Optogenetic EB1 inactivation shortens metaphase spindles by disrupting cortical force-producing interactions with astral microtubules.
Optogenetic EB1 inactivation shortens metaphase spindles by disrupting cortical force-producing interactions with astral microtubules.
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DOI：
10.1016/j.cub.2022.01.017
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发表时间：
2022-03-14
期刊：
影响因子：
--
通讯作者：
Wittmann T
中科院分区：
文献类型：
--
作者：
Dema A;van Haren J;Wittmann T
染色体分离是由有丝分裂纺锤体完成的，纺锤体是一种主要由微管构成的双极微机器。不同的微管群体有助于纺锤体功能：动粒微管附着并传递力到染色体，反平行极间微管支持纺锤体结构，星形微管将纺锤体极连接到细胞皮层。在哺乳动物细胞中，末端结合（EB）蛋白与整个细胞周期中所有生长的微管+末端相关，并作为多种+TIP的衔接子，控制微管动力学和与其他细胞内结构的相互作用。由于许多+TIPs与EB 1的结合以及微管末端的结合被有丝分裂磷酸化关闭，因此对EBs的有丝分裂功能仍然知之甚少。为了分析EB 1和相关的+TIP在不同的纺锤体微管群体中如何促进有丝分裂纺锤体动力学，我们使用了一种光敏EB 1变体π-EB 1，它允许与活细胞中生长的微管末端相关的+TIP的局部、急性和可逆失活。我们发现，急性π-EB 1光失活的结果在快速和可逆的中期纺锤体缩短和短暂的张力松弛整个中央纺锤体。然而，与间期相反，π-EB 1光失活并不抑制微管在中期的生长，而是增加星形微管的长度和数量。然而，在没有EB 1活性的情况下，星形微管不能参与皮质动力蛋白/动力蛋白机制，纺锤体极从π-EB 1光失活的区域移开。总之，我们的光遗传学方法揭示了有丝分裂EB 1功能，这些功能在遗传实验中可能是由于调节脊椎动物纺锤体动力学的补偿分子系统而隐藏的。平衡的胞内力控制有丝分裂纺锤体的形状和位置。Dema等人表明，+TIP EB 1的光遗传学失活破坏了这种平衡，导致快速和可逆的中期纺锤体缩短。结果强调EB 1功能限制星形微管长度和从事向外拉力主轴杆。
Chromosome segregation is accomplished by the mitotic spindle, a bipolar micromachine built primarily from microtubules. Different microtubule populations contribute to spindle function: Kinetochore microtubules attach and transmit forces to chromosomes, antiparallel interpolar microtubules support spindle structure, and astral microtubules connect spindle poles to the cell cortex. In mammalian cells, End Binding (EB) proteins associate with all growing microtubule plus ends throughout the cell cycle and serve as adaptors for diverse +TIPs that control microtubule dynamics and interactions with other intracellular structures. Because binding of many +TIPs to EB1 and thus microtubule-end association is switched off by mitotic phosphorylation the mitotic function of EBs remains poorly understood. To analyze how EB1 and associated +TIPs on different spindle microtubule populations contribute to mitotic spindle dynamics, we use a light sensitive EB1 variant, π-EB1, that allows local, acute and reversible inactivation of +TIP association with growing microtubule ends in live cells. We find that acute π-EB1 photoinactivation results in rapid and reversible metaphase spindle shortening and transient relaxation of tension across the central spindle. However, in contrast to interphase, π-EB1 photoinactivation does not inhibit microtubule growth in metaphase, but instead increases astral microtubule length and number. Yet, in the absence of EB1 activity astral microtubules fail to engage the cortical dynein/dynactin machinery and spindle poles move away from regions of π-EB1 photoinactivation. In conclusion, our optogenetic approach reveals mitotic EB1 functions that remain hidden in genetic experiments likely due to compensatory molecular systems regulating vertebrate spindle dynamics. Balanced intracellular forces control mitotic spindle shape and position. Dema et al. show that optogenetic inactivation of the +TIP EB1 disrupts this balance, leading to rapid and reversible metaphase spindle shortening. Results highlight EB1 functions in limiting astral microtubule length and in engaging outward pulling forces on spindle poles.
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