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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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EM of KLP10AHD induced bundles.(A–D) Gallery of representative EM structures observed when incubating KLP10AHD and MTs. (E) Image of two MTs cross-linked by KLP10AHD molecules (indicated by the red arrows). The area connecting the two microtubules was enhanced by applying a 1/8 nm-1 layer line filter in Fourier space. (F) Interpretation of the observed EM structures. MTs and PFs in ochre; KLP10AHD represented as two color spheres, half red (Kin-Tub-1 side) and half pink (Kin-Tub-2 side).
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pone-0073075-g003: EM of KLP10AHD induced bundles.(A–D) Gallery of representative EM structures observed when incubating KLP10AHD and MTs. (E) Image of two MTs cross-linked by KLP10AHD molecules (indicated by the red arrows). The area connecting the two microtubules was enhanced by applying a 1/8 nm-1 layer line filter in Fourier space. (F) Interpretation of the observed EM structures. MTs and PFs in ochre; KLP10AHD represented as two color spheres, half red (Kin-Tub-1 side) and half pink (Kin-Tub-2 side).

Mentions: To further understand the bundling mechanism, we examined KLP10AHD-MT bundles by electron microscopy (EM). We concentrated our analysis on bundles with few MTs where the connections between MTs could be most easily discerned. However, numerous bundles with many MTs superimposed (not shown) were also observed. A gallery of representative micrographs is shown in Figure 3A–D. All images correspond to MTs incubated with the KLP10AHD WT construct in the presence of ATP, except the one in the bottom of panel 3B that was obtained in the presence of the non-hydrolysable ATP analogue AMPPNP. Panel A shows an example of the typical depolymerization rings observed at MTs ends when MTs are incubated with KLP10A in the presence of ATP [10]. Panel 3B shows examples of the spirals and rings that kinesin-13 constructs form around MTs. Interestingly, we observed the formation of these spirals in the presence of ATP (Figure 3B top), although with less frequency than in the presence of AMPPNP. The fact that these oligomeric rings can also form in the presence of ATP, the natural kinesin substrate, suggests that similar rings could be formed in physiological conditions during MT depolymerization. Note also that the long single PFs observed are not an artifact due to the presence of Taxol or other MT stabilizing agents as they have also been observed in the absence of any tubulin drug [10]. Panel 3C show examples of MTs linked to other MTs through a PF and panels 3C and 3D MTs cross-linked with what appears to be KLP10AHD molecules regularly arranged between MTs. The molecules bridging MTs are regularly spaced every ~8 nm as is expected from kinesin decorated MTs. However, different from other kinesins, KLP10AHD decoration is concentrated to the area between MTs instead of decorating the whole tubulin lattice of each MT. This decoration pattern suggests an increased KLP10A affinity for MTs when both tubulin binding sites can be occupied. Figure 3E shows the area between MTs after applying a filter to reduce noise. The filtered image shows what appears to be a row of KLP10AHD molecules bridging the two MTs, consistent with a model in which a KLP10AHD molecule cross-link two adjacent MTs through Kin-Tub-1 interactions to one MT and Kin-Tub-2 interactions to the other. Figure 3F shows a summary of possible KLP10AHD interactions leading to MT bundling, based on the observed EM structures.


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)

EM of KLP10AHD induced bundles.(A–D) Gallery of representative EM structures observed when incubating KLP10AHD and MTs. (E) Image of two MTs cross-linked by KLP10AHD molecules (indicated by the red arrows). The area connecting the two microtubules was enhanced by applying a 1/8 nm-1 layer line filter in Fourier space. (F) Interpretation of the observed EM structures. MTs and PFs in ochre; KLP10AHD represented as two color spheres, half red (Kin-Tub-1 side) and half pink (Kin-Tub-2 side).
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Related In: Results  -  Collection

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pone-0073075-g003: EM of KLP10AHD induced bundles.(A–D) Gallery of representative EM structures observed when incubating KLP10AHD and MTs. (E) Image of two MTs cross-linked by KLP10AHD molecules (indicated by the red arrows). The area connecting the two microtubules was enhanced by applying a 1/8 nm-1 layer line filter in Fourier space. (F) Interpretation of the observed EM structures. MTs and PFs in ochre; KLP10AHD represented as two color spheres, half red (Kin-Tub-1 side) and half pink (Kin-Tub-2 side).
Mentions: To further understand the bundling mechanism, we examined KLP10AHD-MT bundles by electron microscopy (EM). We concentrated our analysis on bundles with few MTs where the connections between MTs could be most easily discerned. However, numerous bundles with many MTs superimposed (not shown) were also observed. A gallery of representative micrographs is shown in Figure 3A–D. All images correspond to MTs incubated with the KLP10AHD WT construct in the presence of ATP, except the one in the bottom of panel 3B that was obtained in the presence of the non-hydrolysable ATP analogue AMPPNP. Panel A shows an example of the typical depolymerization rings observed at MTs ends when MTs are incubated with KLP10A in the presence of ATP [10]. Panel 3B shows examples of the spirals and rings that kinesin-13 constructs form around MTs. Interestingly, we observed the formation of these spirals in the presence of ATP (Figure 3B top), although with less frequency than in the presence of AMPPNP. The fact that these oligomeric rings can also form in the presence of ATP, the natural kinesin substrate, suggests that similar rings could be formed in physiological conditions during MT depolymerization. Note also that the long single PFs observed are not an artifact due to the presence of Taxol or other MT stabilizing agents as they have also been observed in the absence of any tubulin drug [10]. Panel 3C show examples of MTs linked to other MTs through a PF and panels 3C and 3D MTs cross-linked with what appears to be KLP10AHD molecules regularly arranged between MTs. The molecules bridging MTs are regularly spaced every ~8 nm as is expected from kinesin decorated MTs. However, different from other kinesins, KLP10AHD decoration is concentrated to the area between MTs instead of decorating the whole tubulin lattice of each MT. This decoration pattern suggests an increased KLP10A affinity for MTs when both tubulin binding sites can be occupied. Figure 3E shows the area between MTs after applying a filter to reduce noise. The filtered image shows what appears to be a row of KLP10AHD molecules bridging the two MTs, consistent with a model in which a KLP10AHD molecule cross-link two adjacent MTs through Kin-Tub-1 interactions to one MT and Kin-Tub-2 interactions to the other. Figure 3F shows a summary of possible KLP10AHD interactions leading to MT bundling, based on the observed EM structures.

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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