Abstract
Microtubule dynamic instability is driven by the hydrolysis of the GTP bound to the β-subunit of the α-β tubulin heterodimer. Nucleotide analogues are commonly used to mimic the different steps of the tubulin GTPase cycle, but most of them are poor microtubule nucleators. Usually, microtubule assembly is seeded by guanylyl-(α, β)-methylene-diphosphonate (GMPCPP) or glycerol that can be limiting factors in monitoring the effect of other nucleotide analogs on their polymerization. Here, we describe a protocol that allows the assembly of microtubules in the presence of nucleotide analogues without the need of heterogeneous seeds and at a low final glycerol concentration. Microtubules are first assembled in the presence of the analogue of interest and glycerol to promote assembly. These microtubules are then sonicated to produce seeds that will be used to assemble microtubules in the absence of glycerol. This strategy produces homogeneous nucleotide-bound microtubules that can be further analyzed by biochemical or structural methods such as cryo-electron microscopy.
Keywords: Tubulin, Microtubules, GTP analogues, Microtubule nucleation, Seeded microtubule assembly, Spectrophotometry, Cryo-electron microscopy
Background
Nucleotide analogues are commonly used to investigate the conformational changes that the α-β tubulin heterodimer undergoes during microtubule assembly and hydrolysis of the GTP bound to its exchangeable (E) site. However, apart from GMPCPP, most analogues are poor microtubule nucleators. To overcome this difficulty, GMPCPP-stabilized seeds are classically used to elongate microtubules in the presence of other analogues (Maurer et al., 2011 and 2014; Zhang et al., 2015), resulting in the assembly of mixed nucleotide-bound lattices. Alternatively, glycerol at relatively high concentration (e.g., 25%) can be added to the reaction mix to stimulate tubulin self-assembly. However, high glycerol concentrations might not be desirable, for instance to analyze microtubule structure by cryo-electron microscopy, since this compound matches the protein density and reduces the image contrast.To avoid the use of GMPCPP-seeds or high glycerol concentrations in the final reaction mix, we have developed a protocol that uses seeds assembled in the presence of the same nucleotides that will be used to assemble the microtubules. As a first step, glycerol is used to polymerize microtubules in the presence of the nucleotide analogue. These microtubules are then fragmented by sonication to produce seeds, which are further diluted in the tubulin mix in the absence of glycerol. Microtubules elongated onto these seeds can be analyzed by cryo-electron microscopy without the risk to mistake microtubules assembled in the presence of GMPCPP with those assembled in the presence of the nucleotide analogue of interest.Our protocol describes the step-by-step preparation of the seeds and the assembly of microtubules, which is monitored by turbidimetry at 350 nm. It includes a cooling step at the end of the assembly process to assess the presence of depolymerizing microtubules. These conditions were used to assemble GDP-BeF3--microtubules, whose lattices were further characterized by cryo-electron microscopy (Estévez-Gallego et al., 2020). We have successfully applied this protocol to microtubules assembled in the presence of other analogues such as GTPγS, GDP-AlF3, GMPPCP and GMPCP, all of them being poor microtubule nucleators.
Materials and Reagents
Equipment
Software
Procedure
Data analysis
The LabPower software was used to control the Uvikon XS spectrophotometer and acquire data in autorate mode, which were further saved in Excel (.xls) format. Data were imported into Excel for visualizing the variations in optical density versus time plots. Figures were prepared in Affinity Designer.
Recipes
Acknowledgments
This work was funded by the Agence Nationale de la Recherche (ANR-16-C11-0017-01). This protocol was derived from Estévez-Gallego et al., 2020.
Competing interests
The authors declare no competing interests.
References
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