Vinculin tension probe in neurons
Vinculin tension probe in neurons
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神经元中的纽蛋白张力探针
DOI：
10.1117/12.2578501
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发表时间：
2021
期刊：
影响因子：
--
通讯作者：
Boustany, Nada N.
中科院分区：
文献类型：
--
作者：
Ayad, Marina A.;Mahon, Timothy;Sumetsky, Daniel;Cararo-Lopes, Marina M.;Firestein, Bonnie L.;Boustany, Nada N.
曲马多蛋白是已知的局灶性粘连的关键调节因子;它在与细胞外基质附着的位置经受张力。在这项研究中，我们探索使用黏着斑张力FRET探针，调查神经元内黏着斑张力。神经元生长的一个关键组成部分是迁移，这取决于细胞和细胞外基质之间的机械线索。了解神经元内的张力变化可能有助于我们理解神经发生的机制。为了研究这些力，我们使用先前开发的分子张力传感器，该传感器由弹性连接器TSMod组成，TSMod是插入在蓝绿色荧光蛋白（mTFP 1）和mVenus之间的40个氨基酸长的肽。黏着斑张力传感器VinTS由嵌入黏着斑头部和尾部之间的TSMod组成。当处于张力下时，VinTS将在mTFP 1和mVenus之间表现出较低的荧光共振能量转移（FRET）效率。从胚胎大鼠脑中分离皮质神经元，并在用聚-D-赖氨酸和层粘连蛋白包被的盖玻片上培养。用TSMod（未加载的张力传感器）或VinTS转染神经元。表达TSMod的神经元用作实验的对照组，因为TSMod本身不受粘着斑蛋白张力的影响。171个TSMod和127个VinTS表达神经元的平均FRET效率分别为27.08 ± 4.98%和22.86 ± 3.98%。VinTS的FRET效率显著低于TSMod的FRET效率（通过Welch t检验，p = 6.6e15）。这些结果支持了在神经元中使用VinTS探针的可行性，并提供了对神经元中VinTS FRET效率的首次评估。与TSMod相比，VinTS的FRET效率较低，表明VinTS可能在神经元中处于张力下。然而，需要更多的研究来进一步表征这些结果。
Vinculin is a known key regulator of focal adhesions; it undergoes tension in the locations of attachment to the extracellular matrix. In this study, we explore the use of a vinculin tension FRET probe to investigate vinculin tension within neurons. A critical component of neuronal growth is migration, which is dependent on the mechanical cues between the cells and the extracellular matrix. An understanding of tension variation within the neuron may help us understand mechanisms of neurogenesis. To study these forces, we use a previously developed molecular tension sensor, which consists of an elastic linker, TSMod, a 40-amino-acid-long peptide inserted between teal fluorescent protein (mTFP1) and mVenus. The vinculin tension sensor, VinTS, consists of TSMod embedded between the Vinculin head and tail. When under tension, VinTS will exhibit a lower fluorescence resonance energy transfer (FRET) efficiency between mTFP1 and mVenus. Cortical neurons were isolated from embryonic rat brains and cultured on glass coverslips coated with poly-D-lysine and laminin. The neurons were transfected with TSMod (the unloaded tension sensor) or VinTS. Neurons expressing TSMod are used as the experiment’s control group since TSMod on its own is not affected by vinculin tension. The mean FRET efficiency of 171 TSMod and 127 VinTS expressing neurons was 27.08 ± 4.98%, and 22.86 ± 3.98%, respectively. The FRET efficiency of VinTS was significantly lower than that of TSMod (p = 6.6e15 by Welch’s t-test). These results support the feasibility of using the VinTS probe in neurons and provide a first assessment of VinTS FRET efficiency in neurons. The lower FRET efficiency of VinTS compared with TSMod suggests that VinTS may be under tension in neurons. However, additional studies are required to further characterize these results.
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DOI：
--
发表时间：
2006
期刊：
影响因子：
--
作者：
William A. Ross;A. Morrison‐Saunders;Ross Marshall;Luis E. Sánchez;Joe Weston;E. Au;Richard K. Morgan;Richard Fuggle;Barry Sadler;William A. Ross;A. Morrison‐Saunders;Ross Marshall
通讯作者：
Ross Marshall
CNS神经粘着斑激酶形成与纽蛋白共定位的簇
影响因子：
4.2
作者：
Gerin R. Stevens;Chi Zhang;M. M. Berg;M. Lambert;K. Barber;I. Cantallops;A. Routtenberg;W. Klein
通讯作者：
W. Klein
影响因子：
3.7
作者：
Menaesse, Audrey;Sumetsky, Daniel;Boustany, Nada N.
通讯作者：
Boustany, Nada N.