Coluccio L M, Bretscher A
Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853.
J Cell Biol. 1989 Feb;108(2):495-502. doi: 10.1083/jcb.108.2.495.
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.
肠道上皮细胞具有由众多微绒毛组成的刷状缘膜，每个微绒毛都由15 - 20根肌动蛋白丝组成的交联核心束构成，这些肌动蛋白丝通过横向桥连附着在周围的膜上；横向桥连相对于核心束是倾斜的。分离出的微绒毛核心含有肌动蛋白（42 kD）和其他三种主要蛋白质：丝束蛋白（68 kD）、绒毛蛋白（95 kD）以及110K - 钙调蛋白复合物。先前已表明，向经去污剂处理的分离微绒毛核心中添加ATP会导致横向桥连丧失，并且与核心束相关的110 - kD多肽和钙调蛋白的量相应减少。这提供了首个证据表明这些与膜相连的横向桥连至少部分由与钙调蛋白复合的110 - kD多肽组成。我们现在证明，纯化的110K - 钙调蛋白复合物可以重新添加到经ATP处理的、去除了成分的微绒毛核心中。所得的束状结构呈现出与体内发现的相同的横向桥连的螺旋和周期性排列。在重组实验中，在EGTA中与纯化的丝束蛋白和绒毛蛋白一起孵育的肌动蛋白丝形成了包含数量明显随机的丝的光滑边缘束。添加110K - 钙调蛋白复合物后，通过对负染图像进行电子显微镜观察，束状结构在其整个长度上显示出螺旋排列的突起，其横向桥连的重复间距与体内微绒毛上发现的相同，为33纳米；这些桥连同样相对于束倾斜。因此，用丝束蛋白、绒毛蛋白和110K - 钙调蛋白复合物对肌动蛋白丝进行重组会产生与天然微绒毛核心非常相似的结构。这些数据直接证明110K - 钙调蛋白是桥连蛋白，并表明由丝束蛋白和绒毛蛋白束在一起的肌动蛋白丝具有均匀的极性且排列整齐。束上桥连臂的排列由丝束蛋白和绒毛蛋白固定的核心丝结构决定；不需要膜的作用。
