Bundling of Actin Filaments by ct-Actinin Depends on Its Molecular Length
Bundling of Actin Filaments by ct-Actinin Depends on Its Molecular Length
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ct-肌动蛋白对肌动蛋白丝的捆绑取决于其分子长度
DOI：
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发表时间：
2002
期刊：
影响因子：
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通讯作者：
S. Singer
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文献类型：
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作者：
B. Geiger;A. Dutton;K. Tokuyasu;S. Singer
在相同的反应条件下，用三种不同亚型的哑铃形c-肌动蛋白同源二聚体研究了肌动蛋白细丝(F-肌动蛋白)交联束和网状结构。它们分别是从鸡肌肌、棘阿米巴和网茎线虫中分离得到的。电子显微镜观察表明，每种异构体都能将F-肌动蛋白交联成网络。此外，在肌动蛋白自身聚合成分散细丝的条件下，鸡肌动蛋白和棘阿米巴α-肌动蛋白可以得到F-肌动蛋白束，而不是Dictyostelialu-actinin。这种F-肌动蛋白束的形成严重依赖于α-肌动蛋白与肌动蛋白的适当摩尔比，因此当游离的ct-肌动蛋白从周围介质中撤出时，F-肌动蛋白束立即消失。肌动蛋白结合部位在22℃和37℃半饱和时的表观解离常数(Kos)分别为0.4t~M和1.2/ZM，棘阿米巴和鞭毛虫c-肌动蛋白在22℃时的表观解离常数均为2.7~M。鸡的肌动蛋白细丝主要以反平行方式交联化，而棘阿米巴T~-肌动蛋白细丝优先以平行的方式交联化。游离ot-肌动蛋白的平均分子长度分别为37 nm和35 nm，棘阿米巴分别为46和44 nm，毛盘基菌c-肌动蛋白分别为34和31 nm。在负染的标本中，我们还评估了ct-actinin与肌动蛋白细丝结合时的平均分子长度：鸡胆为36 nm，棘阿米巴ct-actinin为35 nm，分子长度与双链F-actin螺旋的交叉重复序列(即36 nm)大致一致，但Dictyostelio-actinin的分子长度仅为28 nm。此外，在肌动蛋白细丝上，交联型c~-肌动蛋白分子的最小间距接近36 nm，而棘阿米巴o~-肌动蛋白的最小间距仅为31 nm。这一观察表明，α-肌动蛋白同源二聚体的分子长度可能决定其沿肌动蛋白细丝的间距，因此F-肌动蛋白束的形成可能需要a-肌动蛋白分子沿着肌动蛋白细丝“紧密”(即，一个分子接着另一个分子)和“不扭曲”(即，分子的长轴平行于肌动蛋白细丝轴)堆积。I N 1964~-肌动蛋白是从横纹肌中提取的一种蛋白质，在体外可促进肌动蛋白凝胶的收缩并增加F-肌动蛋白溶液的粘度(Ebashi等人，1964)。随着更有效的分离方法的出现，它与肌动蛋白的相互作用得到了更系统的研究(Holmes等人，1971；Goll等人，1972)。因此，在0°C下观察到含有给定量ct-肌动蛋白的F-肌动蛋白溶液的粘度增加最大。在此条件下，当ot-肌动蛋白与肌动蛋白的比例达到最大值时，每个肌动蛋白螺旋的交叉重复(即36 nm)约结合一个α-肌动蛋白二聚体分子。在37℃的溶液中，需要更高的o~-肌动蛋白与肌动蛋白的比例才能产生相同数量的结合o~-肌动蛋白。被阴影的ot-Actinin的电子显微镜显示了一种哑铃形分子，其中两个亚基以并排的方式反向定向，从而具有中心二联体对称轴(例如，Pollard等人，1986)。每个多肽都有一个高度保守的肌动蛋白结合位点，位于多肽链的NH2末端附近，位于分子上的杆状突起上(Mimura和Asano，1987；Imamura等人，1988；Blanchard等人，1989)。因此，o~-肌动蛋白通过每一端与肌动蛋白细丝结合来使F-肌动蛋白交联(podlubnaya等，1975)。在过去的几年里，更多的α-肌动蛋白异构体被分离和鉴定(Feramisco和Burdge，1980；Burbridge和Feramisco，1981；Pollard，1981；Condeelis和Vaeh，1982；Duhaiman和Bamburg，1984；Schleicher等人，1984)，并产生了广泛的交叉反应抗体(Lazarides and Burbridge，1975)。大多数非肌肉异构体的o~-肌动蛋白相互作用是洛克菲勒大学出版社，0021-9525/90/06/2013/12$2.00，《细胞生物学杂志》，第110卷，1999年6月
Cross-linking of actin filaments (F-actin) into bundles and networks was investigated with three different isoforms of the dumbbell-shaped c~-actinin homodimer under identical reaction conditions. These were isolated from chicken gizzard smooth muscle, Acanthamoeba, and Dictyostelium, respectively. Examination in the electron microscope revealed that each isoform was able to cross-link F-actin into networks. In addition, F-actin bundles were obtained with chicken gizzard and Acanthamoeba a-actinin, but not Dictyostelium u-actinin under conditions where actin by itself polymerized into disperse filaments. This F-actin bundle formation critically depended on the proper molar ratio of a-actinin to actin, and hence F-actin bundles immediately disappeared when free ct-actinin was withdrawn from the surrounding medium. The apparent dissociation constants (Kos) at half-saturation of the actin binding sites were 0.4 t~M at 22°C and 1.2/zM at 37°C for chicken gizzard, and 2.7 ~M at 22°C for both Acanthamoeba and Dictyostelium c~-actinin. Chicken gizzard and Dictyostelium a-actinin predominantly cross-linked actin filaments in an antiparallel fashion, whereas Acanthamoeba t~-actinin cross-linked actin filaments preferentially in a parallel fashion. The average molecular length of free ot-actinin was 37 nm for glycerolsprayed/rotary metal-shadowed and 35 nm for negatively stained chicken gizzard; 46 and 44 nm, respectively, for Acanthamoeba; and 34 and 31 nm, respectively, for Dictyostelium c~-actinin. In negatively stained preparations we also evaluated the average molecular length of ct-actinin when bound to actin filaments: 36 nm for chicken gizzard and 35 nm for Acanthamoeba ct-actinin, a molecular length roughly coinciding with the crossover repeat of the twostranded F-actin helix (i.e, 36 nm), but only 28 nm for Dictyostelium o~-actinin. Furthermore, the minimal spacing between cross-linking c~-actinin molecules along actin filaments was close to 36 nm for both smooth muscle and Acanthamoeba o~-actinin, but only 31 nm for Dictyostelium ot-actinin. This observation suggests that the molecular length of the a-actinin homodimer may determine its spacing along the actin filament, and hence F-actin bundle formation may require "tight" (i.e., one molecule after the other) and "untwisted" (i.e., the long axis of the molecule being parallel to the actin filament axis) packing of a-actinin molecules along the actin filaments. I N 1964 ~-actinin was discovered as a protein extracted from striated muscle promoting contraction of actomyosin gels and increasing the viscosity of F-actin solutions in vitro (Ebashi et al., 1964). As more effective separation methods became available, its interaction with actin was more systematically investigated (Holmes et al., 1971; Goll et al., 1972). Accordingly, the largest increase in viscosity of a F-actin solution containing a given amount of ct-actinin was observed at 0°C. Under these conditions the viscosity reached a maximum at an ot-actinin to actin ratio yielding about one a-actinin dimer molecule bound per crossover repeat (i.e., 36 nm) of the actin helix. A much higher o~-actinin to actin ratio was needed to yield the same amount of bound o~-actinin in solutions kept at 37°C. ot-Actinin is a homodimer composed of two polypeptides of ~100 kD each (Suzuki et al., 1976). Electron micrographs of shadowed ot-actinin have revealed a dumbbellshaped molecule with the two subunits being oriented antiparallel in a side-by-side association thus having a central dyad axis of symmetry (e.g., Pollard et al., 1986). Each polypeptide has a highly conserved actin binding site located near the NH2-terminai of the polypeptide chain that on the molecule is located on the "knob-like" protrusion at the end of the rod (Mimura and Asano, 1987; Imamura et al., 1988; Blanchard et al., 1989). As a consequence, o~-actinin crosslinks F-actin by binding with each end to an actin filament (podlubnaya et al., 1975). Over the past few years many more a-actin isoforms have been isolated and characterized (Feramisco and Burridge, 1980; Burridge and Feramisco, 1981; Pollard, 1981; Condeelis and Vahey, 1982; Duhaiman and Bamburg, 1984; Schleicher et al., 1984), and broadly cross-reacting antibodies have been raised (Lazarides and Burridge, 1975). The o~-actinin-actin interaction of most nonmuscle isoforms was © The Rockefeller University Press, 0021-9525/90/06/2013/12 $2.00 The Journal of Cell Biology, Volume 110, June 199
从棘阿米巴中纯化钙敏感肌动蛋白凝胶蛋白。
DOI：
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发表时间：
1981
期刊：
The Journal of biological chemistry
影响因子：
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作者：
Pollard,TD
通讯作者：
Pollard,TD