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Polyamine sharing between tubulin dimers favours microtubule nucleation and elongation via facilitated diffusion.

Mechulam A, Chernov KG, Mucher E, Hamon L, Curmi PA, Pastré D - PLoS Comput. Biol. (2009)

Bottom Line: We suggest for the first time that the action of multivalent cations on microtubule dynamics can result from facilitated diffusion of GTP-tubulin to the microtubule ends.The mechanism of facilitated diffusion requires an attraction force between two tubulins, which can result from the sharing of multivalent counterions.The results presented here show that polyamines can be of particular importance for the regulation of the microtubule network in vivo and provide the basis for further investigations into the effects of facilitated diffusion on cytoskeleton dynamics.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire Structure-Activité des Biomolécules Normales et Pathologiques, Université Evry-Val d'Essonne, Evry, France.

ABSTRACT
We suggest for the first time that the action of multivalent cations on microtubule dynamics can result from facilitated diffusion of GTP-tubulin to the microtubule ends. Facilitated diffusion can promote microtubule assembly, because, upon encountering a growing nucleus or the microtubule wall, random GTP-tubulin sliding on their surfaces will increase the probability of association to the target sites (nucleation sites or MT ends). This is an original explanation for understanding the apparent discrepancy between the high rate of microtubule elongation and the low rate of tubulin association at the microtubule ends in the viscous cytoplasm. The mechanism of facilitated diffusion requires an attraction force between two tubulins, which can result from the sharing of multivalent counterions. Natural polyamines (putrescine, spermidine, and spermine) are present in all living cells and are potent agents to trigger tubulin self-attraction. By using an analytical model, we analyze the implication of facilitated diffusion mediated by polyamines on nucleation and elongation of microtubules. In vitro experiments using pure tubulin indicate that the promotion of microtubule assembly by polyamines is typical of facilitated diffusion. The results presented here show that polyamines can be of particular importance for the regulation of the microtubule network in vivo and provide the basis for further investigations into the effects of facilitated diffusion on cytoskeleton dynamics.

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Effect of spermidine on microtubule elongation.(A) Pseudo-first order rate constant of elongation,                                    Kobs, versus tubulin concentration                                in buffer M. 100 µM spermidine significantly increases the                                elongation rates whatever tubulin concentration. Inset:                                    Log-plot of                                    1−I(t)/I(∞) versus time for                                30 µM tubulin. The slope of this curve is                                    −Kobs. The elongations rate is about three                                times higher with spermidine (except for 40 µM                                    tubulin). (B) A microtubule solution was prepared by                                incubating 30 µM tubulin at 37°c for 1h in 50 mM                                MES-KOH pH 6.8, 1 mM EGTA, 2 mM MgCl2, 20%                                glycerol, 1 mM GTP. At the end of the incubation, 50 µl of                                a solution containing 30 µM of free tubulin dimers without                                or with 100 µM spermidine was then added to 50                                µl of the microtubule solution. The sudden increase of the                                free tubulin concentration allows one to observe elongation of the                                preformed microtubule via light scattering. It turns out that the                                presence of 100 µM spermidine leads to a significant                                increase of the elongation rate as indicated by the slope of the                                assembly curve, which is 2.7 times higher with 100 µM                                spermidine (see dotted lines). The plateau value                                which is more rapidly reached in the presence of polyamines also                                evidences a facilitated elongation.
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pcbi-1000255-g010: Effect of spermidine on microtubule elongation.(A) Pseudo-first order rate constant of elongation, Kobs, versus tubulin concentration in buffer M. 100 µM spermidine significantly increases the elongation rates whatever tubulin concentration. Inset: Log-plot of 1−I(t)/I(∞) versus time for 30 µM tubulin. The slope of this curve is −Kobs. The elongations rate is about three times higher with spermidine (except for 40 µM tubulin). (B) A microtubule solution was prepared by incubating 30 µM tubulin at 37°c for 1h in 50 mM MES-KOH pH 6.8, 1 mM EGTA, 2 mM MgCl2, 20% glycerol, 1 mM GTP. At the end of the incubation, 50 µl of a solution containing 30 µM of free tubulin dimers without or with 100 µM spermidine was then added to 50 µl of the microtubule solution. The sudden increase of the free tubulin concentration allows one to observe elongation of the preformed microtubule via light scattering. It turns out that the presence of 100 µM spermidine leads to a significant increase of the elongation rate as indicated by the slope of the assembly curve, which is 2.7 times higher with 100 µM spermidine (see dotted lines). The plateau value which is more rapidly reached in the presence of polyamines also evidences a facilitated elongation.

Mentions: According to the model of facilitated diffusion to the MT ends (Figure 9), the light scattering curves should reach their plateau value more abruptly in the presence of polyamines. The experiments presented in Figure 7A clearly show that the plateau value is reached quickly in the presence of polyamines. Therefore facilitated diffusion seems to have a beneficial effect on MT elongation for moderate attraction forces between tubulin dimers. However stronger evidences are required to confirm this proposal of facilitated elongation. We thus analyzed the pseudo-first order rate constant of elongation, kobs. This parameter can be obtained by measuring the slope of log [1−(I(t)/I(∞)] as a function of time [35]. Figure 10A shows that the pseudo-first order rates of elongation increase about 3 times in the presence of 100 µM spermidine whatever the tubulin concentration (except for 40 µM, the elongation rate is then lower than expected without spermidine). In addition, we note that the pseudo-first order rate constant of elongation increases with tubulin concentration in the presence of spermidine, as predicted in Regime II.


Polyamine sharing between tubulin dimers favours microtubule nucleation and elongation via facilitated diffusion.

Mechulam A, Chernov KG, Mucher E, Hamon L, Curmi PA, Pastré D - PLoS Comput. Biol. (2009)

Effect of spermidine on microtubule elongation.(A) Pseudo-first order rate constant of elongation,                                    Kobs, versus tubulin concentration                                in buffer M. 100 µM spermidine significantly increases the                                elongation rates whatever tubulin concentration. Inset:                                    Log-plot of                                    1−I(t)/I(∞) versus time for                                30 µM tubulin. The slope of this curve is                                    −Kobs. The elongations rate is about three                                times higher with spermidine (except for 40 µM                                    tubulin). (B) A microtubule solution was prepared by                                incubating 30 µM tubulin at 37°c for 1h in 50 mM                                MES-KOH pH 6.8, 1 mM EGTA, 2 mM MgCl2, 20%                                glycerol, 1 mM GTP. At the end of the incubation, 50 µl of                                a solution containing 30 µM of free tubulin dimers without                                or with 100 µM spermidine was then added to 50                                µl of the microtubule solution. The sudden increase of the                                free tubulin concentration allows one to observe elongation of the                                preformed microtubule via light scattering. It turns out that the                                presence of 100 µM spermidine leads to a significant                                increase of the elongation rate as indicated by the slope of the                                assembly curve, which is 2.7 times higher with 100 µM                                spermidine (see dotted lines). The plateau value                                which is more rapidly reached in the presence of polyamines also                                evidences a facilitated elongation.
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Related In: Results  -  Collection

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pcbi-1000255-g010: Effect of spermidine on microtubule elongation.(A) Pseudo-first order rate constant of elongation, Kobs, versus tubulin concentration in buffer M. 100 µM spermidine significantly increases the elongation rates whatever tubulin concentration. Inset: Log-plot of 1−I(t)/I(∞) versus time for 30 µM tubulin. The slope of this curve is −Kobs. The elongations rate is about three times higher with spermidine (except for 40 µM tubulin). (B) A microtubule solution was prepared by incubating 30 µM tubulin at 37°c for 1h in 50 mM MES-KOH pH 6.8, 1 mM EGTA, 2 mM MgCl2, 20% glycerol, 1 mM GTP. At the end of the incubation, 50 µl of a solution containing 30 µM of free tubulin dimers without or with 100 µM spermidine was then added to 50 µl of the microtubule solution. The sudden increase of the free tubulin concentration allows one to observe elongation of the preformed microtubule via light scattering. It turns out that the presence of 100 µM spermidine leads to a significant increase of the elongation rate as indicated by the slope of the assembly curve, which is 2.7 times higher with 100 µM spermidine (see dotted lines). The plateau value which is more rapidly reached in the presence of polyamines also evidences a facilitated elongation.
Mentions: According to the model of facilitated diffusion to the MT ends (Figure 9), the light scattering curves should reach their plateau value more abruptly in the presence of polyamines. The experiments presented in Figure 7A clearly show that the plateau value is reached quickly in the presence of polyamines. Therefore facilitated diffusion seems to have a beneficial effect on MT elongation for moderate attraction forces between tubulin dimers. However stronger evidences are required to confirm this proposal of facilitated elongation. We thus analyzed the pseudo-first order rate constant of elongation, kobs. This parameter can be obtained by measuring the slope of log [1−(I(t)/I(∞)] as a function of time [35]. Figure 10A shows that the pseudo-first order rates of elongation increase about 3 times in the presence of 100 µM spermidine whatever the tubulin concentration (except for 40 µM, the elongation rate is then lower than expected without spermidine). In addition, we note that the pseudo-first order rate constant of elongation increases with tubulin concentration in the presence of spermidine, as predicted in Regime II.

Bottom Line: We suggest for the first time that the action of multivalent cations on microtubule dynamics can result from facilitated diffusion of GTP-tubulin to the microtubule ends.The mechanism of facilitated diffusion requires an attraction force between two tubulins, which can result from the sharing of multivalent counterions.The results presented here show that polyamines can be of particular importance for the regulation of the microtubule network in vivo and provide the basis for further investigations into the effects of facilitated diffusion on cytoskeleton dynamics.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire Structure-Activité des Biomolécules Normales et Pathologiques, Université Evry-Val d'Essonne, Evry, France.

ABSTRACT
We suggest for the first time that the action of multivalent cations on microtubule dynamics can result from facilitated diffusion of GTP-tubulin to the microtubule ends. Facilitated diffusion can promote microtubule assembly, because, upon encountering a growing nucleus or the microtubule wall, random GTP-tubulin sliding on their surfaces will increase the probability of association to the target sites (nucleation sites or MT ends). This is an original explanation for understanding the apparent discrepancy between the high rate of microtubule elongation and the low rate of tubulin association at the microtubule ends in the viscous cytoplasm. The mechanism of facilitated diffusion requires an attraction force between two tubulins, which can result from the sharing of multivalent counterions. Natural polyamines (putrescine, spermidine, and spermine) are present in all living cells and are potent agents to trigger tubulin self-attraction. By using an analytical model, we analyze the implication of facilitated diffusion mediated by polyamines on nucleation and elongation of microtubules. In vitro experiments using pure tubulin indicate that the promotion of microtubule assembly by polyamines is typical of facilitated diffusion. The results presented here show that polyamines can be of particular importance for the regulation of the microtubule network in vivo and provide the basis for further investigations into the effects of facilitated diffusion on cytoskeleton dynamics.

Show MeSH