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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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Poleward flux rate and KLP10A-MT pole attachment models.(A) Fluorescent live cell images before and after a bleaching beam line was flashed to each side of the mitotic spindle. Poleward flux causes the bleach mark to move towards the spindle poles. The original and current positions of the bleach mark front are indicated by the green and white arrows respectively. Side by side pre-bleach images of the GFP-α-tubulin and mRFP channels are shown. Only the GFP-α-tubulin channel was recorded in the time series after the bleaching flash. (B) Mean poleward flux rates (N=60, 72, 52, 40 respectively for Control, KLP10A-KD, WT-rescue, KT2M-rescue).
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pone-0073075-g005: Poleward flux rate and KLP10A-MT pole attachment models.(A) Fluorescent live cell images before and after a bleaching beam line was flashed to each side of the mitotic spindle. Poleward flux causes the bleach mark to move towards the spindle poles. The original and current positions of the bleach mark front are indicated by the green and white arrows respectively. Side by side pre-bleach images of the GFP-α-tubulin and mRFP channels are shown. Only the GFP-α-tubulin channel was recorded in the time series after the bleaching flash. (B) Mean poleward flux rates (N=60, 72, 52, 40 respectively for Control, KLP10A-KD, WT-rescue, KT2M-rescue).

Mentions: The rate of MT depolymerization at the spindle poles, which drives tubulin poleward flux, is modulated by kinesin-13s and, depending on the cell type, can be a major contributor to anaphase-A chromatid to pole movement [30]. In Drosophila cells, KLP10A inhibition results in an almost complete suppression of poleward flux [31]. To assess whether Kin-Tub-2 mutations affect the ability of kinesin-13s to depolymerize MTs in-vivo, we measured the anaphase-A poleward flux rates of the cell groups described in the previous section using fluorescent live cell imaging (Figure 5, Movie S2). Figure 5A (left) shows side by side mRFP (KLP10A) and GFP (tubulin) channels of mitotic spindles before the onset of anaphase. After induction of mRFP-KLP10A expression either in WT or the KT2M mutant, the protein concentrates at spindle poles and kinetochores, as has been previously described for endogenous and GFP labeled KLP10A [7,21]. KLP10A reduction (KLP10A KD) produces significant decrease of poleward flux rate relative to control (Figure 5B) as previously reported [7,32]. Exogenous expression of mRFP-KLP10A rescues the poleward-flux rates but the values were not significatively different whether the exogenous protein expressed was wild type or the Kin-Tub-2 mutant (WT-rescue vs. KT2M-rescue). These results indicate that disruption of the Kin-Tub-2 site does not interfere with KLP10A localization to the mitotic spindle or with the poleward flux-rate. These are not unexpected results considering that KT2M mutations do not inhibit MT depolymerization activity in-vitro (Figure 2D) and that residues important for cellular localization of KLP10A and other kinesis-13s are found outside the HD [33–36]. On the other hand, the mitotic defects associated with the KT2M mutations (previous section) cannot be attributed to KLP10A mislocalization or impaired depolymerization activity. We instead propose that kinesin-13s have a MT binding role associated with their unique Kin-Tub-2 binding site and that this role is independent of MT depolymerization activity. Disruption of the Kin-Tub-2 site would impair MT binding and produce the phenotype observed in KT2M-rescue cells.


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)

Poleward flux rate and KLP10A-MT pole attachment models.(A) Fluorescent live cell images before and after a bleaching beam line was flashed to each side of the mitotic spindle. Poleward flux causes the bleach mark to move towards the spindle poles. The original and current positions of the bleach mark front are indicated by the green and white arrows respectively. Side by side pre-bleach images of the GFP-α-tubulin and mRFP channels are shown. Only the GFP-α-tubulin channel was recorded in the time series after the bleaching flash. (B) Mean poleward flux rates (N=60, 72, 52, 40 respectively for Control, KLP10A-KD, WT-rescue, KT2M-rescue).
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Related In: Results  -  Collection

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pone-0073075-g005: Poleward flux rate and KLP10A-MT pole attachment models.(A) Fluorescent live cell images before and after a bleaching beam line was flashed to each side of the mitotic spindle. Poleward flux causes the bleach mark to move towards the spindle poles. The original and current positions of the bleach mark front are indicated by the green and white arrows respectively. Side by side pre-bleach images of the GFP-α-tubulin and mRFP channels are shown. Only the GFP-α-tubulin channel was recorded in the time series after the bleaching flash. (B) Mean poleward flux rates (N=60, 72, 52, 40 respectively for Control, KLP10A-KD, WT-rescue, KT2M-rescue).
Mentions: The rate of MT depolymerization at the spindle poles, which drives tubulin poleward flux, is modulated by kinesin-13s and, depending on the cell type, can be a major contributor to anaphase-A chromatid to pole movement [30]. In Drosophila cells, KLP10A inhibition results in an almost complete suppression of poleward flux [31]. To assess whether Kin-Tub-2 mutations affect the ability of kinesin-13s to depolymerize MTs in-vivo, we measured the anaphase-A poleward flux rates of the cell groups described in the previous section using fluorescent live cell imaging (Figure 5, Movie S2). Figure 5A (left) shows side by side mRFP (KLP10A) and GFP (tubulin) channels of mitotic spindles before the onset of anaphase. After induction of mRFP-KLP10A expression either in WT or the KT2M mutant, the protein concentrates at spindle poles and kinetochores, as has been previously described for endogenous and GFP labeled KLP10A [7,21]. KLP10A reduction (KLP10A KD) produces significant decrease of poleward flux rate relative to control (Figure 5B) as previously reported [7,32]. Exogenous expression of mRFP-KLP10A rescues the poleward-flux rates but the values were not significatively different whether the exogenous protein expressed was wild type or the Kin-Tub-2 mutant (WT-rescue vs. KT2M-rescue). These results indicate that disruption of the Kin-Tub-2 site does not interfere with KLP10A localization to the mitotic spindle or with the poleward flux-rate. These are not unexpected results considering that KT2M mutations do not inhibit MT depolymerization activity in-vitro (Figure 2D) and that residues important for cellular localization of KLP10A and other kinesis-13s are found outside the HD [33–36]. On the other hand, the mitotic defects associated with the KT2M mutations (previous section) cannot be attributed to KLP10A mislocalization or impaired depolymerization activity. We instead propose that kinesin-13s have a MT binding role associated with their unique Kin-Tub-2 binding site and that this role is independent of MT depolymerization activity. Disruption of the Kin-Tub-2 site would impair MT binding and produce the phenotype observed in KT2M-rescue cells.

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