Institut National de la Santé et de la Recherche Médicale, UMR, Université Evry-Val d'Essonne, Evry, France.
Biochem J. 2010 Aug 15;430(1):151-9. doi: 10.1042/BJ20091811.
Owing to preferential electrostatic adsorption of multivalent cations on highly anionic surfaces, natural multivalent polyamines and especially quadrivalent spermine can be considered as potential regulators of the complex dynamical properties of anionic MTs (microtubules). Indeed, the C-terminal tails of tubulin display many negative residues in a row which should enable the formation of a correlated liquid-like phase of multivalent counterions on its surface. Although it is known that polyamine counterions promote MT assembly in vitro, little is known about the relevance of this interaction in vivo. In the present study, we have explored the relationship between polyamine levels and MT assembly in HeLa and epithelial NRK (normal rat kidney) cells using DFMO (alpha-difluoromethylornithine), an irreversible inhibitor of ornithine decarboxylase, and APCHA [N-(3-aminopropyl)-N-cyclohexylamine], a spermine synthase inhibitor. Under conditions of intracellular polyamine depletion, the MT network is clearly disrupted and the MT mass decreases. Addition of spermine to polyamine-depleted cells reverses this phenotype and rapidly promotes the extensions of the MT network. Finally, we show that polyamine levels modulate the coating of MTs with MAP4 (MT-associated protein 4), an MT-stabilizing protein, and the spatial distribution of EB1 (end-binding protein 1), an MT plus-end-binding protein. In addition, polyamines favour the formation of gap junctions in NRK cells, a process which requires MT extensions at the cell periphery. The present study provides a basis for a better understanding of the role played by polyamines in MT assembly and establishes polyamine metabolism as a potential cellular target for modulating MT functions.
由于多价阳离子对带高负电荷表面的优先静电吸附作用，天然多价聚胺，尤其是四价的 spermine，可被视为阴离子 MT（微管）复杂动态特性的潜在调节剂。事实上，微管蛋白的 C 末端尾部存在许多连续的负残基，这应该能够在其表面形成多价抗衡离子的相关液态相。虽然已知聚胺抗衡离子能促进 MT 在体外组装，但关于这种相互作用在体内的相关性知之甚少。在本研究中，我们使用不可逆的鸟氨酸脱羧酶抑制剂 DFMO（α-二氟甲基鸟氨酸）和 spermine 合酶抑制剂 APCHA[N-（3-氨基丙基）-N-环己基胺]，探索了 HeLa 和上皮 NRK（正常大鼠肾）细胞中聚胺水平与 MT 组装之间的关系。在细胞内聚胺耗竭的条件下，MT 网络明显被破坏，MT 质量减少。向聚胺耗竭的细胞中添加 spermine 可逆转这种表型，并迅速促进 MT 网络的延伸。最后，我们表明聚胺水平调节 MAP4（微管相关蛋白 4）与 MT 的包裹，MAP4 是一种 MT 稳定蛋白，以及 EB1（末端结合蛋白 1）的空间分布，EB1 是一种 MT 末端结合蛋白。此外，聚胺促进 NRK 细胞中缝隙连接的形成，这一过程需要 MT 在细胞边缘延伸。本研究为更好地理解聚胺在 MT 组装中所起的作用提供了依据，并确立了聚胺代谢作为调节 MT 功能的潜在细胞靶标。
