Modulation of the Polymerization Kinetics of α/β‐Tubulin by Osmolytes and Macromolecular Crowding

Abstract

Tubulin is one of the main components of the cytoskeleton and can be found in nearly all eukaryotic cells. In this study, we explored the effects of kosmotropic and chaotropic osmolytes, such as trimethylamine-N-oxide (TMAO) and the metabolic waste product urea, as well as the crowding agents Ficoll and sucrose on the polymerization reaction of α/β-tubulin. Time-dependent turbidimetry and fluorescence anisotropy experiments were performed to explore the kinetics of the polymerization reaction. Under different solvent conditions, diverse changes in the lag time, the half-life of the polymerization reaction, and the critical concentration of the polymerization reaction were observed. The apparent growth rate of the formation of microtubules was dramatically decreased in the presence of urea but significantly increased in the presence of TMAO. Measurements using mixtures of these two cosolvents showed that TMAO was able to counteract the deteriorating effect of urea on the polymerization reaction of tubulin. To create a more cell-like environment, Ficoll was added as a macromolecular crowding agent. The presence of 10 wt % Ficoll increased the apparent growth rate by one order of magnitude. Our results clearly show that the polymerization of tubulin is very sensitive to the surrounding solvent.

Crowders and Cosolvents—Major Contributors to the Cellular Milieu and Efficient Means to Counteract Environmental Stresses

“…Many crowding studies based on the use of artificial macromolecular agents such as Ficoll, dextran and PEG, reported on the ability of the excluded volume effect to stabilize proteins, stabilize RNA tertiary structure and increase ribozyme activity, enhance protein‐protein associations as well as promote protein polymerization and aggregation . For example, Erlkamp et al.…”

“…[188] Many crowding studies based on the use of artificial macromolecular agentss uch as Ficoll, dextran and PEG, reported on the ability of the excluded volume effect to stabilize proteins, [181,189,190] stabilize RNA tertiary structure and increase ribo-zyme activity, [191][192][193][194] enhance protein-protein associations [195] as well as promote protein polymerization and aggregation. [196][197][198][199][200][201][202][203] For example, Erlkamp et al applied FT-IR spectroscopic, smallangle X-ray scattering and calorimetric measurements to explore the effect of macromolecular crowding (30 wt %F icoll 70) on the temperature-and pressure-dependent stability diagram the protein SNase. [181] As shown in Figure 7b,t he temperature-and pressure-induced equilibrium unfolding of SNase is markedly shifted to highert emperatures and pressures in 30 wt %F icoll solutions,b ut the volumec hange upon unfolding is not affected significantly by crowding.…”

Behaviors of individual microtubules and microtubule populations relative to critical concentrations: Dynamic instability occurs when critical concentrations are driven apart by nucleotide hydrolysis

“…The idea that polymer assembly commences at the critical concentration is now used routinely to design and interpret experiments involving cytoskeletal polymers (e.g. (Amayed et al, 2002;Buey et al, 2005;Wieczorek et al, 2015;Díaz-Celis et al, 2017;Schummel et al, 2017;Concha-Marambio et al, 2017)), and it is a standard topic in cell biology textbooks (e.g., (Alberts et al, 2015;Lodish et al, 2016)). Over time, a set of experimental measurements and definitions of critical concentration have emerged (Table 1, Figure 1), all of which would be equivalent for an equilibrium polymer.…”

“…Undoubtedly, many researchers have an intuitive understanding of the answers to at least some of these questions. However, the observation that even recent literature contains many references to "the" CC for microtubule assembly (e.g., (Wieczorek et al, 2015;Alfaro-Aco and Petry, 2015;Hussmann et al, 2016;Schummel et al, 2017) indicates that this problem deserves attention. While these issues are interesting from a basic science perspective, they also have significant practical relevance: proper design and interpretation of experiments that involve perturbing microtubule dynamics (e.g., characterization of MT-directed drugs or proteins) requires an unambiguous understanding of critical concentrations and how they are measured (e.g., (Verdier-Pinard et al, 2000;Bonfils et al, 2007;Hussmann et al, 2016;Cytoskeleton Inc.).…”

“…For the work presented here, it is important to recognize that the relevant observations are the behaviors of the systems at different scales and the concurrence (or disagreement) between the values of CC that result from various definitions or measurement approaches; the specific numerical CC values observed are simply outcomes of the particular input kinetic rate constants used and so are not by themselves significant. This situation is analogous to physical MTs, where DI parameters and CC values depend on the protein sequences, temperatures, and buffer conditions used (e.g., (Williams et al, 1985;Gildersleeve et al, 1992;Fygenson et al, 1994;Hussmann et al, 2016;Schummel et al, 2017)).…”