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Fluorescence correlation spectroscopy reveals the dynamics of kinesins interacting with organelles during microtubule-dependent transport in cells.
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research ( IF 5.1 ) Pub Date : 2019-10-31 , DOI: 10.1016/j.bbamcr.2019.118572
María Cecilia De Rossi 1 , Nicolás González Bardeci 1 , Yanina Álvarez 1 , Esteban Mocskos 2 , Juan José Romero 1 , Luciana Bruno 3 , Diana Elena Wetzler 1 , Valeria Levi 1
Affiliation  

Microtubule-dependent motors usually work together to transport organelles through the crowded intracellular milieu. Thus, transport performance depends on how motors organize on the cargo. Unfortunately, the lack of methodologies capable of measuring this organization in cells determines that many aspects of the collective action of motors remain elusive. Here, we combined fluorescence fluctuations and single particle tracking techniques to address how kinesins organize on rod-like mitochondria moving along microtubules in cells. This methodology simultaneously provides mitochondria trajectories and EGFP-tagged kinesin-1 intensity at different mitochondrial positions with millisecond resolution. We show that kinesin exchange at the mitochondrion surface is within ~100 ms and depends on the organelle speed. During anterograde transport, the mitochondrial leading tip presents slower motor exchange in comparison to the rear tip. In contrast, retrograde mitochondria show similar exchange rates of kinesins at both tips. Numerical simulations provide theoretical support to these results and evidence that motors do not share the load equally during intracellular transport.

中文翻译:

荧光相关光谱揭示了驱动蛋白在细胞中微管依赖性转运过程中与细胞器相互作用的动力学。

依赖微管的马达通常一起工作,以通过拥挤的细胞内环境转运细胞器。因此,运输性能取决于马达如何组织货物。不幸的是,缺乏能够测量细胞中这种组织的方法学决定了电机集体行动的许多方面仍然难以捉摸。在这里,我们结合了荧光起伏和单颗粒跟踪技术,以解决驱动蛋白如何在沿着细胞中微管运动的棒状线粒体上组织的问题。该方法同时以毫秒级分辨率提供了不同线粒体位置的线粒体轨迹和EGFP标记的kinesin-1强度。我们显示线粒体表面的驱动蛋白交换在〜100毫秒内,并且取决于细胞器速度。在顺行运输过程中,与后部尖端相比,线粒体前部尖端的运动交换较慢。相比之下,逆行线粒体在两个尖端都显示相似的驱动蛋白汇率。数值模拟为这些结果提供了理论支持,并证明了电机在细胞内运输过程中不会平均分担负载。
更新日期:2019-10-31
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