High efficiency penetration of antibody-immobilized nanoneedle thorough plasma membrane for in situ detection of cytoskeletal proteins in living cells.
High efficiency penetration of antibody-immobilized nanoneedle thorough plasma membrane for in situ detection of cytoskeletal proteins in living cells.
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DOI：
10.1186/s12951-016-0226-5
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发表时间：
2016-11-03
影响因子：
10.2
通讯作者：
Nakamura C
中科院分区：
文献类型：
--
作者：
Kawamura R;Shimizu K;Matsumoto Y;Yamagishi A;Silberberg YR;Iijima M;Kuroda S;Fukazawa K;Ishihara K;Nakamura C
活细胞中细胞骨架的结构动力学领域正在引起广泛的兴趣，因为更好地理解细胞骨架的细胞内组织不仅可以为我们提供对基本细胞生物学的见解，而且还可以使再生医学和癌症治疗的新策略的发展成为可能，在这些领域中，依赖于细胞骨架的动力学发挥着关键作用。纳米针技术是一种强大的工具，允许细胞内的调查，因为它可以通过穿透质膜直接插入活细胞，对细胞造成最小的损伤，在使用原子力显微镜的精确操作下。使用抗体对纳米针的修饰允许对各种细胞骨架成分进行精确的机械检测，包括肌动蛋白、微管和中间丝。然而，纳米针通过质膜的成功穿透已经显示在不同的细胞类型和条件之间变化很大。为了克服这一问题并提高纳米针插入活细胞的成功率，我们在这里关注了膜脂质双层的流动性，这可能会阻碍纳米针渗透到细胞溶质环境中。我们的目的是通过增加接近速度或降低实验温度来降低膜的表观流动性。虽然在接近速度的变化没有太大的影响，降低温度被发现大大提高了检测的解束缚力，这表明在质膜流动性的改变导致增加纳米针渗透。在4 °C的较低温度下操作大大提高了以优化的接近速度将纳米针插入活细胞的成功率，同时它不影响固定在纳米针上的抗体与用于机械检测的波形蛋白的结合。由于这些实验参数可以应用于各种细胞类型，因此这些结果可以提高纳米针技术对其他细胞系和平台的通用性。本文的在线版本（doi：10.1186/s12951-016-0226-5）包含补充材料，可供授权用户使用。
The field of structural dynamics of cytoskeletons in living cells is gathering wide interest, since better understanding of cytoskeleton intracellular organization will provide us with not only insights into basic cell biology but may also enable development of new strategies in regenerative medicine and cancer therapy, fields in which cytoskeleton-dependent dynamics play a pivotal role. The nanoneedle technology is a powerful tool allowing for intracellular investigations, as it can be directly inserted into live cells by penetrating through the plasma membrane causing minimal damage to cells, under the precise manipulation using atomic force microscope. Modifications of the nanoneedles using antibodies have allowed for accurate mechanical detection of various cytoskeletal components, including actin, microtubules and intermediate filaments. However, successful penetration of the nanoneedle through the plasma membrane has been shown to vary greatly between different cell types and conditions. In an effort to overcome this problem and improve the success rate of nanoneedle insertion into the live cells, we have focused here on the fluidity of the membrane lipid bilayer, which may hinder nanoneedle penetration into the cytosolic environment. We aimed to reduce apparent fluidity of the membrane by either increasing the approach velocity or reducing experimental temperatures. Although changes in approach velocity did not have much effect, lowering the temperature was found to greatly improve the detection of unbinding forces, suggesting that alteration in the plasma membrane fluidity led to increase in nanoneedle penetration. Operation at a lower temperature of 4 °C greatly improved the success rate of nanoneedle insertion to live cells at an optimized approach velocity, while it did not affect the binding of antibodies immobilized on the nanoneedle to vimentins for mechanical detection. As these experimental parameters can be applied to various cell types, these results may improve the versatility of the nanoneedle technology to other cell lines and platforms. The online version of this article (doi:10.1186/s12951-016-0226-5) contains supplementary material, which is available to authorized users.
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影响因子：
3.9
作者：
Berquand, A;Xia, N;Dufrêne, YF
通讯作者：
Dufrêne, YF
影响因子：
11.2
作者：
Kleeberger, Wolfram;Bova, G. Steven;Berman, David M.
通讯作者：
Berman, David M.
影响因子：
1.6
作者：
HUTTER, JL;BECHHOEFER, J
通讯作者：
BECHHOEFER, J
影响因子：
12.6
作者：
Kihara, Takanori;Yoshida, Narutoshi;Miyake, Jun
通讯作者：
Miyake, Jun
影响因子：
12.6
作者：
Silberberg, Yaron R.;Mieda, Shingo;Nakamura, Chikashi
通讯作者：
Nakamura, Chikashi