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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 polyamines on the maximum slope of microtubule                                assembly.The tubulin concentration (12 µM) was                                purposely fixed to a value near the critical concentration in buffer                                M. (A) Both spermidine and spermine promote microtubule assembly at                                submilimolar concentrations whereas putrescine is unable to improve                                tubulin polymerization even at large concentrations                                    (>1 mM). However, at high concentrations                                of spermidine and spermine (>200                                µM), tubulin aggregates are formed which lowers                                the apparent assembly slope leading to a bell-shape profile. (B) SDS                                page analyses reveal that spermidine gradually increases the pellet                                mass. As observed by AFM, the increase of the pellet mass is due to                                microtubule formation at moderate spermidine concentrations                                    (25–200 µM) but, at higher                                spermidine concentrations (0.5–2 mM),                                oligomers and (or) tubulin aggregates are also observed and                                participate to the increase of the pellet mass. The dashed line                                represents the transition between the zones of microtubule assembly                                and tubulin aggregation.
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pcbi-1000255-g004: Effect of polyamines on the maximum slope of microtubule assembly.The tubulin concentration (12 µM) was purposely fixed to a value near the critical concentration in buffer M. (A) Both spermidine and spermine promote microtubule assembly at submilimolar concentrations whereas putrescine is unable to improve tubulin polymerization even at large concentrations (>1 mM). However, at high concentrations of spermidine and spermine (>200 µM), tubulin aggregates are formed which lowers the apparent assembly slope leading to a bell-shape profile. (B) SDS page analyses reveal that spermidine gradually increases the pellet mass. As observed by AFM, the increase of the pellet mass is due to microtubule formation at moderate spermidine concentrations (25–200 µM) but, at higher spermidine concentrations (0.5–2 mM), oligomers and (or) tubulin aggregates are also observed and participate to the increase of the pellet mass. The dashed line represents the transition between the zones of microtubule assembly and tubulin aggregation.

Mentions: We measured the effects of polyamines on the maximum rate of microtubule assembly by analyzing light scattering curves and the mass of polymerized tubulin by sedimentation (more specific aspects of microtubule assembly, notably nucleation and elongation, will be studied in the following sections). It appears that spermidine (Z = 3) and spermine (Z = 4) promote MT assembly very efficiently while putrescine (Z = 2) has no effect up to 10 mM (Figure 4A). As previously reported, increasing the charge of the counterion favors MT assembly [17]. In agreement with our model, the effect of putrescine is negligible due to a lower energy gain in counterion sharing and an easier replacement by monovalent cations whereas the effects of spermidine and spermine on MT assembly are more pronounced. Above 150–300 µM, the beneficial effects of spermine and spermidine on the maximum polymerization rate decrease while they still induce a high polymerized mass (Figure 4B). However, under these conditions, we detected by AFM the presence of tubulin aggregates or oligomers in agreement with a non-zero value of the light scattering curve at the beginning of assembly (data not shown). Such a strong attraction between tubulin heterodimers is not due to a simple neutralization effect since neutralization does not lead to attraction between heterodimers. It should be triggered by an attraction force via polyamine correlations between tubulin dimers.


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 polyamines on the maximum slope of microtubule                                assembly.The tubulin concentration (12 µM) was                                purposely fixed to a value near the critical concentration in buffer                                M. (A) Both spermidine and spermine promote microtubule assembly at                                submilimolar concentrations whereas putrescine is unable to improve                                tubulin polymerization even at large concentrations                                    (>1 mM). However, at high concentrations                                of spermidine and spermine (>200                                µM), tubulin aggregates are formed which lowers                                the apparent assembly slope leading to a bell-shape profile. (B) SDS                                page analyses reveal that spermidine gradually increases the pellet                                mass. As observed by AFM, the increase of the pellet mass is due to                                microtubule formation at moderate spermidine concentrations                                    (25–200 µM) but, at higher                                spermidine concentrations (0.5–2 mM),                                oligomers and (or) tubulin aggregates are also observed and                                participate to the increase of the pellet mass. The dashed line                                represents the transition between the zones of microtubule assembly                                and tubulin aggregation.
© Copyright Policy
Related In: Results  -  Collection

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

pcbi-1000255-g004: Effect of polyamines on the maximum slope of microtubule assembly.The tubulin concentration (12 µM) was purposely fixed to a value near the critical concentration in buffer M. (A) Both spermidine and spermine promote microtubule assembly at submilimolar concentrations whereas putrescine is unable to improve tubulin polymerization even at large concentrations (>1 mM). However, at high concentrations of spermidine and spermine (>200 µM), tubulin aggregates are formed which lowers the apparent assembly slope leading to a bell-shape profile. (B) SDS page analyses reveal that spermidine gradually increases the pellet mass. As observed by AFM, the increase of the pellet mass is due to microtubule formation at moderate spermidine concentrations (25–200 µM) but, at higher spermidine concentrations (0.5–2 mM), oligomers and (or) tubulin aggregates are also observed and participate to the increase of the pellet mass. The dashed line represents the transition between the zones of microtubule assembly and tubulin aggregation.
Mentions: We measured the effects of polyamines on the maximum rate of microtubule assembly by analyzing light scattering curves and the mass of polymerized tubulin by sedimentation (more specific aspects of microtubule assembly, notably nucleation and elongation, will be studied in the following sections). It appears that spermidine (Z = 3) and spermine (Z = 4) promote MT assembly very efficiently while putrescine (Z = 2) has no effect up to 10 mM (Figure 4A). As previously reported, increasing the charge of the counterion favors MT assembly [17]. In agreement with our model, the effect of putrescine is negligible due to a lower energy gain in counterion sharing and an easier replacement by monovalent cations whereas the effects of spermidine and spermine on MT assembly are more pronounced. Above 150–300 µM, the beneficial effects of spermine and spermidine on the maximum polymerization rate decrease while they still induce a high polymerized mass (Figure 4B). However, under these conditions, we detected by AFM the presence of tubulin aggregates or oligomers in agreement with a non-zero value of the light scattering curve at the beginning of assembly (data not shown). Such a strong attraction between tubulin heterodimers is not due to a simple neutralization effect since neutralization does not lead to attraction between heterodimers. It should be triggered by an attraction force via polyamine correlations between tubulin dimers.

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