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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 concentrationin buffer M. 100 µM spermidine significantly increases theelongation rates whatever tubulin concentration. Inset:Log-plot of1−I(t)/I(∞) versus time for30 µM tubulin. The slope of this curve is−Kobs. The elongations rate is about threetimes higher with spermidine (except for 40 µMtubulin). (B) A microtubule solution was prepared byincubating 30 µM tubulin at 37°c for 1h in 50 mMMES-KOH pH 6.8, 1 mM EGTA, 2 mM MgCl2, 20%glycerol, 1 mM GTP. At the end of the incubation, 50 µl ofa solution containing 30 µM of free tubulin dimers withoutor with 100 µM spermidine was then added to 50µl of the microtubule solution. The sudden increase of thefree tubulin concentration allows one to observe elongation of thepreformed microtubule via light scattering. It turns out that thepresence of 100 µM spermidine leads to a significantincrease of the elongation rate as indicated by the slope of theassembly curve, which is 2.7 times higher with 100 µMspermidine (see dotted lines). The plateau valuewhich is more rapidly reached in the presence of polyamines alsoevidences 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 concentrationin buffer M. 100 µM spermidine significantly increases theelongation rates whatever tubulin concentration. Inset:Log-plot of1−I(t)/I(∞) versus time for30 µM tubulin. The slope of this curve is−Kobs. The elongations rate is about threetimes higher with spermidine (except for 40 µMtubulin). (B) A microtubule solution was prepared byincubating 30 µM tubulin at 37°c for 1h in 50 mMMES-KOH pH 6.8, 1 mM EGTA, 2 mM MgCl2, 20%glycerol, 1 mM GTP. At the end of the incubation, 50 µl ofa solution containing 30 µM of free tubulin dimers withoutor with 100 µM spermidine was then added to 50µl of the microtubule solution. The sudden increase of thefree tubulin concentration allows one to observe elongation of thepreformed microtubule via light scattering. It turns out that thepresence of 100 µM spermidine leads to a significantincrease of the elongation rate as indicated by the slope of theassembly curve, which is 2.7 times higher with 100 µMspermidine (see dotted lines). The plateau valuewhich is more rapidly reached in the presence of polyamines alsoevidences a facilitated elongation.

Mentions: According to the model of facilitated diffusion to the MT ends (Figure 9), the lightscattering curves should reach their plateau value more abruptly in thepresence of polyamines. The experiments presented in Figure 7A clearly show that the plateauvalue is reached quickly in the presence of polyamines. Thereforefacilitated diffusion seems to have a beneficial effect on MT elongation formoderate attraction forces between tubulin dimers. However strongerevidences 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 bymeasuring the slope of log[1−(I(t)/I(∞)] as afunction of time [35]. Figure 10A shows that the pseudo-firstorder rates of elongation increase about 3 times in the presence of 100µM spermidine whatever the tubulin concentration (exceptfor 40 µM, the elongation rate is then lower than expectedwithout spermidine). In addition, we note that the pseudo-firstorder rate constant of elongation increases with tubulin concentration inthe 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 concentrationin buffer M. 100 µM spermidine significantly increases theelongation rates whatever tubulin concentration. Inset:Log-plot of1−I(t)/I(∞) versus time for30 µM tubulin. The slope of this curve is−Kobs. The elongations rate is about threetimes higher with spermidine (except for 40 µMtubulin). (B) A microtubule solution was prepared byincubating 30 µM tubulin at 37°c for 1h in 50 mMMES-KOH pH 6.8, 1 mM EGTA, 2 mM MgCl2, 20%glycerol, 1 mM GTP. At the end of the incubation, 50 µl ofa solution containing 30 µM of free tubulin dimers withoutor with 100 µM spermidine was then added to 50µl of the microtubule solution. The sudden increase of thefree tubulin concentration allows one to observe elongation of thepreformed microtubule via light scattering. It turns out that thepresence of 100 µM spermidine leads to a significantincrease of the elongation rate as indicated by the slope of theassembly curve, which is 2.7 times higher with 100 µMspermidine (see dotted lines). The plateau valuewhich is more rapidly reached in the presence of polyamines alsoevidences a facilitated elongation.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2599886&req=5

pcbi-1000255-g010: Effect of spermidine on microtubule elongation.(A) Pseudo-first order rate constant of elongation,Kobs, versus tubulin concentrationin buffer M. 100 µM spermidine significantly increases theelongation rates whatever tubulin concentration. Inset:Log-plot of1−I(t)/I(∞) versus time for30 µM tubulin. The slope of this curve is−Kobs. The elongations rate is about threetimes higher with spermidine (except for 40 µMtubulin). (B) A microtubule solution was prepared byincubating 30 µM tubulin at 37°c for 1h in 50 mMMES-KOH pH 6.8, 1 mM EGTA, 2 mM MgCl2, 20%glycerol, 1 mM GTP. At the end of the incubation, 50 µl ofa solution containing 30 µM of free tubulin dimers withoutor with 100 µM spermidine was then added to 50µl of the microtubule solution. The sudden increase of thefree tubulin concentration allows one to observe elongation of thepreformed microtubule via light scattering. It turns out that thepresence of 100 µM spermidine leads to a significantincrease of the elongation rate as indicated by the slope of theassembly curve, which is 2.7 times higher with 100 µMspermidine (see dotted lines). The plateau valuewhich is more rapidly reached in the presence of polyamines alsoevidences a facilitated elongation.
Mentions: According to the model of facilitated diffusion to the MT ends (Figure 9), the lightscattering curves should reach their plateau value more abruptly in thepresence of polyamines. The experiments presented in Figure 7A clearly show that the plateauvalue is reached quickly in the presence of polyamines. Thereforefacilitated diffusion seems to have a beneficial effect on MT elongation formoderate attraction forces between tubulin dimers. However strongerevidences 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 bymeasuring the slope of log[1−(I(t)/I(∞)] as afunction of time [35]. Figure 10A shows that the pseudo-firstorder rates of elongation increase about 3 times in the presence of 100µM spermidine whatever the tubulin concentration (exceptfor 40 µM, the elongation rate is then lower than expectedwithout spermidine). In addition, we note that the pseudo-firstorder rate constant of elongation increases with tubulin concentration inthe 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