Anterograde Microtubule Transport Drives Microtubule Bending in LLC-PK1 Epithelial Cells
Anterograde Microtubule Transport Drives Microtubule Bending in LLC-PK1 Epithelial Cells
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DOI：
10.1091/mbc.e08-09-0909
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发表时间：
2009-06-15
影响因子：
3.3
通讯作者：
Odde, David J.
中科院分区：
文献类型：
--
作者：
Bicek, Andrew D.;Tuzel, Erkan;Odde, David J.
微管（MT）已被提出作为机械的压缩支柱，抵抗肌动球蛋白收缩力和自身的聚合力，以机械稳定细胞形状。为了确定MT弯曲的起源，我们直接观察了LLC-PK 1上皮细胞外周的MT弯曲和F-肌动蛋白转运动力学。我们发现，F-肌动蛋白在这些细胞中几乎是静止的，即使MT变形，表明MT弯曲不是由肌动球蛋白收缩驱动的。此外，通过使用blebbistatin抑制肌球蛋白II活性导致微管仍然动态弯曲。此外，通过荧光散斑显微镜测定，MT聚合很少导致弯曲。我们使用诺考达唑抑制动态不稳定性，并且我们观察到MT弯曲动力学没有质的变化。弯曲最常见的原因是近端部分的MT向几乎静止的远端头端的顺行运输。有趣的是，我们发现在体外驱动蛋白-MT滑动测定中，MT以类似的方式弯曲。为了进行定量比较，我们测量了所观察到的MT的曲率分布，发现体内和体外曲率分布在定量上一致。此外，测得的MT曲率分布不是高斯的，如预期的热驱动的半柔性聚合物，表明热力在MT弯曲中起次要作用。我们的结论是，许多已知的MT变形机制，如聚合和肌动蛋白收缩性，在介导LLC-PK 1细胞中的MT弯曲中起着无关紧要的作用，并且基于MT的分子马达可能产生储存在MT晶格中的大部分应变能。结果反对MT在LLC-PK 1细胞中发挥主要机械作用的模型，而是支持机械力控制MT阵列空间分布的模型。
Microtubules (MTs) have been proposed to act mechanically as compressive struts that resist both actomyosin contractile forces and their own polymerization forces to mechanically stabilize cell shape. To identify the origin of MT bending, we directly observed MT bending and F-actin transport dynamics in the periphery of LLC-PK1 epithelial cells. We found that F-actin is nearly stationary in these cells even as MTs are deformed, demonstrating that MT bending is not driven by actomyosin contractility. Furthermore, the inhibition of myosin II activity through the use of blebbistatin results in microtubules that are still dynamically bending. In addition, as determined by fluorescent speckle microscopy, MT polymerization rarely results, if ever, in bending. We suppressed dynamic instability using nocodazole, and we observed no qualitative change in the MT bending dynamics. Bending most often results from anterograde transport of proximal portions of the MT toward a nearly stationary distal tip. Interestingly, we found that in an in vitro kinesin-MT gliding assay, MTs buckle in a similar manner. To make quantitative comparisons, we measured curvature distributions of observed MTs and found that the in vivo and in vitro curvature distributions agree quantitatively. In addition, the measured MT curvature distribution is not Gaussian, as expected for a thermally driven semiflexible polymer, indicating that thermal forces play a minor role in MT bending. We conclude that many of the known mechanisms of MT deformation, such as polymerization and acto-myosin contractility, play an inconsequential role in mediating MT bending in LLC-PK1 cells and that MT-based molecular motors likely generate most of the strain energy stored in the MT lattice. The results argue against models in which MTs play a major mechanical role in LLC-PK1 cells and instead favor a model in which mechanical forces control the spatial distribution of the MT array.