Amieva M R, Litman P, Huang L, Ichimaru E, Furthmayr H
Molecular Mechanisms of Disease Laboratories, Department of Pathology, Stanford University Medical Center, Palo Alto, California 94304-5324, USA.
J Cell Sci. 1999 Jan;112 ( Pt 1):111-25. doi: 10.1242/jcs.112.1.111.
Lamellipodia, filopodia, microspikes and retraction fibers are characteristic features of a dynamic and continuously changing cell surface architecture and moesin, ezrin and radixin are thought to function in these microextensions as reversible links between plasma membrane proteins and actin microfilaments. Full-length and truncated domains of the three proteins were fused to green fluorescent protein (GFP), expressed in NIH3T3 cells, and distribution and behaviour of cells were analysed by using digitally enhanced differential interference contrast (DIC) and fluorescence video microscopy. The amino-terminal (N-)domains of all three proteins localize to the plasma membrane and fluorescence recordings parallel the dynamic changes in cell surface morphology observed by DIC microscopy of cultured cells. Expression of this domain, however, significantly affects cell surface architecture by the formation of abnormally long and fragile filopodia that poorly attach and retract abnormally. Even more striking are abundant irregular, branched and motionless membraneous structures that accumulate during retraction of lamellipodia. These are devoid of actin, endogenous moesin, ezrin and radixin, but contain the GFP-labeled domain. While a large proportion of endogenous proteins can be extracted with non-ionic detergents as in untransfected control cells, >90% of N-moesin and >60% of N-ezrin and N-radixin remain insoluble. The minimal size of the domain of moesin required for membrane localization and change in behavior includes residues 1-320. Deletions of amino acid residues from either end result in diffuse intracellular distribution, but also in normal cell behavior. Expression of GFP-fusions of full-length moesin or its carboxy-terminal domain has no effect on cell behavior during the observation period of 6-8 hours. The data suggest that, in the absence of the carboxy-terminal domain, N-moesin, -ezrin and -radixin interact tightly with the plasma membrane and interfere with normal functions of endogeneous proteins mainly during retraction.
片状伪足、丝状伪足、微刺和收缩纤维是动态且不断变化的细胞表面结构的特征，而膜突蛋白、埃兹蛋白和根蛋白被认为在这些微延伸结构中发挥作用，作为质膜蛋白和肌动蛋白微丝之间的可逆连接。这三种蛋白的全长结构域和截短结构域与绿色荧光蛋白（GFP）融合，在NIH3T3细胞中表达，并通过数字增强型微分干涉相差（DIC）和荧光视频显微镜分析细胞的分布和行为。所有三种蛋白的氨基末端（N-）结构域定位于质膜，荧光记录与通过培养细胞的DIC显微镜观察到的细胞表面形态的动态变化平行。然而，该结构域的表达通过形成异常长且脆弱、附着不良且异常收缩的丝状伪足，显著影响细胞表面结构。更引人注目的是，在片状伪足收缩过程中积累的大量不规则、分支且静止的膜性结构。这些结构缺乏肌动蛋白、内源性膜突蛋白、埃兹蛋白和根蛋白，但含有GFP标记的结构域。虽然与未转染的对照细胞一样，大部分内源性蛋白可用非离子去污剂提取，但>90%的N-膜突蛋白和>60%的N-埃兹蛋白及N-根蛋白仍不溶。膜定位和行为改变所需的膜突蛋白结构域的最小尺寸包括第1至320位残基。从两端删除氨基酸残基会导致细胞内弥漫分布，但细胞行为正常。在6至8小时的观察期内，全长膜突蛋白或其羧基末端结构域的GFP融合蛋白的表达对细胞行为没有影响。数据表明，在没有羧基末端结构域的情况下，N-膜突蛋白、-埃兹蛋白和-根蛋白与质膜紧密相互作用，主要在收缩过程中干扰内源性蛋白的正常功能。
