Reassociation of microvillar core proteins: making a microvillar core in vitro.
Reassociation of microvillar core proteins: making a microvillar core in vitro.
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DOI：
10.1083/jcb.108.2.495
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发表时间：
1989-02
期刊：
影响因子：
--
通讯作者：
Bretscher A
中科院分区：
文献类型：
--
作者：
Coluccio LM;Bretscher A
肠上皮细胞具有许多微绒毛的刷状缘膜，每个微绒毛由15-20根肌动蛋白丝的交联核心束组成，所述核心束通过横向横桥连接到周围的膜;横桥相对于核心束倾斜。分离的微绒毛核心含有肌动蛋白（42 kD）和其他三种主要蛋白质：fimplatin（68 kD），villin（95 kD）和110 K-钙调蛋白复合物。先前已经显示，向去污剂处理的分离的微绒毛核心中添加ATP导致横向交叉桥的损失以及与核心束相关的110-kD多肽和钙调蛋白的量的相应减少。这提供了第一个证据表明，这些横向跨桥的膜至少部分由110 kD的多肽与钙调蛋白复合。我们现在证明，纯化的110 K-钙调素复合物可以重新加入到ATP处理的剥离微绒毛核心。由此产生的束显示相同的螺旋和周期性排列的侧桥是在体内发现的。在重建实验中，肌动蛋白丝在EGTA中与纯化的fimactin和绒毛蛋白一起孵育形成包含明显随机数量的丝的光滑侧束。加入110 K-钙调素复合物后，通过负染色图像的电子显微镜观察，束显示沿着其整个长度螺旋排列的突起，具有与体内微绒毛上发现的横向交叉桥相同的33 nm重复;这些桥同样相对于束倾斜。因此，重构的肌动蛋白丝与fimmellulin，绒毛，和110 K-钙调蛋白复合物的结果在结构上非常相似的天然微绒毛核心。这些数据提供了直接的证据，证明110 K-钙调蛋白是跨桥蛋白，并表明，肌动蛋白丝捆绑的fimmellin和绒毛是一致的极性，并在寄存器中。纤维束上横桥臂的排列方式取决于由绒毛膜和绒毛膜固定的核心纤维的结构;不需要膜的贡献。
Intestinal epithelia have a brush border membrane of numerous microvilli each comprised of a cross-linked core bundle of 15-20 actin filaments attached to the surrounding membrane by lateral cross- bridges; the cross-bridges are tilted with respect to the core bundle. Isolated microvillar cores contain actin (42 kD) and three other major proteins: fimbrin (68 kD), villin (95 kD), and the 110K-calmodulin complex. The addition of ATP to detergent-treated isolated microvillar cores has previously been shown to result in loss of the lateral cross- bridges and a corresponding decrease in the amount of the 110-kD polypeptide and calmodulin associated with the core bundle. This provided the first evidence to suggest that these lateral cross-bridges to the membrane are comprised at least in part by a 110-kD polypeptide complexed with calmodulin. We now demonstrate that purified 110K- calmodulin complex can be readded to ATP-treated, stripped microvillar cores. The resulting bundles display the same helical and periodic arrangement of lateral bridges as is found in vivo. In reconstitution experiments, actin filaments incubated in EGTA with purified fimbrin and villin form smooth-sided bundles containing an apparently random number of filaments. Upon addition of 110K-calmodulin complex, the bundles, as viewed by electron microscopy of negatively stained images, display along their entire length helically arranged projections with the same 33-nm repeat of the lateral cross-bridges found on microvilli in vivo; these bridges likewise tilt relative to the bundle. Thus, reconstitution of actin filaments with fimbrin, villin, and the 110K- calmodulin complex results in structures remarkably similar to native microvillar cores. These data provide direct proof that the 110K- calmodulin is the cross-bridge protein and indicate that actin filaments bundled by fimbrin and villin are of uniform polarity and lie in register. The arrangement of the cross-bridge arms on the bundle is determined by the structure of the core filaments as fixed by fimbrin and villin; a contribution from the membrane is not required.