Atomic force microscopy reveals distinct protofilament-scale structural dynamics in depolymerizing microtubule arrays.
Atomic force microscopy reveals distinct protofilament-scale structural dynamics in depolymerizing microtubule arrays.
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DOI：
10.1073/pnas.2115708119
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发表时间：
2022-02-01
影响因子：
11.1
通讯作者：
Subramanian R
中科院分区：
文献类型：
--
作者：
Wijeratne SS;Marchan MF;Tresback JS;Subramanian R
人们不禁惊叹于微管聚合物在细胞结构中的精确组织，如轴丝和纺锤体。然而，我们对塑造这些阵列的生化机制的理解很大程度上来自于对少量(一两个)微管的体外实验。这有点类似于通过研究单车道街道来研究多车道高速公路的架构。在这里，我们使用原子力显微镜直接可视化在单个微管和原丝分辨率下解聚微管阵列。我们的结果揭示了微管解聚酶活性的差异，并为这些纳米尺度上的酶活性差异如何导致微米尺度上的多微管阵列的不同重塑提供了见解。基于微管的细胞结构的动态重组，如纺锤体和轴丝，从根本上取决于多微管阵列中单个聚合物的动力学。在微管阵列的完全破坏和精细大小控制中涉及的一类主要酶是解聚动蛋白。不同的解聚酶如何以不同的方式重塑微管阵列还知之甚少。解决这一问题的一个主要技术挑战是，现有的光学或电子显微镜方法缺乏时空分辨率来观察较大阵列中单个微管的动力学。在这里，我们使用原子力显微镜(AFM)在单个微管和原丝分辨率下对解聚阵列进行成像。我们通过保守的解聚酶发现了以前未见过的微管阵列失稳模式。我们发现，Kinesin-13MCAK介导了异步型原丝解聚和晶格缺陷扩展，而Kinesin-8Kip3p则促进了同步型原丝解聚。出乎意料的是，MCAK可以解聚高度稳定的轴丝二联体，但Kip3p不能。我们认为，不同的原丝水平的活动是解聚酶功能二分法的基础，导致微管阵列的大规模失稳或长度调节。我们的工作建立了AFM作为一种强大的策略来可视化阵列中的微管动力学，并揭示了纳米级底物专一性如何导致微米级细胞骨架结构的差异化重塑。
One cannot help but marvel at the precise organization of microtubule polymers in cellular structures such as the axoneme and the spindle. However, our understanding of the biochemical mechanisms that sculpt these arrays comes largely from in vitro experiments with a small number (one or two) of microtubules. This is somewhat akin to studying the architecture of multilane highways by studying one-lane streets. Here, we directly visualize depolymerizing microtubule arrays at individual microtubule and protofilament resolution using atomic force microscopy. Our results reveal differences in microtubule depolymerase activity and provide insights into how these differences in enzymatic activity on the nanometer scale can result in the differential remodeling of multimicrotubule arrays on the micron-length scale. The dynamic reorganization of microtubule-based cellular structures, such as the spindle and the axoneme, fundamentally depends on the dynamics of individual polymers within multimicrotubule arrays. A major class of enzymes implicated in both the complete demolition and fine size control of microtubule-based arrays are depolymerizing kinesins. How different depolymerases differently remodel microtubule arrays is poorly understood. A major technical challenge in addressing this question is that existing optical or electron-microscopy methods lack the spatial-temporal resolution to observe the dynamics of individual microtubules within larger arrays. Here, we use atomic force microscopy (AFM) to image depolymerizing arrays at single-microtubule and protofilament resolution. We discover previously unseen modes of microtubule array destabilization by conserved depolymerases. We find that the kinesin-13 MCAK mediates asynchronous protofilament depolymerization and lattice-defect propagation, whereas the kinesin-8 Kip3p promotes synchronous protofilament depolymerization. Unexpectedly, MCAK can depolymerize the highly stable axonemal doublets, but Kip3p cannot. We propose that distinct protofilament-level activities underlie the functional dichotomy of depolymerases, resulting in either large-scale destabilization or length regulation of microtubule arrays. Our work establishes AFM as a powerful strategy to visualize microtubule dynamics within arrays and reveals how nanometer-scale substrate specificity leads to differential remodeling of micron-scale cytoskeletal structures.
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影响因子：
16.8
作者：
通讯作者：
--
微流体辅助小管蛋白清洗探测的微管老化。
影响因子：
3.3
作者：
Duellberg C;Cade NI;Surrey T
通讯作者：
Surrey T
DOI：
10.1073/pnas.211400898
发表时间：
2001-10-23
影响因子：
11.1
作者：
Ando, T;Kodera, N;Toda, A
通讯作者：
Toda, A
影响因子：
21.3
作者：
Gupta, Mohan L., Jr.;Carvalho, Pedro;Pellman, David
通讯作者：
Pellman, David
影响因子：
7.8
作者：
BINDER, LI;ROSENBAUM, JL
通讯作者：
ROSENBAUM, JL