Simultaneous Visualization of the Dynamics of Crosslinked and Single Microtubules In Vitro by TIRF Microscopy.
Simultaneous Visualization of the Dynamics of Crosslinked and Single Microtubules In Vitro by TIRF Microscopy.
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DOI：
10.3791/63377
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发表时间：
2022-02-18
期刊：
影响因子：
--
通讯作者：
Subramanian R
中科院分区：
文献类型：
--
作者：
Mani N;Marchan MF;Subramanian R
微管是αβ-微管蛋白异二聚体的聚合物，在细胞中组织成不同的结构。基于微管的体系结构和网络通常包含动态特性不同的微管阵列的子集。例如，在细胞分裂中，稳定的交联束微管与动态的非交联束微管共存。基于TIRF显微镜的体外重建研究使得能够同时可视化这些不同微管阵列的动力学。在这种分析中，成像室由表面固定的微管组装而成，微管要么以单丝形式存在，要么组织成交联束。通过引入微管蛋白、核苷酸和蛋白质调节剂，可以直接显示相关蛋白质以及单个微管和交联微管的动态特性。此外，可以实时监测动态单个微管组织成束时发生的变化。这里描述的方法允许系统地评估单个蛋白质的活性和定位，以及在相同的实验条件下蛋白质调节剂对两个不同微管亚群的协同效应，从而提供其他方法无法获得的机制洞察力。
Microtubules are polymers of αβ-tubulin heterodimers that organize into distinct structures in cells. Microtubule-based architectures and networks often contain subsets of microtubule arrays that differ in their dynamic properties. For example, in dividing cells, stable bundles of crosslinked microtubules coexist in close proximity to dynamic non-crosslinked microtubules. TIRF-microscopy-based in vitro reconstitution studies enable the simultaneous visualization of the dynamics of these different microtubule arrays. In this assay, an imaging chamber is assembled with surface-immobilized microtubules, which are either present as single filaments or organized into crosslinked bundles. Introduction of tubulin, nucleotides, and protein regulators allows direct visualization of associated proteins and of dynamic properties of single and crosslinked microtubules. Furthermore, changes that occur as dynamic single microtubules organize into bundles can be monitored in real-time. The method described here allows for a systematic evaluation of the activity and localization of individual proteins, as well as synergistic effects of protein regulators on two different microtubule subsets under identical experimental conditions, thereby providing mechanistic insights that are inaccessible by other methods.
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影响因子：
11.8
作者：
Subramanian R;Kapoor TM
通讯作者：
Kapoor TM
DOI：
10.1073/pnas.2115708119
发表时间：
2022-02-01
影响因子：
11.1
作者：
Wijeratne SS;Marchan MF;Tresback JS;Subramanian R
通讯作者：
Subramanian R
影响因子：
3.3
作者：
HYMAN, AA;SALSER, S;MITCHISON, TJ
通讯作者：
MITCHISON, TJ
影响因子：
7.7
作者：
Wijeratne, Sithara;Subramanian, Radhika
通讯作者：
Subramanian, Radhika
影响因子：
64.8
作者：
MITCHISON, T;KIRSCHNER, M
通讯作者：
KIRSCHNER, M