Multiple CaMKII Binding Modes to the Actin Cytoskeleton Revealed by Single-Molecule Imaging.
Multiple CaMKII Binding Modes to the Actin Cytoskeleton Revealed by Single-Molecule Imaging.
复制标题
通过单分子成像揭示的多种CAMKII结合模式与肌动蛋白细胞骨架。
DOI：
10.1016/j.bpj.2016.06.007
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发表时间：
2016-07-26
影响因子：
3.4
通讯作者：
Molloy JE
中科院分区：
文献类型：
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作者：
Khan S;Conte I;Carter T;Bayer KU;Molloy JE
局部化的钙/钙调蛋白依赖性蛋白激酶II（CaMKII）树突棘突触的部分是由肌动蛋白细胞骨架。我们使用全内反射荧光显微镜和单分子跟踪确定了GFP标记的CaMKII与活细胞内标记RFP的肌动蛋白细胞骨架的结合。逐步光漂白表明，CaMKII形成低聚复合物。光活化实验表明，扩散出的倏逝场确定的轨道寿命。Latrunculin治疗引发了肌动蛋白应力纤维和共定位的长寿命CaMKII轨道的耦合损失。以前认为缺乏F-肌动蛋白相互作用的CaMKIIα（α）亚型也显示出结合，但这比CaMKIIβ（β）弱三倍。βE′剪接变体的结合比α弱，表明β的结合主要依赖于结构域间的连接体。模拟自磷酸化状态的突变β T287 D和α T286 D也消除了F-肌动蛋白结合。自磷酸化触发自主CaMKII活性，但不损害GluN 2B结合，另一个重要的突触蛋白的相互作用的CaMKII。通过阻断ATP（β K43 R和α K42 M）或Ca 2 +/钙调蛋白（β A303 R）结合来抑制GluN 2B结合的CaMKII抑制剂tatCN 21或CaMKII突变对与F-肌动蛋白的相互作用没有影响。这些结果为α T286 D突变体的突触棘定位减少提供了第一个基本原理，表明瞬时F-肌动蛋白结合有助于CaMK II α亚型的突触定位。径迹寿命分布有一个拉伸指数的形式一致的非均匀扩散人口。这种异质性表明，CaMKII采用不同的F-肌动蛋白结合模式，这是最容易合理化的CaMKII十二聚体和F-肌动蛋白细胞骨架之间的多个亚基接触，稳定初始弱（微摩尔）单价相互作用。
Localization of the Ca2+/calmodulin-dependent protein kinase II (CaMKII) to dendritic spine synapses is determined in part by the actin cytoskeleton. We determined binding of GFP-tagged CaMKII to tag-RFP-labeled actin cytoskeleton within live cells using total internal reflection fluorescence microscopy and single-molecule tracking. Stepwise photobleaching showed that CaMKII formed oligomeric complexes. Photoactivation experiments demonstrated that diffusion out of the evanescent field determined the track lifetimes. Latrunculin treatment triggered a coupled loss of actin stress fibers and the colocalized, long-lived CaMKII tracks. The CaMKIIα (α) isoform, which was previously thought to lack F-actin interactions, also showed binding, but this was threefold weaker than that observed for CaMKIIβ (β). The βE′ splice variant bound more weakly than α, showing that binding by β depends critically on the interdomain linker. The mutations βT287D and αT286D, which mimic autophosphorylation states, also abolished F-actin binding. Autophosphorylation triggers autonomous CaMKII activity, but does not impair GluN2B binding, another important synaptic protein interaction of CaMKII. The CaMKII inhibitor tatCN21 or CaMKII mutations that inhibit GluN2B association by blocking binding of ATP (βK43R and αK42M) or Ca2+/calmodulin (βA303R) had no effect on the interaction with F-actin. These results provide the first rationale for the reduced synaptic spine localization of the αT286D mutant, indicating that transient F-actin binding contributes to the synaptic localization of the CaMKIIα isoform. The track lifetime distributions had a stretched exponential form consistent with a heterogeneously diffusing population. This heterogeneity suggests that CaMKII adopts different F-actin binding modes, which is most easily rationalized by multiple subunit contacts between the CaMKII dodecamer and the F-actin cytoskeleton that stabilize the initial weak (micromolar) monovalent interaction.