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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 microtubuleassembly.The tubulin concentration (12 µM) waspurposely fixed to a value near the critical concentration in bufferM. (A) Both spermidine and spermine promote microtubule assembly atsubmilimolar concentrations whereas putrescine is unable to improvetubulin polymerization even at large concentrations(>1 mM). However, at high concentrationsof spermidine and spermine (>200µM), tubulin aggregates are formed which lowersthe apparent assembly slope leading to a bell-shape profile. (B) SDSpage analyses reveal that spermidine gradually increases the pelletmass. As observed by AFM, the increase of the pellet mass is due tomicrotubule formation at moderate spermidine concentrations(25–200 µM) but, at higherspermidine concentrations (0.5–2 mM),oligomers and (or) tubulin aggregates are also observed andparticipate to the increase of the pellet mass. The dashed linerepresents the transition between the zones of microtubule assemblyand tubulin aggregation.
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pcbi-1000255-g004: Effect of polyamines on the maximum slope of microtubuleassembly.The tubulin concentration (12 µM) waspurposely fixed to a value near the critical concentration in bufferM. (A) Both spermidine and spermine promote microtubule assembly atsubmilimolar concentrations whereas putrescine is unable to improvetubulin polymerization even at large concentrations(>1 mM). However, at high concentrationsof spermidine and spermine (>200µM), tubulin aggregates are formed which lowersthe apparent assembly slope leading to a bell-shape profile. (B) SDSpage analyses reveal that spermidine gradually increases the pelletmass. As observed by AFM, the increase of the pellet mass is due tomicrotubule formation at moderate spermidine concentrations(25–200 µM) but, at higherspermidine concentrations (0.5–2 mM),oligomers and (or) tubulin aggregates are also observed andparticipate to the increase of the pellet mass. The dashed linerepresents the transition between the zones of microtubule assemblyand tubulin aggregation.

Mentions: We measured the effects of polyamines on the maximum rate of microtubuleassembly by analyzing light scattering curves and the mass of polymerizedtubulin by sedimentation (more specific aspects of microtubuleassembly, notably nucleation and elongation, will be studied in thefollowing sections). It appears that spermidine(Z = 3) and spermine(Z = 4) promote MTassembly very efficiently while putrescine(Z = 2) has no effectup to 10 mM (Figure 4A).As previously reported, increasing the charge of the counterion favors MTassembly [17]. In agreement with our model, the effectof putrescine is negligible due to a lower energy gain in counterion sharingand an easier replacement by monovalent cations whereas the effects ofspermidine and spermine on MT assembly are more pronounced. Above150–300 µM, the beneficial effects of spermine andspermidine on the maximum polymerization rate decrease while they stillinduce a high polymerized mass (Figure 4B). However, under these conditions, we detected by AFMthe presence of tubulin aggregates or oligomers in agreement with a non-zerovalue of the light scattering curve at the beginning of assembly(data not shown). Such a strong attraction betweentubulin heterodimers is not due to a simple neutralization effect sinceneutralization does not lead to attraction between heterodimers. It shouldbe triggered by an attraction force via polyamine correlations betweentubulin 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 microtubuleassembly.The tubulin concentration (12 µM) waspurposely fixed to a value near the critical concentration in bufferM. (A) Both spermidine and spermine promote microtubule assembly atsubmilimolar concentrations whereas putrescine is unable to improvetubulin polymerization even at large concentrations(>1 mM). However, at high concentrationsof spermidine and spermine (>200µM), tubulin aggregates are formed which lowersthe apparent assembly slope leading to a bell-shape profile. (B) SDSpage analyses reveal that spermidine gradually increases the pelletmass. As observed by AFM, the increase of the pellet mass is due tomicrotubule formation at moderate spermidine concentrations(25–200 µM) but, at higherspermidine concentrations (0.5–2 mM),oligomers and (or) tubulin aggregates are also observed andparticipate to the increase of the pellet mass. The dashed linerepresents the transition between the zones of microtubule assemblyand tubulin aggregation.
© Copyright Policy
Related In: Results  -  Collection

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

pcbi-1000255-g004: Effect of polyamines on the maximum slope of microtubuleassembly.The tubulin concentration (12 µM) waspurposely fixed to a value near the critical concentration in bufferM. (A) Both spermidine and spermine promote microtubule assembly atsubmilimolar concentrations whereas putrescine is unable to improvetubulin polymerization even at large concentrations(>1 mM). However, at high concentrationsof spermidine and spermine (>200µM), tubulin aggregates are formed which lowersthe apparent assembly slope leading to a bell-shape profile. (B) SDSpage analyses reveal that spermidine gradually increases the pelletmass. As observed by AFM, the increase of the pellet mass is due tomicrotubule formation at moderate spermidine concentrations(25–200 µM) but, at higherspermidine concentrations (0.5–2 mM),oligomers and (or) tubulin aggregates are also observed andparticipate to the increase of the pellet mass. The dashed linerepresents the transition between the zones of microtubule assemblyand tubulin aggregation.
Mentions: We measured the effects of polyamines on the maximum rate of microtubuleassembly by analyzing light scattering curves and the mass of polymerizedtubulin by sedimentation (more specific aspects of microtubuleassembly, notably nucleation and elongation, will be studied in thefollowing sections). It appears that spermidine(Z = 3) and spermine(Z = 4) promote MTassembly very efficiently while putrescine(Z = 2) has no effectup to 10 mM (Figure 4A).As previously reported, increasing the charge of the counterion favors MTassembly [17]. In agreement with our model, the effectof putrescine is negligible due to a lower energy gain in counterion sharingand an easier replacement by monovalent cations whereas the effects ofspermidine and spermine on MT assembly are more pronounced. Above150–300 µM, the beneficial effects of spermine andspermidine on the maximum polymerization rate decrease while they stillinduce a high polymerized mass (Figure 4B). However, under these conditions, we detected by AFMthe presence of tubulin aggregates or oligomers in agreement with a non-zerovalue of the light scattering curve at the beginning of assembly(data not shown). Such a strong attraction betweentubulin heterodimers is not due to a simple neutralization effect sinceneutralization does not lead to attraction between heterodimers. It shouldbe triggered by an attraction force via polyamine correlations betweentubulin 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