Localization of a class III myosin to filopodia tips in transfected HeLa cells requires an actin-binding site in its tail domain
Localization of a class III myosin to filopodia tips in transfected HeLa cells requires an actin-binding site in its tail domain
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DOI：
10.1091/mbc.e02-10-0656
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发表时间：
2003-10-01
影响因子：
3.3
通讯作者：
Burnside, B
中科院分区：
文献类型：
--
作者：
Erickson, FL;Corsa, AC;Burnside, B
Bass Myo3A 是一种 III 类肌球蛋白，在 HeLa 细胞中以 GFP 融合体的形式表达，以研究其细胞定位。 GFP-Myo3A 定位于细胞质和丝状伪足中 F-肌动蛋白束的尖端，该定位与观察到的感光细胞中 F-肌动蛋白束远端的浓度一致。运动活性位点的突变导致丝状伪足定位丧失，这表明 Myo3A 运动活性是丝状伪足尖端定位所必需的。删除分析表明，丝状伪足定位不需要 NH2 末端激酶结构域，但需要 Myo3A 尾部的 CO2H 末端 22 个氨基酸。该尾部片段的单独表达在整个细胞质和丝状伪足中产生与F-肌动蛋白相关的荧光，并且重组尾部片段在体外与F-肌动蛋白结合。在该尾部片段中鉴定出肌动蛋白结合基序，并且该基序内的突变消除了 Myo3A 的丝状伪足定位和尾部片段单独结合的 F-肌动蛋白结合。当与 Myo3A 共表达时，钙调蛋白定位于丝状末端，但在没有 Myo3A 的情况下则不然，这一观察结果与之前的假设一致，即 III 类肌球蛋白结合钙调蛋白，从而将其定位于某些细胞类型中。
Bass Myo3A, a class III myosin, was expressed in HeLa cells as a GFP fusion in order to study its cellular localization. GFP-Myo3A localized to the cytoplasm and to the tips of F-actin bundles in filopodia, a localization that is consistent with the observed concentration toward the distal ends of F-actin bundles in photoreceptor cells. A mutation in the motor active site resulted in a loss of filopodia localization, suggesting that Myo3A motor activity is required for filopodial tip localization. Deletion analyses showed that the NH2-terminal kinase domain is not required but the CO2H-terminal 22 amino acids of the Myo3A tail are required for filopodial localization. Expression of this tail fragment alone produced fluorescence associated with F-actin throughout the cytoplasm and filopodia and a recombinant tail fragment bound to F-actin in vitro. An actin-binding motif was identified within this tail fragment, and a mutation within this motif abolished both filopodia localization by Myo3A and F-actin binding by the tail fragment alone. Calmodulin localized to filopodial tips when coexpressed with Myo3A but not in the absence of Myo3A, an observation consistent with the previous proposal that class III myosins bind calmodulin and thereby localize it in certain cell types.