Litman P, Amieva M R, Furthmayr H
Molecular Mechanisms of Disease Laboratories, Department of Pathology, Stanford University Medical School, 300 Pasteur Drive, Stanford, CA 94305-5324, USA.
BMC Cell Biol. 2000;1:1. doi: 10.1186/1471-2121-1-1. Epub 2000 Nov 1.
The cell surface undergoes continuous change during cell movement. This is characterized by transient protrusion and partial or complete retraction of microspikes, filopodia, and lamellipodia. This requires a dynamic actin cytoskeleton, moesin, components of Rho-mediated signal pathways, rearrangement of membrane constituents and the formation of focal adhesion sites. While the immunofluorescence distribution of endogenous moesin is that of a membrane-bound molecule with marked enhancement in some but not all microextensions, the C-terminal fragment of moesin co-distributes with filamentous actin consistent with its actin-binding activity. By taking advantage of this property we studied the spontaneous protrusive activity of live NIH3T3 cells, expressing a fusion of GFP and the C-terminal domain of moesin.
C-moesin-GFP localized to stress fibers and was enriched in actively protruding cellular regions such as filopodia or lamellipodia. This localization was reversibly affected by cytochalasin D. Multiple types of cytoskeletal rearrangements were observed that occurred independent of each other in adjacent regions of the cell surface. Assembly and disassembly of actin filaments occurred repeatedly within the same space and was correlated with either membrane protrusion and retraction, or no change in shape when microextensions were adherent.
Shape alone provided an inadequate criterion for distinguishing between retraction fibers and advancing, retracting or stable filopodia. Fluorescence imaging of C-moesin-GFP, however, paralleled the rapid and dynamic changes of the actin cytoskeleton in microextensions. Regional regulatory control is implicated because opposite changes occurred in close proximity and presumably independent of each other. This new and sensitive tool should be useful for investigating mechanisms of localized actin dynamics in the cell cortex.
细胞表面在细胞移动过程中会持续发生变化。其特征表现为微刺、丝状伪足和片状伪足的短暂突出以及部分或完全缩回。这需要动态的肌动蛋白细胞骨架、埃兹蛋白、Rho介导的信号通路成分、膜成分的重排以及粘着斑位点的形成。虽然内源性埃兹蛋白的免疫荧光分布是一种膜结合分子，在一些而非所有微延伸部位有明显增强，但埃兹蛋白的C末端片段与丝状肌动蛋白共分布，这与其肌动蛋白结合活性一致。利用这一特性，我们研究了表达绿色荧光蛋白（GFP）与埃兹蛋白C末端结构域融合蛋白的活NIH3T3细胞的自发突出活性。
C-埃兹蛋白-GFP定位于应力纤维，并在诸如丝状伪足或片状伪足等活跃突出的细胞区域富集。这种定位受到细胞松弛素D的可逆影响。观察到多种类型的细胞骨架重排在细胞表面的相邻区域相互独立地发生。肌动蛋白丝的组装和拆卸在同一空间内反复发生，并且与膜的突出和缩回相关，或者当微延伸部位粘着时形状无变化。
仅形状不足以作为区分缩回纤维与前进、缩回或稳定丝状伪足的标准。然而，C-埃兹蛋白-GFP的荧光成像与微延伸部位肌动蛋白细胞骨架的快速动态变化平行。由于在紧邻区域发生了相反的变化且可能相互独立，这暗示了区域调节控制。这种新的灵敏工具对于研究细胞皮层中局部肌动蛋白动力学机制应是有用的。
