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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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KLP10A mediated MTs pole attachment models.(A) Four possible KLP10A mediated MT pole attachment models based on the structures observed by EM (Figure 3). 1: Multiple pole-attached KLP10As depolymerize and keep the MT end attached to the pole. 2: Multiple KLP10As crosslink pole-unattached MTs to pole-attached ones. 3: Pole-unattached MTs interact with pole-attached ones through PFs with multiple KLP10As bound. 4: Ring of pole attached KLP10As and tubulin form a sleeve around a MT attaching it to the pole. All the MT-pole attachment models proposed could coexist together and analogous mechanisms could contribute to MT-kinetochore attachments. Structures resembling model 1 has been observed in the kinetochores of mammalian cells [54]. (B) Model to explain observed KLP10A KT2M rescue phenotype of S2 cells. Top: In control and WT-rescue rescue cells the MTs comprising each kinetochore fiber (K-fiber) are tightly bundled and attached to poles and kinetochores. Bottom: In KT2M-rescue cells the altered Kin-Tub-2 binding site of KLP10A results in the detachment and separation of MTs from the K-fiber bundles. Random uneven loss of MTs from sister K-fibers results in kinetochore misalignments. The MTs separated from poles and kinetochores lead to the appearance of a larger and less organized spindle. Red/pink spheres: KLP10AHD; Red strings: KLP10A N and C terminal tails; Ochre cylinders and lines: MTs, Orange blob: MT capping proteins; Gray circles: spindle pole matrix in gray; Black ellipses: kinetochores.
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pone-0073075-g006: KLP10A mediated MTs pole attachment models.(A) Four possible KLP10A mediated MT pole attachment models based on the structures observed by EM (Figure 3). 1: Multiple pole-attached KLP10As depolymerize and keep the MT end attached to the pole. 2: Multiple KLP10As crosslink pole-unattached MTs to pole-attached ones. 3: Pole-unattached MTs interact with pole-attached ones through PFs with multiple KLP10As bound. 4: Ring of pole attached KLP10As and tubulin form a sleeve around a MT attaching it to the pole. All the MT-pole attachment models proposed could coexist together and analogous mechanisms could contribute to MT-kinetochore attachments. Structures resembling model 1 has been observed in the kinetochores of mammalian cells [54]. (B) Model to explain observed KLP10A KT2M rescue phenotype of S2 cells. Top: In control and WT-rescue rescue cells the MTs comprising each kinetochore fiber (K-fiber) are tightly bundled and attached to poles and kinetochores. Bottom: In KT2M-rescue cells the altered Kin-Tub-2 binding site of KLP10A results in the detachment and separation of MTs from the K-fiber bundles. Random uneven loss of MTs from sister K-fibers results in kinetochore misalignments. The MTs separated from poles and kinetochores lead to the appearance of a larger and less organized spindle. Red/pink spheres: KLP10AHD; Red strings: KLP10A N and C terminal tails; Ochre cylinders and lines: MTs, Orange blob: MT capping proteins; Gray circles: spindle pole matrix in gray; Black ellipses: kinetochores.

Mentions: The observed phenotypes of the KT2M-rescue cells can be accounted by models in which multiple kinesin-13 tubulin binding sites keep MTs close together and attached to the spindle pole (Figure 6A). In these models pole-bound-Kinesin-13s interacting with MTs using either of their two HD binding sites would keep MTs attached to the poles, even while they are adding or loosing tubulin subunits from their ends. The models are analogous to the type of dynamic attachments that some kinetochore proteins are thought to keep with the MT plus end. Many kinesin-13 HDs could mediate weak interactions with the MT that would keep a grip on the MT end as in a modified Hill type mechanism [37,38]. In addition, the fact that kinesin-13s can form oligomeric rings encircling MTs (Figure 3B) suggests that mechanisms analogous to the one proposed for the yeast Dam1 ring complex [39,40] could also be at play. A direct MT attachment role for Kinesin-13s is supported by in-vitro assays indicating that kinesin-13s are able to hold the end of a depolymerizing MT under tension [41].


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)

KLP10A mediated MTs pole attachment models.(A) Four possible KLP10A mediated MT pole attachment models based on the structures observed by EM (Figure 3). 1: Multiple pole-attached KLP10As depolymerize and keep the MT end attached to the pole. 2: Multiple KLP10As crosslink pole-unattached MTs to pole-attached ones. 3: Pole-unattached MTs interact with pole-attached ones through PFs with multiple KLP10As bound. 4: Ring of pole attached KLP10As and tubulin form a sleeve around a MT attaching it to the pole. All the MT-pole attachment models proposed could coexist together and analogous mechanisms could contribute to MT-kinetochore attachments. Structures resembling model 1 has been observed in the kinetochores of mammalian cells [54]. (B) Model to explain observed KLP10A KT2M rescue phenotype of S2 cells. Top: In control and WT-rescue rescue cells the MTs comprising each kinetochore fiber (K-fiber) are tightly bundled and attached to poles and kinetochores. Bottom: In KT2M-rescue cells the altered Kin-Tub-2 binding site of KLP10A results in the detachment and separation of MTs from the K-fiber bundles. Random uneven loss of MTs from sister K-fibers results in kinetochore misalignments. The MTs separated from poles and kinetochores lead to the appearance of a larger and less organized spindle. Red/pink spheres: KLP10AHD; Red strings: KLP10A N and C terminal tails; Ochre cylinders and lines: MTs, Orange blob: MT capping proteins; Gray circles: spindle pole matrix in gray; Black ellipses: kinetochores.
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getmorefigures.php?uid=PMC3755979&req=5

pone-0073075-g006: KLP10A mediated MTs pole attachment models.(A) Four possible KLP10A mediated MT pole attachment models based on the structures observed by EM (Figure 3). 1: Multiple pole-attached KLP10As depolymerize and keep the MT end attached to the pole. 2: Multiple KLP10As crosslink pole-unattached MTs to pole-attached ones. 3: Pole-unattached MTs interact with pole-attached ones through PFs with multiple KLP10As bound. 4: Ring of pole attached KLP10As and tubulin form a sleeve around a MT attaching it to the pole. All the MT-pole attachment models proposed could coexist together and analogous mechanisms could contribute to MT-kinetochore attachments. Structures resembling model 1 has been observed in the kinetochores of mammalian cells [54]. (B) Model to explain observed KLP10A KT2M rescue phenotype of S2 cells. Top: In control and WT-rescue rescue cells the MTs comprising each kinetochore fiber (K-fiber) are tightly bundled and attached to poles and kinetochores. Bottom: In KT2M-rescue cells the altered Kin-Tub-2 binding site of KLP10A results in the detachment and separation of MTs from the K-fiber bundles. Random uneven loss of MTs from sister K-fibers results in kinetochore misalignments. The MTs separated from poles and kinetochores lead to the appearance of a larger and less organized spindle. Red/pink spheres: KLP10AHD; Red strings: KLP10A N and C terminal tails; Ochre cylinders and lines: MTs, Orange blob: MT capping proteins; Gray circles: spindle pole matrix in gray; Black ellipses: kinetochores.
Mentions: The observed phenotypes of the KT2M-rescue cells can be accounted by models in which multiple kinesin-13 tubulin binding sites keep MTs close together and attached to the spindle pole (Figure 6A). In these models pole-bound-Kinesin-13s interacting with MTs using either of their two HD binding sites would keep MTs attached to the poles, even while they are adding or loosing tubulin subunits from their ends. The models are analogous to the type of dynamic attachments that some kinetochore proteins are thought to keep with the MT plus end. Many kinesin-13 HDs could mediate weak interactions with the MT that would keep a grip on the MT end as in a modified Hill type mechanism [37,38]. In addition, the fact that kinesin-13s can form oligomeric rings encircling MTs (Figure 3B) suggests that mechanisms analogous to the one proposed for the yeast Dam1 ring complex [39,40] could also be at play. A direct MT attachment role for Kinesin-13s is supported by in-vitro assays indicating that kinesin-13s are able to hold the end of a depolymerizing MT under tension [41].

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
Related in: MedlinePlus