Dynamic behavior of GFP-CLIP-170 reveals fast protein turnover on microtubule plus ends.
Dynamic behavior of GFP-CLIP-170 reveals fast protein turnover on microtubule plus ends.
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DOI：
10.1083/jcb.200707203
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发表时间：
2008-02-25
期刊：
影响因子：
--
通讯作者：
Galjart N
中科院分区：
文献类型：
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作者：
Dragestein KA;van Cappellen WA;van Haren J;Tsibidis GD;Akhmanova A;Knoch TA;Grosveld F;Galjart N
微管(MT)加上末端跟踪蛋白(+TIPS)特异性地识别生长中的MT的末端。+TIPS参与多种细胞过程，如细胞分裂、细胞迁移和细胞极性。虽然+TIP跟踪在这些过程中很重要，但哺乳动物+TIPS+末端特异性的潜在机制还不完全清楚。细胞质连接蛋白170(CLIP-170)，其原型+TIP，可能通过与微管蛋白的共聚，以高亲和力结合到MT末端，并在几秒钟后解离。然而，使用基于荧光的方法，我们发现两个+TIP，CLIP-170和末端结合蛋白3(EB3)，在MT末端迅速翻转。由于CLIP-170和EB3与MT+末端的结合，它们的扩散似乎是速率限制的。我们还报告说，生长中的MT的末端含有过剩的Clip-170以相对较低的亲和力结合的位点。我们认为，观察到的正端荧光+尖端的损失并不反映单分子的行为，而是MT端整体结构变化的结果。
Microtubule (MT) plus end–tracking proteins (+TIPs) specifically recognize the ends of growing MTs. +TIPs are involved in diverse cellular processes such as cell division, cell migration, and cell polarity. Although +TIP tracking is important for these processes, the mechanisms underlying plus end specificity of mammalian +TIPs are not completely understood. Cytoplasmic linker protein 170 (CLIP-170), the prototype +TIP, was proposed to bind to MT ends with high affinity, possibly by copolymerization with tubulin, and to dissociate seconds later. However, using fluorescence-based approaches, we show that two +TIPs, CLIP-170 and end-binding protein 3 (EB3), turn over rapidly on MT ends. Diffusion of CLIP-170 and EB3 appears to be rate limiting for their binding to MT plus ends. We also report that the ends of growing MTs contain a surplus of sites to which CLIP-170 binds with relatively low affinity. We propose that the observed loss of fluorescent +TIPs at plus ends does not reflect the behavior of single molecules but is a result of overall structural changes of the MT end.