Affimer proteins for F-actin: novel affinity reagents that label F-actin in live and fixed cells.
Affimer proteins for F-actin: novel affinity reagents that label F-actin in live and fixed cells.
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DOI：
10.1038/s41598-018-24953-4
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发表时间：
2018-04-26
影响因子：
4.6
通讯作者：
Peckham M
中科院分区：
文献类型：
--
作者：
Lopata A;Hughes R;Tiede C;Heissler SM;Sellers JR;Knight PJ;Tomlinson D;Peckham M
细胞中肌动蛋白细胞骨架的成像使用了广泛的方法。通常，小环肽鬼笔环肽的荧光衍生物用于在固定的细胞中成像F-肌动蛋白。Lifeact和F-tractin常用于活细胞中细胞骨架的成像。在这里，我们表征了称为Affimers的新型亲和试剂，其在体外特异性结合F-肌动蛋白，以确定它们是否是合适的替代品作为eGFP融合蛋白，用于标记活细胞中的肌动蛋白，或用于标记固定细胞中的F-肌动蛋白。体外实验表明，所测试的4个Affimers中的3个（Affimers 6、14和24）与纯化的F-肌动蛋白紧密结合，并且似乎具有重叠的结合位点。作为eGFP融合蛋白，相同的3个Affimers标记活细胞中的F-肌动蛋白。FRAP实验表明eGFP-亲和聚体6的行为与F-tractin和Lifeact最相似。然而，它不与mCherry-肌动蛋白共定位在动态皱褶，并可能优先结合稳定的肌动蛋白丝。所有4种Affimers在甲醇固定的细胞中标记F-肌动蛋白，而只有Affimers 14在多聚甲醛固定后标记F-肌动蛋白。eGFP-Affimer 6具有用于选择性地成像活细胞中的稳定肌动蛋白细胞骨架的潜力，而所有4种Affimer都是用于标记固定细胞中的F-肌动蛋白的鬼笔环肽的强替代物。
Imaging the actin cytoskeleton in cells uses a wide range of approaches. Typically, a fluorescent derivative of the small cyclic peptide phalloidin is used to image F-actin in fixed cells. Lifeact and F-tractin are popular for imaging the cytoskeleton in live cells. Here we characterised novel affinity reagents called Affimers that specifically bind to F-actin in vitro to determine if they are suitable alternatives as eGFP-fusion proteins, to label actin in live cells, or for labeling F-actin in fixed cells. In vitro experiments showed that 3 out of the 4 Affimers (Affimers 6, 14 and 24) tested bind tightly to purified F-actin, and appear to have overlapping binding sites. As eGFP-fusion proteins, the same 3 Affimers label F-actin in live cells. FRAP experiments suggest that eGFP-Affimer 6 behaves most similarly to F-tractin and Lifeact. However, it does not colocalise with mCherry-actin in dynamic ruffles, and may preferentially bind stable actin filaments. All 4 Affimers label F-actin in methanol fixed cells, while only Affimer 14 labels F-actin after paraformaldehyde fixation. eGFP-Affimer 6 has potential for use in selectively imaging the stable actin cytoskeleton in live cells, while all 4 Affimers are strong alternatives to phalloidin for labelling F-actin in fixed cells.
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影响因子：
2.7
作者：
Spracklen, Andrew J.;Fagan, Tiffany N.;Lovander, Kaylee E.;Tootle, Tina L.
通讯作者：
Tootle, Tina L.
影响因子：
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作者：
Nemethova, Maria;Auinger, Sonja;Small, J. Victor
通讯作者：
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影响因子：
48
作者：
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通讯作者：
Wedlich-Soldner, Roland
影响因子：
1.5
作者：
Nagasaki, Akira;Kijima, Saku T.;Uyeda, Taro Q. P.
通讯作者：
Uyeda, Taro Q. P.
影响因子：
4.8
作者：
Heissler, Sarah M.;Chinthalapudi, Krishna;Sellers, James R.
通讯作者：
Sellers, James R.