Mechanical effects of EB1 on microtubules depend on GTP hydrolysis state and presence of paclitaxel

Abstract

Using the nonhydrolyzable GTP analog GMPCPP and the slowly hydrolyzable GTPγS, we polymerize microtubules that recapitulate the end binding behavior of the plus end interacting protein (+TIP) EB1 along their entire length, and use these to investigate the impact of EB1 binding on microtubule mechanics. To measure the stiffness of single filaments, we use a spectral analysis method to determine the ensemble of shapes adopted by a freely diffusing, fluorescently labeled microtubule. We find that the presence of EB1 can stiffen microtubules in a manner that depends on the hydrolysis state of the tubulin-bound nucleotide, as well as the presence of the small-molecule stabilizer paclitaxel. We find that the magnitude of the EB1-induced stiffening is not proportional to the EB1-microtubule binding affinity, suggesting that the stiffening effect does not arise purely from an increase in the total amount of bound EB1. Additionally, we find that EB1 binds cooperatively to microtubules in manner that depends on tubulin-bound nucleotide state.

Molecular control of stress transmission in the microtubule cytoskeleton

“…One study tested addition of paclitaxel to GMPCPP-MTs after polymerization and found no change in stiffness [114]. Another study confirmed this result while also finding that when paclitaxel is present during polymerization of GMPCPP-microtubules, the stiffness is significantly reduced to nearly half that of the no paclitaxel case, showing that even order of addition (during or after polymerization) is an important factor affecting microtubule flexural rigidity [115]. GTPγS-polymerized microtubules are less stable than GMPCPP-microtubules so paclitaxel is generally included for stabilization.…”

“…It is clear that microtubule stiffness is sensitive to a large number of parameters: tubulin source [112], polymerization rate [113], presence of GTP or GTP analog [114,115], small molecule stabilizers [115][116][117][118][119][120], and microtubule associating proteins (MAPs) [96,114,115,[121][122][123][124]. While there appear to be consistent stiffness values reported within a particular research group for a fixed condition, the values vary significantly among different laboratories, even under seemingly similar MT preparation conditions.…”

“…For a particular MT preparation, the experimental values of stiffness reported, even by a single laboratory, show a broad and non-Gaussian distribution [115,120], which some groups have described as log-normal [112]. The distribution is typically wide, with as much as an order of magnitude variation from maximal to minimal values, much larger than the errors expected from the image processing and spectral analysis steps, which has been estimated to be less than 10% [44].…”

“…Studies have consistently shown that when MTs are polymerized with GMPCPP they are ~3× stiffer as compared to GTP-polymerized MTs stabilized with paclitaxel [17,114,115]. Unlike GTP-polymerized microtubules, GMPCPP-polymerized microtubules are stable with or without paclitaxel, presenting an opportunity to test the effects of paclitaxel on MT mechanics.…”

“…GTPγS-polymerized microtubules are less stable than GMPCPP-microtubules so paclitaxel is generally included for stabilization. The stiffness of GTPγS-polymerized microtubules (in the presence of paclitaxel) has been found to be comparable to the stiffness of GTP-polymerized microtubules [114,115].…”