Niethammer Philipp, Kronja Iva, Kandels-Lewis Stefanie, Rybina Sonja, Bastiaens Philippe, Karsenti Eric
Cell Biology and Biophysics Department, European Molecular Biology Laboratory, Heidelberg, Germany.
PLoS Biol. 2007 Feb;5(2):e29. doi: 10.1371/journal.pbio.0050029.
The cytoplasm of eukaryotic cells is thought to adopt discrete "states" corresponding to different steady states of protein networks that govern changes in subcellular organization. For example, in Xenopus eggs, the interphase to mitosis transition is induced solely by activation of cyclin-dependent kinase 1 (CDK1) that phosphorylates many proteins leading to a reorganization of the nucleus and assembly of the mitotic spindle. Among these changes, the large array of stable microtubules that exists in interphase is replaced by short, highly dynamic microtubules in metaphase. Using a new visual immunoprecipitation assay that quantifies pairwise protein interactions in a non-perturbing manner in Xenopus egg extracts, we reveal the existence of a network of interactions between a series of microtubule-associated proteins (MAPs). In interphase, tubulin interacts with XMAP215, which is itself interacting with XKCM1, which connects to APC, EB1, and CLIP170. In mitosis, tubulin interacts with XMAP215, which is connected to EB1. We show that in interphase, microtubules are stable because the catastrophe-promoting activity of XKCM1 is inhibited by its interactions with the other MAPs. In mitosis, microtubules are short and dynamic because XKCM1 is free and has a strong destabilizing activity. In this case, the interaction of XMAP215 with EB1 is required to counteract the strong activity of XKCM1. This provides the beginning of a biochemical description of the notion of "cytoplasmic states" regarding the microtubule system.
真核细胞的细胞质被认为会呈现出离散的“状态”，这些状态对应着控制亚细胞组织变化的蛋白质网络的不同稳态。例如，在非洲爪蟾卵中，从间期到有丝分裂的转变仅由细胞周期蛋白依赖性激酶1（CDK1）的激活诱导，CDK1使许多蛋白质磷酸化，导致细胞核重组和有丝分裂纺锤体的组装。在这些变化中，间期存在的大量稳定微管在中期被短的、高度动态的微管所取代。利用一种新的可视化免疫沉淀测定法，该方法以非干扰方式定量非洲爪蟾卵提取物中的成对蛋白质相互作用，我们揭示了一系列微管相关蛋白（MAPs）之间存在相互作用网络。在间期，微管蛋白与XMAP215相互作用，而XMAP215本身又与XKCM1相互作用，XKCM1与APC、EB1和CLIP170相连。在有丝分裂中，微管蛋白与XMAP215相互作用，XMAP215与EB1相连。我们表明，在间期，微管是稳定的，因为XKCM1与其他MAPs的相互作用抑制了其促进微管解聚的活性。在有丝分裂中，微管短且动态，因为XKCM1是游离的，具有很强的去稳定活性。在这种情况下，XMAP215与EB1的相互作用是抵消XKCM1的强活性所必需的。这为关于微管系统的“细胞质状态”概念提供了生化描述的开端。
