Microtubules are cytoskeletal protein polymers relevant to a wide range of cell functions. In order to polymerize, the constituent tubulin subunits need to bind the nucleotide GTP, but its subsequent hydrolysis to GDP in the microtubule lattice induces depolymerization. The resulting behaviour of stochastic switching between growth and shrinkage is called dynamic instability. Both dynamic instability and microtubule mechanical properties are integral to many cell functions, yet are poorly understood. The present study uses thermal fluctuation measurements of grafted microtubules with different nucleotide contents to extract stiffnesses, relaxation times, and drag coefficients with an unprecedented precision. Both the stiffness and the relaxation time data indicate that stiffness is a function of length for GDP microtubules stabilized with the chemotherapy drug taxol. By contrast, measurements on microtubules polymerized with the non-hydrolizable GTP-analogue GMPCPP show a significantly higher, but constant, stiffness. The addition of taxol is shown to not significantly affect the properties of these microtubules, but a lowering of the GMPCPP content restores the length-dependent stiffness seen for taxol microtubules. The data are interpreted on the basis of a recent biopolymer model that takes into account the anisotropic architecture of microtubules which consist of loosely coupled protofilaments arranged in a tube. Using taxol microtubules and GMPCPP microtubules as the respective analogues of the GDP and GTP state of microtubules, evidence is presented that shear coupling between neighbouring protofilaments is at least two orders of magnitude stiffer in the GTP state than in the GDP state. Previous studies of nucleotide effects on tubulin have focussed on protofilament bending, and the present study is the first to be able to show a dramatic effect on interprotofilament bonds. The finding’s profound implications for dynamic instability are discussed. In addition, internal friction is found to dominate over hydrodynamic drag for microtubules shorter than ∼ 4 μm and, like stiffness, to be affected by the bound nucleotide, but not by taxol. Furthermore, the thermal shape fluctuations of free microtubules are imaged, and the intrinsic curvatures of microtubules are shown for the first time to follow a spectrum reminiscent of thermal bending. Regarding the extraction of mechanical data, this assay, though previously described in the literature, is shown to suffer from systematic flaws.

Microtubules are cytoskeletal protein polymers relevant to a wide range of cell functions. In order to polymerize, the constituent tubulin subunits need to bind the nucleotide GTP, but its subsequent hydrolysis to GDP in the microtubule lattice induces depolymerization. The resulting behaviour of stochastic switching between growth and shrinkage is called dynamic instability. Both dynamic instability and microtubule mechanical properties are integral to many cell functions, yet are poorly understood. The present study uses thermal fluctuation measurements of grafted microtubules with different nucleotide contents to extract stiffnesses, relaxation times, and drag coefficients with an unprecedented precision. Both the stiffness and the relaxation time data indicate that stiffness is a function of length for GDP microtubules stabilized with the chemotherapy drug taxol. By contrast, measurements on microtubules polymerized with the non-hydrolizable GTP-analogue GMPCPP show a significantly higher, but constant, stiffness. The addition of taxol is shown to not significantly affect the properties of these microtubules, but a lowering of the GMPCPP content restores the length-dependent stiffness seen for taxol microtubules. The data are interpreted on the basis of a recent biopolymer model that takes into account the anisotropic architecture of microtubules which consist of loosely coupled protofilaments arranged in a tube. Using taxol microtubules and GMPCPP microtubules as the respective analogues of the GDP and GTP state of microtubules, evidence is presented that shear coupling between neighbouring protofilaments is at least two orders of magnitude stiffer in the GTP state than in the GDP state. Previous studies of nucleotide effects on tubulin have focussed on protofilament bending, and the present study is the first to be able to show a dramatic effect on interprotofilament bonds. The finding’s profound implications for dynamic instability are discussed. In addition, internal friction is found to dominate over hydrodynamic drag for microtubules shorter than ∼ 4 μm and, like stiffness, to be affected by the bound nucleotide, but not by taxol. Furthermore, the thermal shape fluctuations of free microtubules are imaged, and the intrinsic curvatures of microtubules are shown for the first time to follow a spectrum reminiscent of thermal bending. Regarding the extraction of mechanical data, this assay, though previously described in the literature, is shown to suffer from systematic flaws.

Microtubules are a part of the cell cytoskeleton that performs different functions, such as providing the mechanical support for the shape of a cell, acting as tracks along which the motor protein move organelles from one part of the cell to another, or the forming mitotic spindle during the cell division. The microtubules are dynamic structures, namely they can grow and shrink. The phase of microtubule growth alternates with the phase of shrinkage that results in the dynamic microtubule network in the cell. However, to form stable and spatially well-defined structures, such as a mitotic spindle, the cell needs to control this stochastic process. This is done by microtubule-associated proteins (MAPs). One class of MAPs is the proteins of XMAP216/Dis1 family, which are microtubule polymerases. The founding member of this family is X. laevis XMAP215. XMAP215 is a processive polymerase acting on the microtubule plus end. XMAP215 binds either directly or reaches the microtubule plus end by the diffusion along the microtubule lattice. Being at the microtubule plus-end XMAP215 stays there transiently and helps to incorporate up to 25 tubulin dimers into microtubule lattice before it dissociates and, therefore, it processively tracks the growing microtubule end during polymerization. There are two hypothesis of microtubule assembly promotion: (i) XMAP215 repeatedly releases an associated tubulin dimer into the microtubule growing plus end or (ii) structurally stabilizes a polymerized tubulin intermediate at the growing plus end and, therefore, preventing depolymerization events. The first way results into the increase of on-rate of tubulin dimers at the microtubule end, whereas the second way results into the decrease of off-rate of tubulin dimers at the microtubule end. Here, I show the study of the mechanism of microtubule growth acceleration by XMAP215 and the dependence of XMAP215 polymerization activity on the applied force. To answer these questions, I investigated the addition of tubulin dimers to the plus end of the microtubule by XMAP215 and how this addition depends on the applied force. XMAP215 remains at the microtubule end for several rounds of tubulin addition surfing both growing and shrinking microtubule ends. Therefore, if one could track the position of the XMAP215 molecules at the very tip of a microtubule with sufficient resolution, it would provide the information about the dynamics of the microtubule end. The technique, which can detect the position of the object of interest with high spatial and temporal resolution in addition to being able to exert a force, is an optical trap. A calibrated optical trap not only provides a good measure of displacement but also enables force measurements. To monitor the position of the molecules of interest, the molecules of interest are usually attached to a microsphere. Hence, I tethered XMAP215 to a microsphere held by an optical trap, and used XMAP215 as a handle to interact with the microtubule tip. When the microtubule grows, the XMAP215 coated microsphere will move in the optical trap and this movement can be detected with high temporal and spatial resolution. My work demonstrates that cooperatively working XMAP215 molecules can not only polymerize microtubule but also harness the energy of microtubule polymerization or depolymerization to transport some cargo. There is an evidence that orthologues of XMAP215 in budding yeasts, fission yeasts and Drosophila localize on the kinetochores. Therefore, the ability of the bearing some load during microtubule polymerization could be potentially important for the XMAP215 functioning during cell division. I also showed the influence of external force applied to the XMAP215 molecules. Pointing toward microtubule growth, a force of 0.5 pN applied to the microtubule tip-coupled XMAP215-coated microsphere increases XMAP215 polymerization activity. However, the force of the same magnitude but applied against microtubule growth does not affect XMAP215 polymerization activity. This result can be explained by the fact, that the force acting in the direction of microtubule growth constrains XMAP215 to be at the very microtubule tip. Hence, XMAP215 can not diffuse away from plus-end and there is higher chance to incorporate tubulin dimers into the microtubule plus-end. The on- and off-rate of tubulin dimers at the microtubule end are both decreased when the external force applied either in direction of microtubule growth or opposite to it. The external force affects the off-rate slightly stronger than on-rate of tubulin dimer. Taking together, my study gives new insights into the mechanism of microtubule polymerization by XMAP215 and shows some novel properties of this protein.

Microtubules are a part of the cell cytoskeleton that performs different functions, such as providing the mechanical support for the shape of a cell, acting as tracks along which the motor protein move organelles from one part of the cell to another, or the forming mitotic spindle during the cell division. The microtubules are dynamic structures, namely they can grow and shrink. The phase of microtubule growth alternates with the phase of shrinkage that results in the dynamic microtubule network in the cell. However, to form stable and spatially well-defined structures, such as a mitotic spindle, the cell needs to control this stochastic process. This is done by microtubule-associated proteins (MAPs). One class of MAPs is the proteins of XMAP216/Dis1 family, which are microtubule polymerases. The founding member of this family is X. laevis XMAP215. XMAP215 is a processive polymerase acting on the microtubule plus end. XMAP215 binds either directly or reaches the microtubule plus end by the diffusion along the microtubule lattice. Being at the microtubule plus-end XMAP215 stays there transiently and helps to incorporate up to 25 tubulin dimers into microtubule lattice before it dissociates and, therefore, it processively tracks the growing microtubule end during polymerization. There are two hypothesis of microtubule assembly promotion: (i) XMAP215 repeatedly releases an associated tubulin dimer into the microtubule growing plus end or (ii) structurally stabilizes a polymerized tubulin intermediate at the growing plus end and, therefore, preventing depolymerization events. The first way results into the increase of on-rate of tubulin dimers at the microtubule end, whereas the second way results into the decrease of off-rate of tubulin dimers at the microtubule end. Here, I show the study of the mechanism of microtubule growth acceleration by XMAP215 and the dependence of XMAP215 polymerization activity on the applied force. To answer these questions, I investigated the addition of tubulin dimers to the plus end of the microtubule by XMAP215 and how this addition depends on the applied force. XMAP215 remains at the microtubule end for several rounds of tubulin addition surfing both growing and shrinking microtubule ends. Therefore, if one could track the position of the XMAP215 molecules at the very tip of a microtubule with sufficient resolution, it would provide the information about the dynamics of the microtubule end. The technique, which can detect the position of the object of interest with high spatial and temporal resolution in addition to being able to exert a force, is an optical trap. A calibrated optical trap not only provides a good measure of displacement but also enables force measurements. To monitor the position of the molecules of interest, the molecules of interest are usually attached to a microsphere. Hence, I tethered XMAP215 to a microsphere held by an optical trap, and used XMAP215 as a handle to interact with the microtubule tip. When the microtubule grows, the XMAP215 coated microsphere will move in the optical trap and this movement can be detected with high temporal and spatial resolution. My work demonstrates that cooperatively working XMAP215 molecules can not only polymerize microtubule but also harness the energy of microtubule polymerization or depolymerization to transport some cargo. There is an evidence that orthologues of XMAP215 in budding yeasts, fission yeasts and Drosophila localize on the kinetochores. Therefore, the ability of the bearing some load during microtubule polymerization could be potentially important for the XMAP215 functioning during cell division. I also showed the influence of external force applied to the XMAP215 molecules. Pointing toward microtubule growth, a force of 0.5 pN applied to the microtubule tip-coupled XMAP215-coated microsphere increases XMAP215 polymerization activity. However, the force of the same magnitude but applied against microtubule growth does not affect XMAP215 polymerization activity. This result can be explained by the fact, that the force acting in the direction of microtubule growth constrains XMAP215 to be at the very microtubule tip. Hence, XMAP215 can not diffuse away from plus-end and there is higher chance to incorporate tubulin dimers into the microtubule plus-end. The on- and off-rate of tubulin dimers at the microtubule end are both decreased when the external force applied either in direction of microtubule growth or opposite to it. The external force affects the off-rate slightly stronger than on-rate of tubulin dimer. Taking together, my study gives new insights into the mechanism of microtubule polymerization by XMAP215 and shows some novel properties of this protein.