. (D) Explanation of evaluated spindle metrics. P: pole, E: equatorial plane. SL: spindle length; SW: spindle width; KW: kinetochore area width; SD: standard deviation of the distribution of kinetochore distances to the equatorial plane. AvgDist: mean distance of kinetochores to the equatorial plane. (E) Distribution of metaphase kinetochore positions for all the cells analyzed (31 in each case). (F) Kinetochore distributions mean SD and AvgDist (N=31). (G) Mean SW, KW and SL (N = 39, 42, 39, 41 respectively for Control, KLP10A-KD, WT-rescue, KT2M-rescue). (H) Mean frequency (%) of cells with lagging kinetochores during anaphase-A, monopolar and multipolar spindles (Number of repeat experiments, N=3 for each case).
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pone-0073075-g004: Effect of KLP10A Kin-Tub-2 mutations on S2 cells during mitosis.(A) Western blot of cell line transfected with mRFP-WT-KLP10A (WT cell line). Lanes: WT-rescue: Cells after dsRNAi treatment against endogenous KLP10A untranslated RNA region and induction of exogenous mRFP-WT-KLP10A expression; KLP10A KD: dsRNAi treatment against KLP10A coding sequence; Control: mock dsRNAi treatment; No-label: molecular weight markers ladder. (B) Relative amount of KLP10A proteins in cell lines containing plasmids for mRFP-WT-KLP10A (top) and mRFP-KT2M-KLP10A (bottom) (N=3). (C) Fluorescent images (GFP-α-tubulin) of representative mitotic spindles. (D) Explanation of evaluated spindle metrics. P: pole, E: equatorial plane. SL: spindle length; SW: spindle width; KW: kinetochore area width; SD: standard deviation of the distribution of kinetochore distances to the equatorial plane. AvgDist: mean distance of kinetochores to the equatorial plane. (E) Distribution of metaphase kinetochore positions for all the cells analyzed (31 in each case). (F) Kinetochore distributions mean SD and AvgDist (N=31). (G) Mean SW, KW and SL (N = 39, 42, 39, 41 respectively for Control, KLP10A-KD, WT-rescue, KT2M-rescue). (H) Mean frequency (%) of cells with lagging kinetochores during anaphase-A, monopolar and multipolar spindles (Number of repeat experiments, N=3 for each case).

Mentions: The results of the previous section indicate that the unique Kin-Tub-2 site of kinesin-13 has a significant influence on the way that kinesin-13 interact with MTs in-vitro. However, whether these interactions are important for kinesin-13 function in-vivo is not clear. To investigate whether the positively charged kinesin-13 Kin-Tub-2 site has functional importance in-vivo, we used a knock-down and rescue approach to compare cells during mitosis with or without mutations in the Kin-Tub-2 site of KLP10A. We generated D. melanogaster S2 cells lines transfected with plasmids coding for KLP10A labeled with monomeric red fluorescent protein (mRFP) under the control of a copper-inducible promoter (methods). Two cell lines were made containing plasmids with either the KLP10A WT sequence (WT cell line) or KLP10A with the same Kin-Tub-2 residue changes as the KT2M mutant described in the previous section (KT2M cell line). The relative amounts of endogenous and exogenous KLP10A in the cell lines after distinct dsRNAi treatments and protein expression induction regimens were estimated by western blot analysis (Figure 4A, B). Figure 4C shows representative mitotic spindles images of fixed cells during metaphase and anaphase. Control corresponds to cells treated with dsRNA against no D. melanogaster protein (mock dsRNAi treatment); KLP10A-KD to cells treated with dsRNA against the KLP10A coding sequence (i.e. targeting endogenous and exogenous KLP10A expression). The phenotype of cells after mock dsRNA treatment was similar for WT and the KT2M cell lines so data from these two cell lines were pooled together under a single control category. For the same reason, data from the two cell lines dsRNA treated against the coding sequence of KLP10A were pooled together under a single KLP10A-KD category. WT-rescue and KT2M-rescue correspond to cells dsRNA treated against KLP10A mRNA untranslated regions (UTR, i.e. targeting only endogenous KLP10A expression) followed by induction of exogenous KLP10A expression. In the WT-rescue and KT2M-rescue cells, exogenous KLP10A (WT or KT2M) represents more than 83% of the total KLP10A expressed (Figure 4B). Therefore, differences between these two cell groups can reveal cellular processes where the positively charged Kin-Tub-2 site plays a role.


A second tubulin binding site on the kinesin-13 motor head domain is important during mitosis.

Zhang D, Asenjo AB, Greenbaum M, Xie L, Sharp DJ, Sosa H - PLoS ONE (2013)

Effect of KLP10A Kin-Tub-2 mutations on S2 cells during mitosis.(A) Western blot of cell line transfected with mRFP-WT-KLP10A (WT cell line). Lanes: WT-rescue: Cells after dsRNAi treatment against endogenous KLP10A untranslated RNA region and induction of exogenous mRFP-WT-KLP10A expression; KLP10A KD: dsRNAi treatment against KLP10A coding sequence; Control: mock dsRNAi treatment; No-label: molecular weight markers ladder. (B) Relative amount of KLP10A proteins in cell lines containing plasmids for mRFP-WT-KLP10A (top) and mRFP-KT2M-KLP10A (bottom) (N=3). (C) Fluorescent images (GFP-α-tubulin) of representative mitotic spindles. (D) Explanation of evaluated spindle metrics. P: pole, E: equatorial plane. SL: spindle length; SW: spindle width; KW: kinetochore area width; SD: standard deviation of the distribution of kinetochore distances to the equatorial plane. AvgDist: mean distance of kinetochores to the equatorial plane. (E) Distribution of metaphase kinetochore positions for all the cells analyzed (31 in each case). (F) Kinetochore distributions mean SD and AvgDist (N=31). (G) Mean SW, KW and SL (N = 39, 42, 39, 41 respectively for Control, KLP10A-KD, WT-rescue, KT2M-rescue). (H) Mean frequency (%) of cells with lagging kinetochores during anaphase-A, monopolar and multipolar spindles (Number of repeat experiments, N=3 for each case).
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pone-0073075-g004: Effect of KLP10A Kin-Tub-2 mutations on S2 cells during mitosis.(A) Western blot of cell line transfected with mRFP-WT-KLP10A (WT cell line). Lanes: WT-rescue: Cells after dsRNAi treatment against endogenous KLP10A untranslated RNA region and induction of exogenous mRFP-WT-KLP10A expression; KLP10A KD: dsRNAi treatment against KLP10A coding sequence; Control: mock dsRNAi treatment; No-label: molecular weight markers ladder. (B) Relative amount of KLP10A proteins in cell lines containing plasmids for mRFP-WT-KLP10A (top) and mRFP-KT2M-KLP10A (bottom) (N=3). (C) Fluorescent images (GFP-α-tubulin) of representative mitotic spindles. (D) Explanation of evaluated spindle metrics. P: pole, E: equatorial plane. SL: spindle length; SW: spindle width; KW: kinetochore area width; SD: standard deviation of the distribution of kinetochore distances to the equatorial plane. AvgDist: mean distance of kinetochores to the equatorial plane. (E) Distribution of metaphase kinetochore positions for all the cells analyzed (31 in each case). (F) Kinetochore distributions mean SD and AvgDist (N=31). (G) Mean SW, KW and SL (N = 39, 42, 39, 41 respectively for Control, KLP10A-KD, WT-rescue, KT2M-rescue). (H) Mean frequency (%) of cells with lagging kinetochores during anaphase-A, monopolar and multipolar spindles (Number of repeat experiments, N=3 for each case).
Mentions: The results of the previous section indicate that the unique Kin-Tub-2 site of kinesin-13 has a significant influence on the way that kinesin-13 interact with MTs in-vitro. However, whether these interactions are important for kinesin-13 function in-vivo is not clear. To investigate whether the positively charged kinesin-13 Kin-Tub-2 site has functional importance in-vivo, we used a knock-down and rescue approach to compare cells during mitosis with or without mutations in the Kin-Tub-2 site of KLP10A. We generated D. melanogaster S2 cells lines transfected with plasmids coding for KLP10A labeled with monomeric red fluorescent protein (mRFP) under the control of a copper-inducible promoter (methods). Two cell lines were made containing plasmids with either the KLP10A WT sequence (WT cell line) or KLP10A with the same Kin-Tub-2 residue changes as the KT2M mutant described in the previous section (KT2M cell line). The relative amounts of endogenous and exogenous KLP10A in the cell lines after distinct dsRNAi treatments and protein expression induction regimens were estimated by western blot analysis (Figure 4A, B). Figure 4C shows representative mitotic spindles images of fixed cells during metaphase and anaphase. Control corresponds to cells treated with dsRNA against no D. melanogaster protein (mock dsRNAi treatment); KLP10A-KD to cells treated with dsRNA against the KLP10A coding sequence (i.e. targeting endogenous and exogenous KLP10A expression). The phenotype of cells after mock dsRNA treatment was similar for WT and the KT2M cell lines so data from these two cell lines were pooled together under a single control category. For the same reason, data from the two cell lines dsRNA treated against the coding sequence of KLP10A were pooled together under a single KLP10A-KD category. WT-rescue and KT2M-rescue correspond to cells dsRNA treated against KLP10A mRNA untranslated regions (UTR, i.e. targeting only endogenous KLP10A expression) followed by induction of exogenous KLP10A expression. In the WT-rescue and KT2M-rescue cells, exogenous KLP10A (WT or KT2M) represents more than 83% of the total KLP10A expressed (Figure 4B). Therefore, differences between these two cell groups can reveal cellular processes where the positively charged Kin-Tub-2 site plays a role.

Bottom Line: To address this issue, we investigated the in-vitro and in-vivo effects of mutating Kin-Tub-2 family conserved residues on the Drosophila melanogaster kinesin-13, KLP10A.Disruption of the Kin-Tub-2 site, despite not having a deleterious effect on MT depolymerization, results in abnormal mitotic spindles and lagging chromosomes during mitosis in Drosophila S2 cells.The results suggest that the additional Kin-Tub-2 tubulin biding site plays a direct MT attachment role in-vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, New York, United States of America ; State Key Lab of Reproductive Medicine, College of Basic Medicine, Nanjing Medical University, Nanjing, Jiangsu, China.

ABSTRACT
Kinesin-13s are microtubule (MT) depolymerases different from most other kinesins that move along MTs. Like other kinesins, they have a motor or head domain (HD) containing a tubulin and an ATP binding site. Interestingly, kinesin-13s have an additional binding site (Kin-Tub-2) on the opposite side of the HD that contains several family conserved positively charged residues. The role of this site in kinesin-13 function is not clear. To address this issue, we investigated the in-vitro and in-vivo effects of mutating Kin-Tub-2 family conserved residues on the Drosophila melanogaster kinesin-13, KLP10A. We show that the Kin-Tub-2 site enhances tubulin cross-linking and MT bundling properties of KLP10A in-vitro. Disruption of the Kin-Tub-2 site, despite not having a deleterious effect on MT depolymerization, results in abnormal mitotic spindles and lagging chromosomes during mitosis in Drosophila S2 cells. The results suggest that the additional Kin-Tub-2 tubulin biding site plays a direct MT attachment role in-vivo.

Show MeSH