The Experts below are selected from a list of 264 Experts worldwide ranked by ideXlab platform
Hernando Sosa - One of the best experts on this subject based on the ideXlab platform.
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exploring the mechanism of microtubule depolymerization by the Kinesin 13 klp10a and it s phosphoregulation
Biophysical Journal, 2017Co-Authors: Matthieu P M H Benoit, Ana B Asenjo, Gary J Gerfen, David J Sharp, Daniel J Diazvalencia, Hernando SosaAbstract:Kinesins-13s are non-motile molecular motors that regulate microtubule (MT) dynamics by promoting MT depolymerization activity at the MT tips. We have used cryo-electron microscopy and functional assays to investigate the mechanism of action and regulation of the Drosophila melanogaster Kinesin-13 KLP10A. Previous work has shown that phosphorylation of serine 573 in the KLP10A motor domain down regulates depolymerization activity. We have obtained cryo-EM sub-nanometer resolution maps of constructs that include the KLP10A motor and N-terminal neck domains bound to MTs and to tubulin depolymerization intermediates. Comparison of these structures revealed key features of the depolymerization process. To study the down-regulation mechanism by phosphorylation we have combined a variety of cell biology and biophysical methods and compared the behavior of phosphomutant (S573E) and wild-type (WT) KLP10A. Surprisingly, we found that the binding affinity and ATPase activity stimulated by curved tubulin polymers (mimicking MT ends) of WT-KLP10A and S573E-KLP10A were relatively similar (kcat of 1.8 s−1 and 2.5 s−1 respectively). In contrast, we found significant differences in their MT-lattice stimulated ATPases (kcat of 0.56 s−1 and 0.14 s−1 respectively). We also found that the mutant S573E had shorter residence time relative to WT (τ of 5 s and 28 s respectively) while undergoing one-dimensional diffusion over the MT lattice. These results suggest a model in which phosphorylation of S573 reduce KLP10 MT depolymerization activity by decreasing the probability of reaching the MT ends by one dimensional diffusion. Consistent with this model we found by fluorescent live cell imaging that KLP10A-S573E MRFP labeled constructs accumulate significantly less at the tip of growing MTs than WT-KLP10A MRFP labeled constructs.
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distinct interaction modes of the Kinesin 13 motor domain with the microtubule
Biophysical Journal, 2016Co-Authors: Chandrima Chatterjee, Matthieu P M H Benoit, Vania Depaoli, Juan D Diazvalencia, Ana B Asenjo, Gary J Gerfen, David J Sharp, Hernando SosaAbstract:Kinesins-13s are members of the Kinesin superfamily of motor proteins that depolymerize microtubules (MTs) and have no motile activity. Instead of generating unidirectional movement over the MT lattice, like most other Kinesins, Kinesins-13s undergo one-dimensional diffusion (ODD) and induce depolymerization at the MT ends. To understand the mechanism of ODD and the origin of the distinct Kinesin-13 functionality, we used ensemble and single-molecule fluorescence polarization microscopy to analyze the behavior and conformation of Drosophila melanogaster Kinesin-13 KLP10A protein constructs bound to the MT lattice. We found that KLP10A interacts with the MT in two coexisting modes: one in which the motor domain binds with a specific orientation to the MT lattice and another where the motor domain is very mobile and able to undergo ODD. By comparing the orientation and dynamic behavior of mutated and deletion constructs we conclude that 1) the Kinesin-13 class specific neck domain and loop-2 help orienting the motor domain relative to the MT. 2) During ODD the KLP10A motor-domain changes orientation rapidly (rocks or tumbles). 3) The motor domain alone is capable of undergoing ODD. 4) A second tubulin binding site in the KLP10A motor domain is not critical for ODD. 5) The neck domain is not the element preventing KLP10A from binding to the MT lattice like motile Kinesins.
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exploring the mechanisms of a phosphorylation induced inhibition of microtubule depolymerization in the Kinesin 13 klp10a
Biophysical Journal, 2016Co-Authors: Matthieu P M H Benoit, Ana B Asenjo, Gary J Gerfen, David J Sharp, Daniel J Diaz, Hernando SosaAbstract:Kinesins 13 are a class of non-motile molecular motors able to regulate microtubule dynamics through their depolymerization activity at microtubule tips. A previous study in our lab on Drosophila Melanogaster KLP10A found a phosphorylation site within the motor domain that reduces microtubule depolymerization activity. This led us to study the inhibition mechanisms related to this phosphorylation site distant from the ATPase site. We have employed an integrated approach to compare the behavior of phosphomutant and wild-type KLP10A constructs with cell biology experiments and a variety of in-vitro biophysical methods. Imaging insect cells having only fluorescently labelled KLP10A variants revealed that the phosphomutant is distributed homogeneously on microtubules (lattice and tips) in contrast with the wild-type or a non-phosphorylatable mutant showing higher Kinesin concentration on microtubule tips. Wild-type and phosphomutant neck-motor Kinesins constructs have similar properties with isolated structures that mimic these microtubule tips: they can both stabilize curved microtubule protofilaments, an important property for microtubule depolymerization, and have similar affinity and ATPase activity on curved tubulin. However the phosphomutation strongly reduces the microtubule lattice stimulated ATPase activity despite non distinguishable microtubule lattice affinities. Single molecule fluorescence polarization microscopy revealed nucleotide dependent differences in the lattice diffusive behavior between the constructs. EPR and cryo-EM experiments are used to compare the Kinesins on the microtubule lattice vs curved protofilaments. The data are compatible with a reduced microtubule depolymerization activity of the phosphomutant caused by a reduction of the lattice stimulated ATPase activity. This reduced activity constrains the Kinesin to be in nucleotide states which restrict it from reaching the microtubule ends in comparison to the unphosphorylated Kinesin.
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a second tubulin binding site on the Kinesin 13 motor head domain is important during mitosis
PLOS ONE, 2013Co-Authors: Ana B Asenjo, David J Sharp, Dong Zhang, Michaela Greenbaum, Luping Xie, Hernando SosaAbstract: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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structural model for tubulin recognition and deformation by Kinesin 13 microtubule depolymerases
Cell Reports, 2013Co-Authors: Ana B Asenjo, Chandrima Chatterjee, Vania Depaoli, Dongyan Tan, William J Rice, Ruben Diazavalos, Mariena Silvestry, Hernando SosaAbstract:Summary To elucidate the structural basis of the mechanism of microtubule depolymerization by Kinesin-13s, we analyzed complexes of tubulin and the Drosophila melanogaster Kinesin-13 KLP10A by electron microscopy (EM) and fluorescence polarization microscopy. We report a nanometer-resolution (1.1 nm) cryo-EM three-dimensional structure of the KLP10A head domain (KLP10AHD) bound to curved tubulin. We found that binding of KLP10AHD induces a distinct tubulin configuration with displacement (shear) between tubulin subunits in addition to curvature. In this configuration, the Kinesin-binding site differs from that in straight tubulin, providing an explanation for the distinct interaction modes of Kinesin-13s with the microtubule lattice or its ends. The KLP10AHD-tubulin interface comprises three areas of interaction, suggesting a crossbow-type tubulin-bending mechanism. These areas include the Kinesin-13 family conserved KVD residues, and as predicted from the crossbow model, mutating these residues changes the orientation and mobility of KLP10AHDs interacting with the microtubule.
Ana B Asenjo - One of the best experts on this subject based on the ideXlab platform.
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exploring the mechanism of microtubule depolymerization by the Kinesin 13 klp10a and it s phosphoregulation
Biophysical Journal, 2017Co-Authors: Matthieu P M H Benoit, Ana B Asenjo, Gary J Gerfen, David J Sharp, Daniel J Diazvalencia, Hernando SosaAbstract:Kinesins-13s are non-motile molecular motors that regulate microtubule (MT) dynamics by promoting MT depolymerization activity at the MT tips. We have used cryo-electron microscopy and functional assays to investigate the mechanism of action and regulation of the Drosophila melanogaster Kinesin-13 KLP10A. Previous work has shown that phosphorylation of serine 573 in the KLP10A motor domain down regulates depolymerization activity. We have obtained cryo-EM sub-nanometer resolution maps of constructs that include the KLP10A motor and N-terminal neck domains bound to MTs and to tubulin depolymerization intermediates. Comparison of these structures revealed key features of the depolymerization process. To study the down-regulation mechanism by phosphorylation we have combined a variety of cell biology and biophysical methods and compared the behavior of phosphomutant (S573E) and wild-type (WT) KLP10A. Surprisingly, we found that the binding affinity and ATPase activity stimulated by curved tubulin polymers (mimicking MT ends) of WT-KLP10A and S573E-KLP10A were relatively similar (kcat of 1.8 s−1 and 2.5 s−1 respectively). In contrast, we found significant differences in their MT-lattice stimulated ATPases (kcat of 0.56 s−1 and 0.14 s−1 respectively). We also found that the mutant S573E had shorter residence time relative to WT (τ of 5 s and 28 s respectively) while undergoing one-dimensional diffusion over the MT lattice. These results suggest a model in which phosphorylation of S573 reduce KLP10 MT depolymerization activity by decreasing the probability of reaching the MT ends by one dimensional diffusion. Consistent with this model we found by fluorescent live cell imaging that KLP10A-S573E MRFP labeled constructs accumulate significantly less at the tip of growing MTs than WT-KLP10A MRFP labeled constructs.
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distinct interaction modes of the Kinesin 13 motor domain with the microtubule
Biophysical Journal, 2016Co-Authors: Chandrima Chatterjee, Matthieu P M H Benoit, Vania Depaoli, Juan D Diazvalencia, Ana B Asenjo, Gary J Gerfen, David J Sharp, Hernando SosaAbstract:Kinesins-13s are members of the Kinesin superfamily of motor proteins that depolymerize microtubules (MTs) and have no motile activity. Instead of generating unidirectional movement over the MT lattice, like most other Kinesins, Kinesins-13s undergo one-dimensional diffusion (ODD) and induce depolymerization at the MT ends. To understand the mechanism of ODD and the origin of the distinct Kinesin-13 functionality, we used ensemble and single-molecule fluorescence polarization microscopy to analyze the behavior and conformation of Drosophila melanogaster Kinesin-13 KLP10A protein constructs bound to the MT lattice. We found that KLP10A interacts with the MT in two coexisting modes: one in which the motor domain binds with a specific orientation to the MT lattice and another where the motor domain is very mobile and able to undergo ODD. By comparing the orientation and dynamic behavior of mutated and deletion constructs we conclude that 1) the Kinesin-13 class specific neck domain and loop-2 help orienting the motor domain relative to the MT. 2) During ODD the KLP10A motor-domain changes orientation rapidly (rocks or tumbles). 3) The motor domain alone is capable of undergoing ODD. 4) A second tubulin binding site in the KLP10A motor domain is not critical for ODD. 5) The neck domain is not the element preventing KLP10A from binding to the MT lattice like motile Kinesins.
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exploring the mechanisms of a phosphorylation induced inhibition of microtubule depolymerization in the Kinesin 13 klp10a
Biophysical Journal, 2016Co-Authors: Matthieu P M H Benoit, Ana B Asenjo, Gary J Gerfen, David J Sharp, Daniel J Diaz, Hernando SosaAbstract:Kinesins 13 are a class of non-motile molecular motors able to regulate microtubule dynamics through their depolymerization activity at microtubule tips. A previous study in our lab on Drosophila Melanogaster KLP10A found a phosphorylation site within the motor domain that reduces microtubule depolymerization activity. This led us to study the inhibition mechanisms related to this phosphorylation site distant from the ATPase site. We have employed an integrated approach to compare the behavior of phosphomutant and wild-type KLP10A constructs with cell biology experiments and a variety of in-vitro biophysical methods. Imaging insect cells having only fluorescently labelled KLP10A variants revealed that the phosphomutant is distributed homogeneously on microtubules (lattice and tips) in contrast with the wild-type or a non-phosphorylatable mutant showing higher Kinesin concentration on microtubule tips. Wild-type and phosphomutant neck-motor Kinesins constructs have similar properties with isolated structures that mimic these microtubule tips: they can both stabilize curved microtubule protofilaments, an important property for microtubule depolymerization, and have similar affinity and ATPase activity on curved tubulin. However the phosphomutation strongly reduces the microtubule lattice stimulated ATPase activity despite non distinguishable microtubule lattice affinities. Single molecule fluorescence polarization microscopy revealed nucleotide dependent differences in the lattice diffusive behavior between the constructs. EPR and cryo-EM experiments are used to compare the Kinesins on the microtubule lattice vs curved protofilaments. The data are compatible with a reduced microtubule depolymerization activity of the phosphomutant caused by a reduction of the lattice stimulated ATPase activity. This reduced activity constrains the Kinesin to be in nucleotide states which restrict it from reaching the microtubule ends in comparison to the unphosphorylated Kinesin.
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a second tubulin binding site on the Kinesin 13 motor head domain is important during mitosis
PLOS ONE, 2013Co-Authors: Ana B Asenjo, David J Sharp, Dong Zhang, Michaela Greenbaum, Luping Xie, Hernando SosaAbstract: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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structural model for tubulin recognition and deformation by Kinesin 13 microtubule depolymerases
Cell Reports, 2013Co-Authors: Ana B Asenjo, Chandrima Chatterjee, Vania Depaoli, Dongyan Tan, William J Rice, Ruben Diazavalos, Mariena Silvestry, Hernando SosaAbstract:Summary To elucidate the structural basis of the mechanism of microtubule depolymerization by Kinesin-13s, we analyzed complexes of tubulin and the Drosophila melanogaster Kinesin-13 KLP10A by electron microscopy (EM) and fluorescence polarization microscopy. We report a nanometer-resolution (1.1 nm) cryo-EM three-dimensional structure of the KLP10A head domain (KLP10AHD) bound to curved tubulin. We found that binding of KLP10AHD induces a distinct tubulin configuration with displacement (shear) between tubulin subunits in addition to curvature. In this configuration, the Kinesin-binding site differs from that in straight tubulin, providing an explanation for the distinct interaction modes of Kinesin-13s with the microtubule lattice or its ends. The KLP10AHD-tubulin interface comprises three areas of interaction, suggesting a crossbow-type tubulin-bending mechanism. These areas include the Kinesin-13 family conserved KVD residues, and as predicted from the crossbow model, mutating these residues changes the orientation and mobility of KLP10AHDs interacting with the microtubule.
David J Sharp - One of the best experts on this subject based on the ideXlab platform.
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exploring the mechanism of microtubule depolymerization by the Kinesin 13 klp10a and it s phosphoregulation
Biophysical Journal, 2017Co-Authors: Matthieu P M H Benoit, Ana B Asenjo, Gary J Gerfen, David J Sharp, Daniel J Diazvalencia, Hernando SosaAbstract:Kinesins-13s are non-motile molecular motors that regulate microtubule (MT) dynamics by promoting MT depolymerization activity at the MT tips. We have used cryo-electron microscopy and functional assays to investigate the mechanism of action and regulation of the Drosophila melanogaster Kinesin-13 KLP10A. Previous work has shown that phosphorylation of serine 573 in the KLP10A motor domain down regulates depolymerization activity. We have obtained cryo-EM sub-nanometer resolution maps of constructs that include the KLP10A motor and N-terminal neck domains bound to MTs and to tubulin depolymerization intermediates. Comparison of these structures revealed key features of the depolymerization process. To study the down-regulation mechanism by phosphorylation we have combined a variety of cell biology and biophysical methods and compared the behavior of phosphomutant (S573E) and wild-type (WT) KLP10A. Surprisingly, we found that the binding affinity and ATPase activity stimulated by curved tubulin polymers (mimicking MT ends) of WT-KLP10A and S573E-KLP10A were relatively similar (kcat of 1.8 s−1 and 2.5 s−1 respectively). In contrast, we found significant differences in their MT-lattice stimulated ATPases (kcat of 0.56 s−1 and 0.14 s−1 respectively). We also found that the mutant S573E had shorter residence time relative to WT (τ of 5 s and 28 s respectively) while undergoing one-dimensional diffusion over the MT lattice. These results suggest a model in which phosphorylation of S573 reduce KLP10 MT depolymerization activity by decreasing the probability of reaching the MT ends by one dimensional diffusion. Consistent with this model we found by fluorescent live cell imaging that KLP10A-S573E MRFP labeled constructs accumulate significantly less at the tip of growing MTs than WT-KLP10A MRFP labeled constructs.
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distinct interaction modes of the Kinesin 13 motor domain with the microtubule
Biophysical Journal, 2016Co-Authors: Chandrima Chatterjee, Matthieu P M H Benoit, Vania Depaoli, Juan D Diazvalencia, Ana B Asenjo, Gary J Gerfen, David J Sharp, Hernando SosaAbstract:Kinesins-13s are members of the Kinesin superfamily of motor proteins that depolymerize microtubules (MTs) and have no motile activity. Instead of generating unidirectional movement over the MT lattice, like most other Kinesins, Kinesins-13s undergo one-dimensional diffusion (ODD) and induce depolymerization at the MT ends. To understand the mechanism of ODD and the origin of the distinct Kinesin-13 functionality, we used ensemble and single-molecule fluorescence polarization microscopy to analyze the behavior and conformation of Drosophila melanogaster Kinesin-13 KLP10A protein constructs bound to the MT lattice. We found that KLP10A interacts with the MT in two coexisting modes: one in which the motor domain binds with a specific orientation to the MT lattice and another where the motor domain is very mobile and able to undergo ODD. By comparing the orientation and dynamic behavior of mutated and deletion constructs we conclude that 1) the Kinesin-13 class specific neck domain and loop-2 help orienting the motor domain relative to the MT. 2) During ODD the KLP10A motor-domain changes orientation rapidly (rocks or tumbles). 3) The motor domain alone is capable of undergoing ODD. 4) A second tubulin binding site in the KLP10A motor domain is not critical for ODD. 5) The neck domain is not the element preventing KLP10A from binding to the MT lattice like motile Kinesins.
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exploring the mechanisms of a phosphorylation induced inhibition of microtubule depolymerization in the Kinesin 13 klp10a
Biophysical Journal, 2016Co-Authors: Matthieu P M H Benoit, Ana B Asenjo, Gary J Gerfen, David J Sharp, Daniel J Diaz, Hernando SosaAbstract:Kinesins 13 are a class of non-motile molecular motors able to regulate microtubule dynamics through their depolymerization activity at microtubule tips. A previous study in our lab on Drosophila Melanogaster KLP10A found a phosphorylation site within the motor domain that reduces microtubule depolymerization activity. This led us to study the inhibition mechanisms related to this phosphorylation site distant from the ATPase site. We have employed an integrated approach to compare the behavior of phosphomutant and wild-type KLP10A constructs with cell biology experiments and a variety of in-vitro biophysical methods. Imaging insect cells having only fluorescently labelled KLP10A variants revealed that the phosphomutant is distributed homogeneously on microtubules (lattice and tips) in contrast with the wild-type or a non-phosphorylatable mutant showing higher Kinesin concentration on microtubule tips. Wild-type and phosphomutant neck-motor Kinesins constructs have similar properties with isolated structures that mimic these microtubule tips: they can both stabilize curved microtubule protofilaments, an important property for microtubule depolymerization, and have similar affinity and ATPase activity on curved tubulin. However the phosphomutation strongly reduces the microtubule lattice stimulated ATPase activity despite non distinguishable microtubule lattice affinities. Single molecule fluorescence polarization microscopy revealed nucleotide dependent differences in the lattice diffusive behavior between the constructs. EPR and cryo-EM experiments are used to compare the Kinesins on the microtubule lattice vs curved protofilaments. The data are compatible with a reduced microtubule depolymerization activity of the phosphomutant caused by a reduction of the lattice stimulated ATPase activity. This reduced activity constrains the Kinesin to be in nucleotide states which restrict it from reaching the microtubule ends in comparison to the unphosphorylated Kinesin.
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a second tubulin binding site on the Kinesin 13 motor head domain is important during mitosis
PLOS ONE, 2013Co-Authors: Ana B Asenjo, David J Sharp, Dong Zhang, Michaela Greenbaum, Luping Xie, Hernando SosaAbstract: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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Kinesin HD electrostatic surface potentials in tubulin binding areas.
2013Co-Authors: Dong Zhang, Ana B Asenjo, David J Sharp, Michaela Greenbaum, Luping Xie, Hernando SosaAbstract:(A) Ribbon representation of the KLP10AHD-tubulin-MT spiral complex model (PDB IC: 3J2U [10]) showing the two tubulin binding sites at opposite sides of the HD. The Kin-Tub-1 area corresponds to the putative MT binding site, common to all Kinesins, but in this case it mediates binding to a curved tubulin protofilament. The Kin-Tub-2 area mediates binding of the Kinesin-13-HD-curved-tubulin complex to the MT and the formation of spirals wrapping the MT. Mutating Kinesin-13 class conserved positively charged residues in the Kin-Tub-2 area (KLP10A residues K306, K350, and K399 highlighted as blue atom spheres) disrupt these interactions and prevents the spirals from wrapping around MTs [13]. The location of the Kinesin-13 Loop-2 (L2) is indicated. (B) Electrostatic surface potential comparison of Kin-Tub-1 and Kin-Tub-2 areas of a Kinesin-1 (HsKIF5B) and a Kinesin-13 (DmKLP10A). Color scale inset: -10 (red) to +10 (blue) kcal/mol·e. (C) Kin-Tub-2 area of several Kinesin families. The corresponding Kinesin family is indicated below each HD structure. (D) Kin-Tub-2 area of several Kinesin-13s (all within the Kinesin-13B/MCAK subfamily [14]). In B-D the name corresponding to each Kinesin sequence are indicated with an italics two letter prefix corresponding to the organism origin (Ce: Caenorhabditis elegans; Cg: Cricetulus griseus; Dr: Danio rerio; Ds: Drosophila melanogaster; Hs: Homo Sapiens; Mm: Mus musculus; Xl: Xenopus laevis) and the PDB IC below (references [46–52]. When no atomic structures were available, a homology based model (HBM) was calculated using the program Modeller [53].
Melanie H Cobb - One of the best experts on this subject based on the ideXlab platform.
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ras regulates Kinesin 13 family members to control cell migration pathways in transformed human bronchial epithelial cells
Oncogene, 2014Co-Authors: Elma Zaganjor, Jihan K Osborne, Lauren M Weil, Laura A Diazmartinez, Joshua X Gonzales, Stina M Singel, Jill E Larsen, Luc Girard, John D Minna, Melanie H CobbAbstract:Ras regulates Kinesin 13 family members to control cell migration pathways in transformed human bronchial epithelial cells
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ras regulates Kinesin 13 family members to control cell migration pathways in transformed human bronchial epithelial cells
Oncogene, 2014Co-Authors: Elma Zaganjor, Jihan K Osborne, Lauren M Weil, Laura A Diazmartinez, Joshua X Gonzales, Stina M Singel, Jill E Larsen, Luc Girard, John D Minna, Melanie H CobbAbstract:We show that expression of the microtubule depolymerizing Kinesin KIF2C is induced by transformation of immortalized human bronchial epithelial cells (HBEC) by expression of K-Ras(G12V) and knockdown of p53. Further investigation demonstrates that this is due to the K-Ras/ERK1/2 MAPK pathway, as loss of p53 had little effect on KIF2C expression. In addition to KIF2C, we also found that the related Kinesin KIF2A is modestly upregulated in this model system; both proteins are expressed more highly in many lung cancer cell lines compared to normal tissue. As a consequence of their depolymerizing activity, these Kinesins increase dynamic instability of microtubules. Depletion of either of these Kinesins impairs the ability of cells transformed with mutant K-Ras to migrate and invade matrigel. However, depletion of these Kinesins does not reverse the epithelial to mesenchymal transition (EMT) caused by mutant K-Ras. Our studies indicate that increased expression of microtubule destabilizing factors can occur during oncogenesis to support enhanced migration and invasion of tumor cells.
Jonathon Howard - One of the best experts on this subject based on the ideXlab platform.
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depolymerizing Kinesins kip3 and mcak shape cellular microtubule architecture by differential control of catastrophe
Cell, 2011Co-Authors: Melissa K Gardner, Christopher Gell, Marija Zanic, Volker Bormuth, Jonathon HowardAbstract:Microtubules are dynamic filaments whose ends alternate between periods of slow growth and rapid shortening as they explore intracellular space and move organelles. A key question is how regulatory proteins modulate catastrophe, the conversion from growth to shortening. To study this process, we reconstituted microtubule dynamics in the absence and presence of the Kinesin-8 Kip3 and the Kinesin-13 MCAK. Surprisingly, we found that, even in the absence of the Kinesins, the microtubule catastrophe frequency depends on the age of the microtubule, indicating that catastrophe is a multistep process. Kip3 slowed microtubule growth in a length-dependent manner and increased the rate of aging. In contrast, MCAK eliminated the aging process. Thus, both Kinesins are catastrophe factors; Kip3 mediates fine control of microtubule length by narrowing the distribution of maximum lengths prior to catastrophe, whereas MCAK promotes rapid restructuring of the microtubule cytoskeleton by making catastrophe a first-order random process.
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the Kinesin 13 mcak has an unconventional atpase cycle adapted for microtubule depolymerization
The EMBO Journal, 2011Co-Authors: Claire T. Friel, Jonathon HowardAbstract:Unlike other Kinesins, members of the Kinesin-13 subfamily do not move directionally along microtubules but, instead, depolymerize them. To understand how Kinesins with structurally similar motor domains can have such dissimilar functions, we elucidated the ATP turnover cycle of the Kinesin-13, MCAK. In contrast to translocating Kinesins, ATP cleavage, rather than product release, is the rate-limiting step for ATP turnover by MCAK; unpolymerized tubulin and microtubules accelerate this step. Further, microtubule ends fully activate the ATPase by accelerating the exchange of ADP for ATP. This tuning of the cycle adapts MCAK for its depolymerization activity: lattice-stimulated ATP cleavage drives MCAK into a weakly bound nucleotide state that reaches microtubule ends by diffusion, and end-specific acceleration of nucleotide exchange drives MCAK into a strongly bound state that promotes depolymerization. This altered cycle accounts well for the different mechanical behaviour of this Kinesin, which depolymerizes microtubules from their ends, compared to translocating Kinesins that walk along microtubules. Thus, the Kinesin motor domain is a nucleotide-dependent engine that can be differentially tuned for transport or depolymerization functions.
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mcak Kinesin 13 has an unconventional atp hydrolysis cycle adapted for microtubule depolymerization
Biophysical Journal, 2010Co-Authors: Claire T. Friel, Jonathon HowardAbstract:Unlike members of the Kinesin-1 subfamily, the microtubule-depolymerising Kinesin-13, MCAK, has no translocation activity. Rather it diffuses on the microtubule lattice to accelerate targeting to both ends, where it carries out ATP-dependent catalytic depolymerisation. The ATP hydrolysis cycle of MCAK has been largely overlooked. However, it may hold the key to the strikingly different behavior of Kinesin-13 proteins compared to the conventional translocating Kinesins that move directionally on the lattice. We have elucidated the ATP hydrolysis cycle of MCAK in solution and in the presence of both free tubulin dimers and microtubules. In contrast to most other Kinesins and also myosins, for which product release is rate-limiting, ATP cleavage limits the hydrolysis cycle of MCAK in solution. Therefore MCAK meets the microtubule from solution in the ATP-containing state which binds tightly. Lattice-stimulated ATP cleavage drives MCAK into a weakly-bound nucleotide state, which diffuses on the lattice to target the microtubule end. An end-specific feature of the microtubule acts as a nucleotide exchange factor, promoting exchange of ADP for ATP by increasing the rate constant for ADP dissociation by more than 20-fold over the equivalent process in solution. Nucleotide exchange triggers tight binding of ATP-MCAK at the microtubule end, deforming the bound tubulin dimer causing lattice destabilization, leading to depolymerization. Tubulin-stimulated ATP hydrolysis is required to allow dissociation of tubulin-MCAK complexes released from the MT end, thereby allowing catalytic depolymerization. The altered ATP hydrolysis cycle of MCAK, relative to Kinesin-1, tailors its affinity for tubulin to produce the characteristic weakly-bound diffusive interaction with the microtubule lattice and the strong microtubule end-dependent binding that promotes depolymerization.
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yeast Kinesin 8 depolymerizes microtubules in a length dependent manner
Nature Cell Biology, 2006Co-Authors: Vladimir Varga, Jonne Helenius, Kozo Tanaka, Anthony A Hyman, Tomoyuki U Tanaka, Jonathon HowardAbstract:The microtubule cytoskeleton and the mitotic spindle are highly dynamic structures, yet their sizes are remarkably constant, thus indicating that the growth and shrinkage of their constituent microtubules are finely balanced. This balance is achieved, in part, through Kinesin-8 proteins (such as Kip3p in budding yeast and KLP67A in Drosophila) that destabilize microtubules. Here, we directly demonstrate that Kip3p destabilizes microtubules by depolymerizing them--accounting for the effects of Kinesin-8 perturbations on microtubule and spindle length observed in fungi and metazoan cells. Furthermore, using single-molecule microscopy assays, we show that Kip3p has several properties that distinguish it from other depolymerizing Kinesins, such as the Kinesin-13 MCAK. First, Kip3p disassembles microtubules exclusively at the plus end and second, remarkably, Kip3p depolymerizes longer microtubules faster than shorter ones. These properties are consequences of Kip3p being a highly processive, plus-end-directed motor, both in vitro and in vivo. Length-dependent depolymerization provides a new mechanism for controlling the lengths of subcellular structures.
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the depolymerizing Kinesin mcak uses lattice diffusion to rapidly target microtubule ends
Nature, 2006Co-Authors: Jonne Helenius, Gary J Brouhard, Yannis Kalaidzidis, Stefan Diez, Jonathon HowardAbstract:Single-molecule microscopy reveals that the Kinesin-13 protein MCAK undergoes a one-dimensional random walk on the microtubule surface, unlike the unidirectional movement of other Kinesins. The microtubule cytoskeleton is a dynamic structure in which the lengths of the microtubules are tightly regulated. One regulatory mechanism is the depolymerization of microtubules by motor proteins in the Kinesin-13 family1. These proteins are crucial for the control of microtubule length in cell division2,3,4, neuronal development5 and interphase microtubule dynamics6,7. The mechanism by which Kinesin-13 proteins depolymerize microtubules is poorly understood. A central question is how these proteins target to microtubule ends at rates exceeding those of standard enzyme–substrate kinetics8. To address this question we developed a single-molecule microscopy assay for MCAK, the founding member of the Kinesin-13 family9. Here we show that MCAK moves along the microtubule lattice in a one-dimensional (1D) random walk. MCAK–microtubule interactions were transient: the average MCAK molecule diffused for 0.83 s with a diffusion coefficient of 0.38 µm2 s-1. Although the catalytic depolymerization by MCAK requires the hydrolysis of ATP, we found that the diffusion did not. The transient transition from three-dimensional diffusion to 1D diffusion corresponds to a “reduction in dimensionality”10 that has been proposed as the search strategy by which DNA enzymes find specific binding sites11. We show that MCAK uses this strategy to target to both microtubule ends more rapidly than direct binding from solution.
