Department of Biochemistry, University of Geneva, Geneva, Switzerland.
Department of Computer Sciences, Universidad de Valencia, Valencia, Spain.
Elife. 2017 Oct 12;6:e25197. doi: 10.7554/eLife.25197.
Dynamin is a large GTPase that forms a helical collar at the neck of endocytic pits, and catalyzes membrane fission (Schmid and Frolov, 2011; Ferguson and De Camilli, 2012). Dynamin fission reaction is strictly dependent on GTP hydrolysis, but how fission is mediated is still debated (Antonny et al., 2016): GTP energy could be spent in membrane constriction required for fission, or in disassembly of the dynamin polymer to trigger fission. To follow dynamin GTP hydrolysis at endocytic pits, we generated a conformation-specific nanobody called dynab, that binds preferentially to the GTP hydrolytic state of dynamin-1. Dynab allowed us to follow the GTPase activity of dynamin-1 in real-time. We show that in fibroblasts, dynamin GTP hydrolysis occurs as stochastic bursts, which are randomly distributed relatively to the peak of dynamin assembly. Thus, dynamin disassembly is not coupled to GTPase activity, supporting that the GTP energy is primarily spent in constriction.
动力蛋白是一种大型 GTP 酶，在胞吞陷窝的颈部形成一个螺旋状的领子，并催化膜的分裂（Schmid 和 Frolov，2011；Ferguson 和 De Camilli，2012）。动力蛋白的分裂反应严格依赖于 GTP 的水解，但分裂是如何介导的仍有争议（Antonny 等人，2016）：GTP 的能量可能用于分裂所需的膜收缩，或者用于解聚动力蛋白聚合物以触发分裂。为了在胞吞陷窝中跟踪动力蛋白的 GTP 水解，我们生成了一种构象特异性的纳米抗体，称为 dynab，它优先结合到动力蛋白-1 的 GTP 水解状态。Dynab 使我们能够实时跟踪 dynamin-1 的 GTP 酶活性。我们表明，在成纤维细胞中，动力蛋白的 GTP 水解以随机爆发的形式发生，与动力蛋白组装的峰值相比，随机分布。因此，动力蛋白的解聚与 GTP 酶活性没有偶联，这支持 GTP 的能量主要用于收缩。
